WELCOME BACK TO THE ASSAY
WORKSHOP FOR HIGH-THROUGHPUT AND
LEADER DISCOVERY.
WE'RE EXCITED TO BE YOU SO, I
KEEP REMINDING EVERYONE TO
CONNECT WITH US SO THERE'S
DIFFERENT WAYS YOU CAN CONNECT
WITH US AND YOU CAN CONNECT
THROUGH TWITTER AND USING THE
HANDLE @ASSAY GUIDANCE.
WE ARE ON LinkedIn SO YOU
CAN JOIN OUR LinkedIn GROUP.
WE POST OUR EVENTS ON NIH NCATS
EVENTS WEBSITE AS WELL AS WE
HAVE A WEBSITE THAT WE ARE
ACTUALLY UPDATING SO IT WILL BE
ONE STOP FOR YOU TO GET ALL OUR
RESOURCES IN THE FUTURE.
I JUST WANT TO ALSO REWIND YOU
THAT WE HAVE AN AWESOME MANUAL,
ELECTRONIC BOOK CALLED ASSAY
GUIDANCE MANUAL THAT CARRIES
MULTIPLE CHAPTERS ON ALL
STAGES -- EARLY STAGES OF SMALL
MOLECULE TRACK DISCOVERY AND WE
WERE TALKING ABOUT IT ALL DAY
YESTERDAY AND WE'LL TALK ABOUT
IT TODAY.
YOU CAN ACCESS THIS FOR FREE
THROUGH NLM NCBI AND I'M SHOWING
YOU THE LINK HERE AND AGAIN,
WE'LL SHARE WITH YOU ALL THESE
LINKS AND WE'LL SHARE MOST OF
THE STRIDES AS WELL OF COURSE AT
THE DISCRETION OF THE SPEAKERS
AT THE END OF THE WORKSHOP.
ONE MORE THING, WE ARE OFFERING
DIGITAL BADGES IN COLLABORATION
WITH THE EDUCATION BRANCH TO
EARN A DIGITAL BADGE DESCRIBING
HOW THE LESSONS LEARNED FROM
THIS WORKSHOP CAN BE INTEGRATED
AND IMPROVE YOUR RESEARCH.
WE'LL FOLLOW YOU UP WITH
REGARDING WITH YOU AFTER THE
WORKSHOP.
AS WELL.
OK, SO, TODAY'S AGENDA, WE'RE
REALLY EXCITED TO HAVE FANTASTIC
TALKS TODAY AS WELL SO WE'LL
HAVE TALKS DNA AND 3D MODELS,
DATA ANALYSIS SO REALLY WE'LL
COVER A LOT OF DIFFERENT SPACE
HERE AND YOU WILL ACKNOWLEDGE
HOW EXCITING AND INTERESTING OR
NOT THIS TALKS ARE GOING TO BE
AND PROVIDE A LOT OF KNOWLEDGE
FOR YOU ALL AND HOPEFULLY HELP
YOUR RESEARCH.
SO, COUPLE OF THINGS WE'LL HAVE
A BREAK AT 10:30 AND A BREAK IN
THE AFTERNOON AT 3:00 AND WE'LL
HAVE A LUNCH BREAK A 12:30 AND T
5:00 WE'LL HAVE OPEN DISCUSSION
SESSION.
THESE ARE THE TIMES AVAILABLE
FOR YOU TO INTERACT WITH OUR
SPEAKERS TO TALK ABOUT YOUR
INDIVIDUAL RESEARCH NEEDS AND
SEEK HELP AS WELL.
THEY'RE HERE TO TALK TO YOU SO,
YOU KNOW, I RECOMMEND TAKING
ADVANTAGE OF THAT OPPORTUNITY.
SO, WITH THIS I'D LIKE TO
INTRODUCE OUR FIRST SPEAKERS OF
TODAY, SO Dr. TRAVIS
MATTHEWSON AND Dr. LANE
MILMIDED.TRAVIS WITHIN THE COMPD
MANAGEMENT AND DISTRIBUTION AT
PFIZER AND LANE IS A SENIOR
SCIENTIST SUPPORTING LABORATORY
AUTO MAKE AND LIQUID HANDLING
WORKFLOWS AND INNSTATION FOR
COMPOUND MANAGEMENT AND
DISTRIBUTION AT PFIZER.
THEIR TALK IS ENTITLED "BEST
PRACTICES IN COMPOUND
MANAGEMENT" AND I'M REALLY
EXCITED TO HAVE THEM HERE TODAY.
WELCOME TRAVIS AND LANE AND THE
FLOOR IS YOURS.
>>I DO WANT TO MENTION THAT
UNFORTUNATELY, TRAVIS AND LANE
THEY CONDITION MAKE IT TODAY SO
THEY WILL BE PRESENTING
VIRTUALLY.
>>HELLO I HOPE YOU CAN HEAR ME.
IT'S ECHOING.
>>HANG ON JUST A SECOND.
TRAVIS AND LANE CAN YOU HEAR ME?
WE'RE HAVING A FEW TECHNICAL
DIFFICULTIES, SORRY.
I DIDN'T DO ANYTHING.
IT WASN'T ME.
[LAUGHTER]
WHAT IS THAT?
DID YOU HEAR THAT EVERYONE, IT'S
NOT ME.
[LAUGHTER]
SOMETIMES IT IS ME.
>>CAN YOU HEAR ME NOW?
>>WE CAN HEAR YOU NOW BUT WE'RE
WORKING ON THE VIDEO PART OF
THIS TRANSACTION.
THERE'S SOMETHING VERY PROFOUND
ABOUT THIS IMAGE.
THERE'S SOMETHING ABOUT MODERN
TECHNOLOGY TODAY.
WE SEE YOU AND WE HEAR YOU.
CAN YOU SAY SOMETHING.
GREAT.
>>HELLO.
GOOD MORNING.
GOOD TO GO.
CAN YOU HEAR ME AND SEE THE
SLIDES.
>>YES.
>>OK.
IF YOU GO INTO PRESENTATION
MODE.
IS THAT BETTER?
>>NOT YET.
>>FANTASTIC.
NOT YET.
NOT YET IN PRESENTATION MODE.
YOU CAN HIT THE PRESENTATION
ICON OR F5 WORKS IF YOU ARE ON A
PC.
>>ARE YOU USING A TWO-SCREEN
SET UP?
>>YEAH.
LET ME JUST UNPLUG THIS.
TECHNICAL DIFFICULTIES ALL
AROUND.
ARE WE THERE YET?
IS IT WORKING RIGHT.
>>NOT QUITE YET.
HOW ABOUT THE ICON AT THE
BOTTOM.
YES, THAT ONE, EXACTLY.
WE CAN JUST ROLL WITH THIS.
YOU COULD ALSO TRY THE SLIDER,
THERE, OH, NO.
THANK YOU FOR YOUR PATIENCE, I
APPRECIATE IT.
TROY SHATRY SHARE AGAIN.THEN TRD
LET'S SEE IF IT WORKS.
NO, OK.
ARE WE GOOD?
>>WE'RE NOT BUT LET'S ROLL WITH
THIS AND WE'LL GET A LITTLE
SNEAK PREVIEW OF UPCOMING
SLIDES.
YOU ARE GETTING THE OTHER
SLIDES.
WE ONLY HAVE ONE SCREEN NOW.
WE JUST REMOVED THE OTHER ONE.
SORRY ABOUT THIS EVERYONE.
SORRY WE COULDN'T BE THERE
IN-PERSON.
WE'LL JUST TROY WE'LL DO THE
BEST WE CAN, SORRY ABOUT THAT.
HI, MY NAME IS TRAVIS AND AS YOU
HEARD I LEAD THE AUTOMATION DATA
SUPPORT IN LIQUID FUNCTIONS FOR
COM POUND MANAGEMENT AND I HAVE
LANE MY AUTOMATION EXPERT AND
WE'LL TALK ABOUT BEST PRACTICES
FOR COMPOUND MANAGEMENT.
SOME OF THE TOPICS WE'RE GOING
TO COVER IN IN PRESENTATION ARE
STORAGE CONDITIONS, COMPOUND QC
AND JUST DATA FLOWS IN GENERAL.
SO BACK IN 2004, WHEN MARK
SUCKER BURG WAS COMING OUT WITH
Facebook, THE BIGGEST KEN WAS
THAT FREE STOP CYCLES WERE
HAVING THE GREATEST IMPACT ON
COMPOUND DEROGATION.
WHAT WE FOUND INSTEAD WAS THAT A
FRO CELL CYCLE AFTER 10 CYCLES,
HAD VERY LITTLE IMPACT ON THE
COMPOUNDS THEMSELVES AND
INSTEAD, MORE IMPORTANTLY, WE
FOUND THAT A REFRIGERATOR WAS
THE FIRST CONDITION YOU COULD
STORE YOUR COMPOUNDS AT.
WE SAW THINGS WHERE BREAKING
DOWN QUICKLY AND WE WERE GETTING
HIGHER LEVELS OF DEGRADATION TO
THE RECOMMENDATION AT THE TIME
WAS KEEP THINGS AT ROOM
TEMPERATURE FOR UP TO SIX MONTHS
OR KEEP THEM FROZEN.
AND IN 2019, WE DID WHAT WE
CALLED OUR CANARY STUDY AND WHAT
THAT WAS WAS A SELECT GROUP OF
ABOUT 3,000 COMPOUNDS THAT WE
BASICALLY, WE WEREN'T SO
CONCERNED ABOUT FRO CELL CYCLES
AT THE TIME WE WERE MORE
CONCERNED ABOUT THE STORAGE
CONDITIONS THEMSELVES AND FROM
FROZEN, WE WENT TOUT A YEAR THE
COMPOUNDS WERE PRETTY STABLE AND
IT WAS CONSISTENT AND SO THAT IS
WHERE WE HAD OUR RECOMMENDATIONS
THAT IF YOU KEEP THEM AT ROOM
TEMPERATURE, KEEP THEM IN THERE
YOU HAVE UP TO A YEAR.
WE USUALLY DON'T COPE THEM THOSE
CONDITIONS FOR MORE THAN SIX
MONTHS AND THEN THEY GO INTO
FROZEN.
THE REASON WE DID THIS IS
BECAUSE -- YOU ARE STILL ON THE
TITLE SLIDE HERE.
I WAS ON SLIDE THREE.
I'LL JUST SHARE MY ENTIRE
SCREEN.
YEAH, MAYBE.
DO YOU SEE THE FILE?
>>NOW WE SEE THE FILE INTEGRITY
SLIDE.
DO YOU -- OH YEAH!
YEAH!
>>NOW WE'LL FULL SCREEN AND IF
YOU COULD JUST ROLL IT BACK A
LITTLE BIT, MAYBE START -- HOW
ABOUT START THERE AGAIN.
SO BASICALLY YOU KNOW, JUST
LETTING YOU KNOW, THESE ARE THE
TOPICS WORE PLANNING ON
COVERING, STORAGE, COMPOUND QC
AND DATA FLOW THESE ARE ALL
WORKED INTO OUR COMPOUND
MANAGEMENT SYSTEMS.
SO BACK TO THIS, SORRY WE
WEREN'T ON THE SAME SLIDE.
THE ONE WE LEFT WAS WHEN
Facebook STARTED IN 2004 AND
AS I WAS SAYING, AT THE TIME
PEOPLE WERE VERY CONCERNED ABOUT
FREE CELL CYCLES HAVING A
SIGNIFICANT IMPACT ON COMPOUND
DEGRADATION.
WE FOUND FROM THIS DATA WAS THAT
IT REALLY WAS THE STORAGE
CONDITIONS THEM ELVES HAD A
GREATER IMPACT THAN THE FREE
CELL CYCLES AND WE DID 10 FREE
CELL CYCLES, WE DID THIS FOR SIX
MONTHS, AND THE RECOMMENDATION
THAT CAME OUT OF THIS WAS IF YOU
HAVE THEM AT ROOM TEMPERATURE,
DO IT FOR NO MORE THAN SIX
MONTHS AND TROY TO KEEP THEM
FROZEN AND NEVER PUT ANYTHING IN
A REFRIGERATOR AT 4C SINCE IT
HAD THE GREATEST IMPACT ON THE
COMPOUNDS THEMSELVES.
IN 2019, AT THE TIME, MORE
CONCERNED ABOUT JUST THE STORAGE
CONDITIONS THEMSELVES, WE DID
ANOTHER SPECIAL STUDY ON ABOUT
3,000 COMPOUNDS AND DIVERSE SET
OF COMPOUNDS AND THE REAL POINT
OF THIS STUDY WAS BECAUSE OUR
SYSTEM TEMPERATURE, HAD NO
INDEGRADATION ISSUES AND OUR
COMPOUNDS WERE STORED AT ROOM
TEMPERATURE FOR GREATER THAN A
YEAR ON OUR SYSTEM FOR ORDER
FULFILLMENT.
THIS GOES OUT TO A YEAR.
WE DIDN'T SEE SIGNIFICANT
DEGRADATION WITHIN A YEAR SO
THAT WAS FINE.
WE HAVE DATA THAT GOES TO -- UP
TO TWO YEARS.
AT LEAST WE'VE GONE BEYOND THIS.
AGAIN, THE RECOMMENDATION IS
DON'T PUT ANYTHING IN A 4° ROW
FRIDGE RATER AND THAT'S PRETTY
MUCH THE WORSE THING CAN YOU DO
TO YOUR COMPOUNDS BUT OBVIOUSLY,
THE BEST THING YOU CAN DO IS
COPE KEEPTHEM FROZEN.
IF THEY HAVE TO BE AT ROOM
TEMPERATURE, TRY TO KEEP THEM IN
THEIR CONDITIONS.
WE ALSO DID AT THE TIME, SO WE
STARTED LOOKING AT THE NEW
ACOUSTICS TUBE TECHNOLOGY AND IN
THESE TUBES WE WANTED TO KNOW
HOW FAST IS THE DMSO ABSORBING
WATER AND WE KNOW DMSO IS HYDRO
SCOPIC AND THE ONE ON THE LEFT,
THAT WAS OVER THE COURSE A
REGULAR BUSINESS DAY SO YOU CAN
SEE EVEN IN JUST A REGULAR
EIGHT-HOUR DAY YOU WOULD GET
CLOSE TO EQUILIBRIUM IF THEY'RE
EXPOSED IN THE LAB CONDITIONS
FOR THAT TIME.
THE ONE ON THE RIGHT WE STRETCH
OUT BECAUSE THAT DRAMATIC LINE
IN THE LEFT WAS OVERNIGHT SO
WHAT YOU CAN SEE THERE IS THAT
AS YOU APPROACH EQUILIBRIUM.
THE REAL POINT, EVEN IN THESE
ACOUSTIC TUBES WITH A SMALL
CENSUS AREA, 100% DSMO IS
ABSORBING WATER AT A VERY RAPID
RATE.
SO JUST SOME GENERAL THOUGHTS
FOR BEST PRACTICES IF YOU ARE AT
ROOM TEMPERATURE THREE MONTHS
DON'T PUT ANYTHING AT 4C WE GO
UP TO SIX MONTHS AT ROOM
TEMPERATURE AND THAT IS BEEN
PRETTY GOOD FOR OUR COMPOUNDS.
KEEP THEM DRIVEWAY THEM DRY.
IF LONG-TERM STORAGE MAKE SURE
THEY'RE SEALED AND FROZEN -20.
NEVER, NEVER AT 4°.
ALL THE IMAGES ON THE RIGHT SIDE
THERE, BASICALLY REFLECT WHAT
HAPPENS WITH DMSO, TRY TO ALWAYS
DEEP YOUR DILUTION AT DMSO FOR
100% AND GO A KEY US AND YOU
NEED TO USE IT WITHIN 24 HOUR
AND LEAVE THOSE HANGING AROUND
AQUEOUS FOR LONGER THAN THAT.
TO REITERATE SOME CONSIDERATIONS
HERE.
WE HAD QC FAIL AND THIS IS THE
CASE ACROSS THE BOARD.
YOU WILL NEVER HAVE A PERFECT
FILE.
WE EXPECT TO ROUND 10% TO 15%
THAT HAS SOME SORT OF ISSUE.
SO JUST BE VERY FAMILIAR WITH
THAT THAT THAT'S WHAT YOU ARE
DEALING WITH.
YOU SHOULD ALWAYS TRY TO WHERE
YOUR QUALITY OF YOUR COMPOUNDS
AND THE STEWARDSHIP ON YOUR
COMPOUNDS AND IF YOU HAVE ANY
KIND OF COM POUND QUALITY ISSUES
THAT COULD INTERFERE WITH THE
QUALITY OF THE COMPASS FOR ASSAY
IT'S GOOD TO KNOW AND UTILIZE
YOUR RESOURCES SO YOU REALLY
UNDERSTAND WHAT YOU ARE WORKING
WITH.
MAKE SURE YOU ARE ALWAYS VERY
PROACTIVE.
YOU UNDERSTAND THAT THE ASSAY
AND ANY SENSITIVITY TO ANY
COMPOUND QUALITY ISSUES IS
IMPORTANT.
IF YOU HAVE TO DO A RISK
ASSESSMENT AS FAR AS THE
QUALITY, INTEGRITY AND MAYBE YOU
NEED TO DO A DEEPER
CHARACTERIZATION OF THE
COMPOUNDS SO SEE WHAT THEY'RE
ACTUALLY DOING, WHAT WE
BASICALLY ARE FROM DRY TO
WHATEVER CONCENTRATION YOU ARE
STORING AT LIQUID, THAT'S WERE
THE BIGGEST ERROR TIMES COME IN
SO THEY'RE NOMINAL
CONCENTRATION.
IT'S PLUS OR MINUS.
JUST TO GO INTO OUR QC PROCESS,
SO WE HAVE OUR AUTOMATED QC
PROCESS.
FOR OUR NEELY SYNTHESIZED
COMPOUNDS WE HAVE OUR PHRASES,
IF YOU SCREEN IT WE'LL QC IT.
WE HAVE A 30-DAY WINDOW IN WHICH
A COMPOUND IF IT GETS ORDERED IT
WILL GO TO QC UNLESS THERE'S A
QC VALUE PRESENT.
WE USE THIS CRITERIA AND WE HAVE
A PASS-FAIL SUSPECT RESULT TO
EACH OF THE COMPOUNDS AND WE
ALSO CHECK TO MAKE SURE THAT THE
MASS WAS FOUND AND THAT THE
PURITY IS GREATER THAN 80%.
WE HAVE THIS CATCH-ALL CATEGORY
CALLED SUSPECT WHICH CAUSES
CONFUSION AND BASICALLY THAT IS
EXACTLY THAT.
IT'S A CATCH-ALL.
THE METHOD FAILED.
THE INSTRUMENT FAILED.
IT COULD BE ANYTHING.
I TROY TO TELL PEOPLE IF YOU SEE
A SUSPECT, ROW SEND IT FOR QC OR
IT JUST DOESN'T WORK IN OUR
METHOD AND YOU WON'T GET A QC
AND SOMETHING ELSE HAS TO BE
DONE.
IT HAS THE CONNOTATION OF IT'S
SUSPECT THERE'S SOMETHING WRONG.
IT COULD BE FINE.
AND THEN, I WANTED TO JUST POINT
OUT, IT'S UV215 HAS TO BE 80% OR
GREATER.
THIS IS A HARD LINE.
IT'S GOT TO UNDERSTAND THAT DATA
BECAUSE IT'S VERY POSSIBLE TO
GET A COMPOUND THAT WILL BE 79
AND A HALF, THE FIRST TIME YOU
RUN IT AND 80 THE NEXT TIME AND
YES, THE FIRST ONE WILL BE
LABELED A FAIL AND THE NEXT
RESULT WILL BE LABELED A PASS.
IT'S IMPORTANT TO UNDERSTAND HOW
THAT DATA WORKS AND HOW TO APPLY
THAT TO THE COMPOUNDS THAT WE'RE
LOOKING AT.
THERE'S AN EXAMPLE, SO, ON THE
TOP, IT'S PROBABLY HARD TO SEE
BUT BASICALLY WE HAVE A SYSTEM
SET UP, WHERE WHEN WE DELIVER
PLATES TO OUR CUSTOMERS, OUR
SCREENERS, THEY HAVE THE TOOL
THEY CAN CLICK ON AND SCAN THE
BAR CODE AND IT WILL GIVE THEM A
PICTURE OF THEIR PLATE AND ANY
OF THE MOST RECENT QC DATA ON
THE TOP.
YOU CAN SEE YOUR PASS, FAILS AND
SUSPECTS.
IF THERE'S NO VALUE THERE, THE
RESULT HASN'T BEEN PUBLISHED OR
RUN YET SO MAYBE THEY NEED TO
GET IT ROW RUN AND THEN IN THE
BOTTOM, WE HAVE PRECIPITATE
DATA.
WE USE THIS TWO BORDER WE HAVE
AND IT TAKES A PICTURE OF THE
COMPOUNDS SOL R IN TUBES AND IT
DETERMINES IF IT THIS IS THERE'S
MY PRECIPITATE IN THE TUBES.
WE DON'T PUBLISH A NO
INTENTIONALLY.
WE ONLY PUBLISH A YES AND IT'S
NOT A YES IT'S PRECIPITATE YOU
SHOULD BE QUESTIONING IF YOU SEE
A YES.
WE HAVE A PROCEDURE WHERE IF
SOMEONE REALLY WANTS
CONFIRMATION, WE CAN ACTUALLY GO
PULL THE SOURCE TUBE AND LOOK AT
IT AND MICK A JUDGMENT CALL.
THIS GIVES EVERYBODY RAPID
ACCESS TO ALL THE DATA THAT THEY
CAN JUST MARRY UP TO WHAT THEY
HAVE AND HAVE THIS INFORMATION
PREPARED WHEN THEY DO THE
SCREENING.
SO WHEN THINKING ABOUT KIND OF
TRIAGE AND COMPOUND QC ONE THING
IS IMPORTANT TO BE AWARE THERE
ARE BATCH-TO-BATCH DIFFERENCES
SO WE HAVE SEVERAL BATCHES OF
OUR COMPOUNDS AND OFTEN TIMES
ORDERS COME IN FROM A PARENT
STRUCTURE AND NO ONE CARES ABOUT
WHAT BATCHES AT THE INITIAL TIME
FOR A HIGH SCREEN PER SE BUT
IT'S IMPORTANT TO KNOW THERE ARE
OTHER BATCHES SO IF YOU ARE
GETTING SOME STRANGE DATA,
THERE'S ALWAYS AN OPPORTUNITY TO
MAYBE GO FIND AN ALTERNATE BATCH
AND ALWAYS CHECK COMPOUND QC, IF
THERE ISN'T A RESENT QC DATA
VALUE IT'S POSSIBLE TO SEND IT
TO YOUR QC SCREEN AND GET AN
UPDATED VALUE ON THE COMPOUNDS
YOU ARE RUNNING SO YOU CAN TAKE
A LOOK AT THAT AND IT'S
IMPORTANT TO PRETTY MUCH CHECK
EVERY OPTION YOU HAVE BEFORE YOU
TRY TO COMMIT SYNTHETIC CHEM IS
TREMENDOUS RESOURCES TO HAVE IT
THE SIZE.
THIS IS VERY IMPORTANT.
I MEAN, I KNOW IT'S THE LAST
RESORT FOR US WE LOOK AT WHAT IS
AVAILABLE AND DUE AND WHAT OTHER
SOURCES OF THE COMPOUND ARE IN
THERE.
SO PULLING THIS TOGETHER AND
APPLYING TO YOUR HTS SCREENING
FUNNEL AND IT'S VERY STANDARD.
YOU RUN YOUR PRIMARY HTS AND
KEEP YOUR STOCKS AT 100% DMSO
AND DON'T GO AQUEOUS AND RUN
THEM WITHIN THAT DAY IT'S
IMPORTANT AND WHEN YOU GET YOUR
HITS BACK, IF YOU, FOR WHATEVER
REASON, YOU HAVE AN UNEXPECTED
LILYLOW RATE, RETEST BUT MAYBE U
SHOULD LOOK FOR YOUR ANALYTICAL
DATA AND USE THAT AS PART OF
YOUR TRIAGE TO SEE IF THE QC
DATA WAS BAD OR THERE'S
SOMETHING AN ALTERNATE BATCH YOU
NEED TO LOOK AT AND THEN, AS YOU
MOVE INTO YOUR FOLLOW-UP AND
YOUR IC50s, AGAIN, SAME THING,
DON'T PREPARE YOUR IC50s
AQUEOUS KEEP EVERYTHING AT 100%
DMSO AND GO INTO YOUR ASSAY
PLATE BEFORE YOU DELAWSUIT
AQUEOUS AND RUN THEM THE SAME
DAY.
AGAIN, IT'S IMPORTANT TO TAKE A
LOOK AT ANY ANALYTICAL
CHARACTERIZATION DATA THAT YOU
HAVE AND ININCLUDED THAT AS PART
OF YOUR FILTERING FOR YOUR HITS
AND THEN VERIFY EVERYTHING
BEFORE GOING BACK TO RESENT
ASSISTS.
SO AGAIN, BEST PRACTICES,
ESPECIALLY FOR -- NEVER USE OLD
DMSO UNDER LAB CONDITIONS YOU
WILL GET THE EQUILIBRIUM
QUICKLY.
DON'T LEAVE THE CAP OFF THE DMSO
DO LEAVE IT OUT AND DON'T TRUST
IT SITTING ON THE DECK ALL DAY.
MAKE SURE YOU SWAP THOSE OUT.
KEEPING IT FRESH.
ONE OF OUR RECOMMENDATIONS WHEN
WE GET DRY, WE RECOMMEND NOT
ORDERING LESS THAN TWO MIGS OF A
SAMPLE AT A TIME BECAUSE THE
VARIATION IN CALIBRATION.
IT'S WHERE THE BIGGEST ERROR
COMES IN IF THEY ASK FOR ONE
MIG, YOU CAN HAVE VARIATION AND
YOU WILL BE OFF.
IT'S ALSO IMPORTANT TO CHECK
YOUR LIQUID HANDLING EQUIPMENT
AND MAKE SURE THAT YOU ARE
FOLLOWING ALL THE PROPER
MAINTENANCE, REGULAR
MAINTENANCE, GETTING PMs DONE
AND MAKING SURE THEY'RE
CALIBRATED AND YOU QC THEM AS
WELL.
SO, YOU HEARD ME TALK BEFORE
ABOUT THE ACOUSTIC TUBES.
THIS IS THE CUTTING EDGE
TECHNOLOGY FOR COMPOUND
MANAGEMENT STORAGE.
WE HAVE THE MIDDLE, YOU CAN SEE
THIS BIG PICTURE THAT'S A LARGE
SAMPLE STORE WE HAVE THAT WE
HOLD ALL OF OUR TUBES AND IN
THERE ARE THESE VERY SMALL
ACOUSTIC TUBES.
THEY'RE VERY INTERESTING, THEY
ALL HAVE INDIVIDUALLY SEALS WITH
SCREW CAP.
YOU WILL SEE THESE FOUR TINY BAR
CODES ON THE BOTTOM AND THIS IS
CLEVER WHAT THEY DID.
THEY FOUND THAT THE BAR CODES
ITSELF ON THE BOTTOM INDER
FEARED WITH THE ACOUSTIC
TECHNOLOGY.
THERE'S A DIAGRAM OF HOW IT
WORKS OVER HERE.
IT ACTUALLY INTERFERED WITH THE
ACOUSTIC SO THEY SPLIT IT NO
FOUR.
WHY FOUR?
IT'S DOUBLE REDUNDANT.
IT'S TWO HALF OF THE BAR CUED
SPLIT IN COUPE LA INDICATE SO
EVEN IF DAMAGE HAPPENS TO TWO OF
THESE, AS LONG AS THEY'RE NOT ON
THE DIAGONAL IT CAN READ THE BAR
CODE WITHOUT ISSUES.
THESE ARE ALL INTER 96 RACK.
SO WE CAN PUT SIX OF THESE RACKS
ON A TRAY.
THEY GET LOADED AND RE FORMATTED
INTO A D TRAY AND IT'S JUST TO
INCREASE THE STORAGE INSIDE THE
STORAGE.
BY DOING THAT IN THE SAME SPACE
THAT WE HAVE SIX RACKS, WE'RE
FITTING 1,000 AND 8 TUBES IN THE
HC RACK SO THIS PICTURE IS OUR
NEW SYSTEM THAT WE USE WITH THE
NEW ECHOES, SUCCESS IT THAT CAN
USE THESE AS A SOURCE OR A
COMPOUND AND THE TUBES AND IT
KEEPS ALL SAMPLES FROZEN AND NOT
IN USE AND ONLY EXPOS SAMPLES TO
SIGN AND ROOM TEMPERATURE
CONDITIONS WHEN THEY'RE BEING
USED TO FULFILL AN ORDER AND THE
ECHO SO THEY GO IN THE ECHO IT
DETECTS THE BOTTOM AND EJECTS
THE 2.5 DROPS INTO THE TARGET
PLATES.
COMPARED TA OUR OLD SYSTEM WE
HAD TO DO OUR CANARY STUDY
BECAUSE OUR PLATES BASICALLY SIT
AT ROOM TEMPERATURE OUT ON RACKS
AND AGAIN, IT'S BECAUSE THEY'RE
IN PLATES IF YOU ORDER ONE
SAMPLE FROM A PLATE, IT HAS TO
BE UNSEALED.
THE WHOLE PLATE OF COMPOUNDS IS
EXPOSED WHILE THAT ONE COMPOUND
IS BEING TRANSFERRED SO THIS
SOLVES THAT PROBLEM AND THOSE
COMPOUNDS WILL STAY FROZEN AND
UNTOUCHED WHEN COMPOUND ORDER
FULFILLMENT IS BEING DONE.
YOU WILL HEAR US EXPLAIN THE
WORKFLOWS.
IN ARP IT'S THE COMPOUND IN 100%
DMSO A SINGLE POINT OR ZERO
DILUTION AND WHEN IT GETS INTO
THE HANDS OF THE SCREENER THAT'S
WHERE TH REAGENTS ARE ADD AND IT
GOES INTO THE ASSAY.
AN NARP IS FOR ANYTHING IT COULD
BE SORT OF A PLATE THAT GETS
TRANSFERRED INTO A DIFFERENT
PLATE THAT IS THEN
TRACTOR-TRAILER ASSAY READY
FINAL PLAY OR MAYBE IT GETS THE
BUFFER AND EVERYTHING ADDED
BEFORE IT'S TRANSFERRED AND WITH
THAT I'LL HAPPENED HAND IT OVER.
>>IN OUR COMPOUND MANAGEMENT
WORK FLOW WE START OUT IN A
1.4-MILLILITER TUBE THAT IS
STORED IN A HAMILTON VERSE OWE
STORE IN COMPOUND MANAGEMENT
ORGANIZATIONS THERE MAY BE
DIFFERENT TYPES OF EQUIPMENT
THAT HAVE THE ORGANIZATION FINDS
FITS WELL UNDER THEIR WORK FLOW.
WE FOUND THAT WE'VE BEEN ABLE TO
STANDARDIZED WITH THE HAMILTON
STAR AS THE TIP-BASED PLATFORM
AND THE ECHO PRODUCTS FOR ACCUSE
TICK DISPENSING INTO ASSAY READY
PLATES.
TYPICALLY FOR NARP PROCESS IT
WOULD BE A STEP FOR ANY ASSAY
READY PLATE PRODUCTION STEP, YOU
CAN SEE THAT IT ALWAYS WOULD
START WITH A TIP-BASED STEP ONE
STARTING OUT FROM THE 1.4ML TO.
WHEN WE MOVED TO THE ACOUSTIC
TUBE IT WOULD REMOVE THE STEP
FROM OUR ASSAY PRODUCTION
WORKFLOWS.
THERE'S ALWAYS A TIME WHEN USING
A TIP-BASED TO DID A TRANSFER
MAKES MORE SENSOR LESS THAN AN
ACOUSTIC TRANSFER WE CALL THIS
THE SOUND CHOICE.
SO, ONE FACTS OR TO CONSIDER IS
THE TIME IT TAKES TO DISPENSE SO
WITH AN ECHO LIQUID HANDLER,
DISPENSE NORTH 2.5 NANO LITER
INCREMENTS THE HIGHER VOLUME YOU
DISPENSE INCREASES THE TIME SO
THE GRAPH ON THE TOP SHOWS THE
DIFFERENCE IN TIME FOR ACCUSE
TICK IN THE GREEN DOTS AND FOR
TYPICAL BY PETTER THE TIME TO
DISTANCE LIQUIDS IS CONSTANT.
THE TARGET VOLUME ALSO EFFECTS
THE CV THAT YOU CAN EXPERIENCE
SO THE LOWER GRAPH SHOWS THAT
WITH A TIP-BASED DIS SENSE YOU U
CAN LOWER YOUR CV YOU WOULD SEE
IN THOSE DISPENSES AT ABOUT ONE
MIKE RE LITER YOU CAN SEE THOSE
CROSS WHERE THE ACOUSTIC
DISPENSER IS A CONSTANT AROUND
3% WITH SOME VARIATIONS BUT THE
TIP-BASED DISPENSER WILL DROP
BELOW THAT IF YOU INCREASE THAT
VOLUME.
AND THE CONTAINERS AS WELL ARE
LIMITATIONS SO YOU ARE ALWAYS
REQUIRED TO USE ECHO QUALIFIED
CONTAINERS WHICH HAVE SPECIFIC
DEAD VOLUMES.
SO, DEPENDING ON THE DENSITY OF
YOUR CONTAINER, YOU CAN HAVE AS
LOW AS A ONE MICRO LEADER DEAD
VOLUME FOR A CONTAINER FOR AN
ACOUSTIC DISPENSER BUT IT CAN
INCREASE FROM THERE UP TO 15
MICRO LITERS FOR THE ACOUSTIC
TUBES.
ONE THING ENABLED BY ACOUSTIC
DISPENSING IS TYPICAL SERIAL DIE
SOLUTION TRANSFERRING FROM ONE
WELL OR CONTAINER TO THE NEXT
AND A CONSTANT VOLUME WHEREAS
DIRECTION DILUTION ALLOWS YOU TO
DISTANCE THE ACTUAL AMOUNT OF
VOLUME NEEDED TO ACHIEVE THE
DESIRED CONCENTRATION IN A WELL
AND USING A VARIABLE AMOUNT OF
BUFFER TO BRING THAT UP TO THE
DESIRED COMPOUND.
THE BIGGEST IMPACT HERE IS WE
DOUGH CRDECREASE THE ERROR.
THERE ARE LIMITATIONS.
THE TWO AND A HALF NANO LITER
DROP SIZE DOES CAUSE A POTENTIAL
DEVIATION FROM YOUR TARGET
VOLUME, WHICH DOES CREATE A
DEVIATION FROM YOUR
CONCENTRATION OVER YOUR DILUTION
RANGE.
WE ARE ALSO LIMITED AND OUR
DILUTION RANGE IN HOW LOW WE CAN
ACHIEVE USING A SINGLE INTER
MEDIATE PLATE.
SOME PROCESSES MAY USE A SECOND
PLATE BUT WE OPTED OUT OF THAT
FOR SIMPLICITY IN OUR PROCESSES
AND LIMIT THE LOWEST
CONCENTRATION THAT WE CAN
ACHIEVE FOR OUR DOWNSTREAM
PROCESSES.
DILUTION FACTOR IS SOMETHING TO
CONSIDER, 3.162 IS A HALF LOG
AND TYPICALLY IT'S SOMETHING
THAT IS USED FREQUENTLY IN
SCREENING BUT WE FOUND ACTUALLY
USING WHOLE NUMBERS DUE TO THE
2.5 NANO LITER DROP SIZE IS MORE
ADD VADVANTAGEOUS.
THE VOLUME HERE IS A FACTOR AS
WELL SO THE LARGER VOLUMES DO
TAKE LONGER TO TRANSFER.
SO IT'S SOMETHING TO CONSIDER
WHEN WE WORK WITH OUR DOWN
STREAM SCREENERS WHEN SETTING UP
THEIR SUBMITALS IS THE NUMBER OF
DILUTION POINTS AND DILUTION
FACTORS SO AS I MENTIONED, YOU
CAN SEE THAT THERE'S A
DIFFERENCE HERE IN THE WHOLE
NUMBERS ON THE LEFT AND RIGHT
AND THE HALF LOG DILUTION YOU
CAN SEE A MUCH BOUNCIER
DEVIATION FROM THE TARGET
CONCENTRATION.
SO YOUR TAKE HOMES HERE ARE
REALLY THAT ONE THING IS THAT A
10-POINT DILUTION IS BETTER THAN
11, ESPECIALLY IF YOU ARE AT A
HIGHER DILUTION FACTOR AND YOUR
WHOLE NUMBER GIVES YOU GENERALLY
A CLOSER TO YOUR TARGET.
WHEN YOU ARE LOOKING AT YOUR TOP
CONCENTRATION AND THE VOLUME OF
YOUR DELIVERABLE, THESE THINGS
GO HAND AND HAND AS WELL AND
WHETHER YOU ARE WORKING WITH A
FIXED CONCENTRATION INVENTORY OR
YOU ARE ABLE TO DILUTE YOUR
SOURCE TO THE TOP CONCENTRATION,
YOU CAN ALSO EFFECT YOUR ABILITY
TO HIT THOSE LE LOWER
CONCENTRATIONS IN YOUR DILUTION.
YOU CAN INCREASE YOUR VOLUME TO
ACCOMMODATE THAT AND TO GET TO
THOSE LOWER CONCENTRATIONS AND
FOR A THREEFOLD DILUTION, THE
MULTIPLE OF 22.5 NANO LITER IS
BEST AND 40 AND I DERIVED THAT
FROM THE GUIDELINE CALCULATION
UP AT THE TOP WHICH DICK TAKES A
GOOD TARTING PLACE THAT A ASSAY
READY PLATE MIGHT BE READY TO
ACHIEVE.
IT'S VERY IMPORTANT FOR US TO
MAINTAIN WE USING SINGLE SHELF
AND WE HAVE A HOME-BUILT SERIAL
DILUTION THAT WE DO THAT IS
EXCEL BASED AND VALUES AND CAN
GIVE US THE HEALTH VALUE IN HOW
WELL OUR SYSTEMS ARE MAKING
SERIAL AND DIRECT DILUTION
DELIVERABLES.
YOU CAN SEE HERE, IN THIS GRAPH
AND EXAMPLE OF WHERE WE
IDENTIFIED A PROBLEM, SO YOU CAN
SEE OVER TIME, WE'RE ABLE TO
LOOK AT QC VALUES FROM OUR ECHOS
AND YOU CAN SEE ON THE TOP, A
NICE TREND OVER TIME WHERE WE'RE
NOT SEEING A LOT OF VARIATION
AND ON THE BOTTOM, AN EXAMPLE
FROM A HAMILTON WHERE AND YOU
CAN SEE THE VALUES BACK TO A
HISTORICAL NORMAL VALUE AFTER
THAT SERVICE WAS CREED.
WITHIN COMPOUND MANAGEMENT, WE
TRACK ALL OF OUR PROCESSES AND
USING ONE AND TWO D BAR CODES
THROUGH OUT EVERY STEP OF THE
WAY AND WE USE LABORATORY
INFORMATION MANAGEMENT SYSTEMS
TO TRACK THOSE IN EVERY STEP OF
THE PROCESS AND YOU CAN SEE AN
EXAMPLE HERE OF A USER INTERFACE
AND ONE OF OUR SYSTEMS THAT
ALLOWS US TO ESSENTIALLY TURN
EVERY STEP OF THE OF OUR PROCESS
INTO A BUSINESSARD THAT CAN BE
GIVEN BY ANY MEMBER OF THE LAB
AND ONE THING WE FOUND WITH THIS
IS REALLY REDUCES OUR TRAINING
BURDEN AND WHEN WE HAVE STAFF
TURNOVER AND WE'RE ABLE TO
QUICKLY BRING OUR STAFF UP TO
SPEED AND MAKE THEM ABLE TO
PROCESS OUR DELIVERABLES.
WE HAVE PROCESS ANOMALY THAT'S
WE HAVE TO TROUBLE SHOOT SO
OCCASIONALLY, A DOWNSTREAM
CUSTOMER COMES AND SAYS THEY
FOUND SOMETHING OUT OF ORDINARY
AND WE USED TOOLS WITHIN OUR
TOOLBOX TO ANALYZE THE PROCESS
AND DETERMINE WHERE IT OCCURRED.
ONE GREAT EXAMPLE IS THE ECHO
TRANSFER LOB AND WE'LL USE OUR
LIMB SYSTEM TO DETERMINE WHICH
ECHO THE PROCESS OCCURRED ON AND
WE CAN THEN GO LOCK AT THAT LOG
TO POTENTIALLY DETERMINE A
FAILURE.
WE CAN ALSO CHECK QC AND
DETERMINE THE PERFORMANCE OF
THAT INSTRUMENT AT THAT TIME AS
WELL.
SO, JUST TO WRAP THINGS UP,
SAMPLE STORAGE IS SOMETHING THAT
IS VERY IMPORTANT AND IN
COMPOUND MANAGEMENT AND TO
PROLONG THE LIFE OF THE
COLLECTION AND AS LONG AS
POSSIBLE AND IN ORDER TO
DETERMINE HOW LONG OUR
COLLECTION CAN SURVIVE, SAMPLE
QC IS SOMETHING THAT WE HAVE TO
DO REGULARLY AS WELL AS
MAINTAINING AND QCs OUR
INNSTATION TO ENSURE
"HIGH-QUALITY DELIVERABLES AND
THE DATA HANDLING ALONG THE
WHOLE PROCESS IN ORDER TO TRACK
THE COMPOUNDS ALONG THE WAY.
SO, I'D LIKE TO THANK SOME OTHER
MEMBERS OF OUR TEAM, OUR
COMPOUND MANAGEMENT AND
DISTRIBUTION TEAM.
I WON'T LIST ALL OF THEM BUT
EVERYONE HAS CONTRIBUTED TO OUR
EXPERTISE HERE AND OUR
ADDITIONAL COLLABORATORS, ROSE,
THE FORMER DIRECTOR OF OUR GROUP
AND HOLLY McKEE, WHO WAS.
SO WITH THAT WE'D LIKE TO INVITE
QUESTIONS THAT YOU MIGHT HAVE.
>>THANK YOU, VERY MUCH.
TRAVIS AND LANE.
[APPLAUSE]
WE HAVE QUESTIONS.
I'M GOING TO TAKE THE MICROPHONE
TO THE FIRST QUESTION.
>>THANK YOU FOR THE NICE
PRESENTATION.
I'M JUST CURIOUS, DURING
AUTOMATIC HANDLING OF THE
COMPOUND, HOW DOES IT HANDLE THE
STICKY COMPOUND TO TRANSFER THE
EXACT AMOUNT AND ALSO IT'S
DIFFICULT TO HANDLE THE VERY
STICKY COMPOUND AND ANOTHER
THING WHEN WE HAVE DIFFERENT
COMPOUNDS, SAME COMPOUND DO WE
KEEP THAT COMPOUND IN THE SAME
PART OR IT'S A DIFFERENT BECAUSE
IT COULD BE LOOKS THE SAME.
THANK YOU.
>>THIS IS A TRICKY ONE.
I THINK THERE'S MANY SCHOOLS OF
THOUGHT ON THAT AND OURS IS
TYPICALLY THAT IF IT'S TOO
STICKY TO DISSOLVE IT ALL IN
DMSO THAT THAT IS SOMETHING THAT
MIGHT BE CAUGHT IN OUR QC
PROCESS AND THAT IS FLAGGED.
TYPICALLY, I THINK WE FIND THAT
THOSE COMPOUNDS JUST EXPERIENCE
A LOSS OF CONCENTRATION AND SO
WORE ABLE TO PROCESS THOSE
THROUGH MOSTLY THROUGH OUR
NORMAL PROCESSES AND THROUGH
DOWNSTREAM ANALYSIS DETERMINED
WHAT WE NEED TO.
>>SO, AGAIN, TO SORT OF RELATES
TO THE STICKY COMPOUNDS AS WELL
RIGHT.
SO IF YOU ARE EXPECTING SOME
SORT OF VALUE IN YOUR ASSAY FROM
THAT COMPOUND AND YOU ARE
GETTING IT, THAT'S WHERE WE
WOULD BACKTRACK THROUGH OUR
PROCESS.
A LOT OF TIMES I THINK, THE ECHO
WOULD IT TRANSFER OR NOT
TRANSFER?
TYPICALLY IT WOULD BE --
>>THE ECHO WE MAY SEE AN
INFRACTION AND WE'RE ABLE TO
FLAG IT IF THE ECHO WILL
DETERMINE THAT THERE'S SOME
ANOMALY WITH THE LIQUID AND
UNABLE TO DISPENSE IT.
ONE THING OUR LIMP SYSTEM IS IT
INTEG GATES THE PROFILE FROM THE
ECHO AND THE ECHO REPORTS THAT
IT WAS UNABLE TO MAKE A TRANSFER
FOR PARTICULAR COMPOUNDS, WE DO
CAPTURE THAT AND WE PUBLISH THAT
BACK TO THE SYSTEMS SO THAT THE
RECIPIENT OF THOSE COMPOUNDS
KNOWS THAT THAT COMPOUND IS NOT
ACTUALLY PRESENT IN THAT WELL
WHERE THEY EXPECTED IT AND WE
HAVE DIFFERENT WAYS OF HANDLING
IT.
AS FAR AS BATCH TO BATCH
VARIATION, WE HAVE DIFFERENT
WAYS OF HANDLING IT, JUST TO
REFLECT BACK TO THAT OTHER SIDE
THAT LANE SHOWED YOU WITH THE
21.4 TUBE, THE UPSTREAM
PURIFICATION PROCESS HAS
BASICALLY STANDARDIZED ON THAT
CONTAINER SO ALL OF OUR NEW
COMPOUNDS COME IN AND THAT
CONTAINER AND IF A SYNTHESIZED
COMPOUND HAS TWO DIFFERENT
BATCHES IN THOSE TUBES IN THAT
SYSTEM WE CAN RUN ADDITIONAL
ANALYSIS AND THEY SAY THIS IS A
PROBLEM, AND THEY CAN ACTUALLY
REQUEST TO FULL AND WE CAN PULL
THAT TUBE AND RETURN IT TO THEM
AND WE CAN THROW IT OUT SO WE DO
HAVE A PROCESS IN PLACE FOR
THAT, HOWEVER, WHEN THOSE
COMPOUNDS HAVE GONE INTO OUR
LONG-TERM STORAGE, THEY'RE NOW
SPREAD ACROSS MANY CONTAINERS
AND WE'VE HAD THIS ISSUE AS
WELL.
WE'VE HAD A CASE WHERE CHEMIST
SAY THIS IS NOT GOOD CAN YOU
REMOVE IT?
WE CAN'T PHYSICALLY REMOVE IT
BECAUSE IT'S SPREAD ACROSS A
THOUSAND CONTAINERS, I'M
EXAGGERATING BUT WE HAVE A
MECHANISM TO DELETE IT
ELECTRONICALLY.
AS WE RECOMMEND TO OUR
SCREENERS, ALWAYS CHECK THE QC
DATA.
IT COULD BE THAT IT'S NOT
ELECTRONICALLY DELETED THAT
MATCH IS IN OUR FILE, THEY
RECEIVED IT AND THE QC DATA SAYS
FAIL, FAIL, FAIL.
ONE THING TO NOTE ALSO WITH THE
PURITY, WE USE THE PURITY CUT
OFF OF 80%.
THERE'S BEEN OTHER SIDE STUDIES
THAT HAVE SHOWN THAT BIOCHEMICAL
ASSAYS CAN GET RELY ABLE RESULTS
RATHER THE CELL BASED ASSAYS ARE
A LOT MORE SENSITIVE TO THAT AND
ALTERNATIVELY, OUR MASS SPECK
END POINT CAN TOLERATE A 50%
PURE COMP FOUND SO IT'S NOT JUST
READING THE PASS-FAIL SUSPECT
BUT GET THE REAL DATA AND KNOW
WHAT THAT PURIFICATION IS OF
THAT COMPOUND SO THERE'S A LOT
OF DIGGING YOU NEED TO DO BUT WE
HAVE A MECHANISM FOR IF THERE'S
DEFINITELY A BAD BATCH, WE HAVE
A WAY OF EITHER PHYSICALLY
REMOVING IT OR ELECTRONICALLY
REMOVING IT FROM OUR COLLECTION.
>>TWO QUICK QUESTIONS, ONE IS
WITH REGARD TO YOUR WEIGHING
GREATER THAN TWO MIGS, WHAT IS
THE NUMBER YOU WEIGH TOO?
WE DON'T HANDLE DRIVES IN OUR
WORK FLOW, WE ACTUALLY ONLY,
WELL WE DO PHYSICALLY HAVE A
BALANCE BUT WE TELL PEOPLE WE
DON'T.
ALL OF OUR STAFF IS BASICALLY
OUTSOURCES TO SIGMA AND THEY
HAVE, THEY DO HAVE BALANCE
THAT'S CAN GO DOWN TO ONE MIG
AND LOWER BUT WE'VE EXPERIENCED
ISSUES ON THAT AND THAT IS WE
STANDARDIZED ON THE TWO PIGS --
>>IS IT 1.25MIGS OR?
>>IT'S TWO.
SO DUE WEIGH POWDER SO YOU CAN
WORK WITH OIL AS LONG AS YOU
KNOW THE WEIGHT?
>>WE DON'T REWEIGH.
>>CORRECT.
>>HAVE YOU RIDE TAKING THE
COMPOUNDS OUT AT ROOM
TEMPERATURE AND DO YOU SEE A
PROBLEM?
>>WE DIDN'T PUT IT IN HERE.
WE HAD THEM AT ROM TEMPERATURE
FOR TWO YEARS AND IT WAS AT
WHERE THE REAL PROBLEMS REALLY
STARTED TO SHOW UP MOST
SIGNIFICANTLY WAS 18 MONTHS AND
OUR RECOMMENDATION WAS TO
BASICALLY RE FRESH THEM AFTER A
YEAR SO OUR CURRENT SAMPLES THAT
WE HAVE SO WE DON'T GET INTO
THAT TROUBLE ZONE BUT WHEN WE
HAVE OUR SAMPLES AT ROOM
TEMPERATURE ON OUR CURRENT
SYSTEM, WE REFRESH THEM ONCE A
YEAR SO, THAT'S WHERE WE'RE AT
NOW.
>>THERE'S NO TWIST.
>>YEAH.
>>IT'S DEGRADATION.
>>AT ABOUT 18 MONTHS IT STARTS
TO BE MUCH MORE NOTICEABLE AND
AT TWO YEARS, WE ACTUALLY DID
ONE STUDY UP TO THROW YE THREE T
IT WAS WORST-CASE SCENARIO.
OUR RECOMMENDATION IS USE A YEAR
IF YOU HAVE TO PUSH IT OUT THEN
DON'T GO PASSED 18 MONTHS.
>>AND WHAT DO YOU THINK ABOUT
JUST SATURATING YOUR DMSO TO
BEGIN WITH SINCE IT SAT RATES SO
QUICKLY FOR EXPOSURE.
WHAT DO YOU THINK ABOUT THAT?
>>SO --
[LAUGHTER]
IN ONE OF MY PAST POSITIONS AT
PFIZER, WE DID DO THAT.
FOR THAT WRACK FLOW, IT DID WORK
PRETTY WELL.
THE THING THERE IS YOU DO HAVE
THE DIFFERENT KINDS OF -- THE
WAY THAT DMSO PERFORMS SO YOU
WON'T FREEZE IT SO EQUILIBRIUM
EVEN AT NEGATIVE 140C YOU WILL
GET A GEL.
WE DON'T HAVE A LOT OF DATA ON
THAT.
ONE THING WE USED TO DO IS WE
USED TO ADD AN YOU HAVE TO PLAY
WITH IT AND BASED ON THE
RECOMMENDATION AND STUDY AND HOW
WE'VE AND I DON'T KNOW IF YOU'VE
SEEN CHRIS LA PIN SKI'S FUEL AND
ALL THE OTHER THINGS BUT PULLING
THINGS TOGETHER IT WAS TO KEEP
IT IN 100% DMSO FOR AS LONG AS
POSSIBLE AND THE KEEP THE WATER
OUT OF IT.
>>ONE THING I WOULD MENTION
ABOUT THAT IN PARTICULAR IS THE
ECHO LIMIT OF DETECTION IS 70%
AND SO IF YOU ARE AT EQUILIBRIUM
YOU WILL LIKELY EXPERIENCE MANY
MORE FAILURES AND UTILIZING
ACOUSTICS.
>>IN THE PAST WHEN IT WAS ALL
TIP BASED THAT WASN'T SUCH A BIG
CONCERN AND I THINK IT WAS NICE
TO HAVE EVERYTHING UNIFORM BUT
IN THE ACOUSTIC WORLD THAT'S
PROBABLY A DISADVANTAGE.
>>THAT'S A GOOD POINT.
JUST ONE LAST QUESTION, WHEN YOU
ARE DOING YOUR SERIAL SOLUTION
WITH THE ECHO DO YOU BACK FILL
WITH DSMO TO KEEP IT CONSTANT
WITH THE ECHO AS WELL?
>>IT DEPENDS ON THE SYSTEM
WE'RE USING AND THE VOLUME
REQUIRED FOR THE BACK FILL SO
DEPENDING ON THE DELIVERABLE
VOLUME IT MAY BE AN EX POLICE
AN EXPLICITDMSO PLATE THAT GET E
ECHO TO DO THOSE BACK FILLS OR
IF IT'S A HIGH HAVE IT MAY BE
DONE WITH MULTI-DROP COM BNL OR
OTHER SOME NANO LEADER DISPEN
DISPENSER.
>>I KNOW THEY HAVE BEEN THIS
CONCERN AROUND THE HIGHEST
CONCENTRATION OF THE DMSO TO USE
FOR CELL BASED ASSAYS AND DO YOU
HAVE ANY SUGGESTION ON THAT?
>>MOST PEOPLE DEFAULT TO NOT
GOING HIGHER THAN .5% DMSO IN
CELL BASED AND I KNOW SOME,
WE'VE SEEN DATA WHERE THEY CAN
TOLERATE UP TO 1.5% BUT WE
USUALLY RECOMMEND ALWAYS LESS
THAN 1% AND CERTAINLY THAT'S AN
EXPERIMENT THAT YOU CAN RUN
BECAUSE THERE'S DIFFERENCES
BETWEEN THE CELL LINE.
>>EXACTLY, YEAH.
>>I KNOW THE DEFAULT WITHOUT
DOING THAT EXPERIMENT IS TRYING
TO GET IT TO .5% FINAL IN THE
ASSAY.
>>THANK YOU.
[APPLAUSE]
>>THANK YOU, VERY MUCH, TRAVIS
AND LANE FOR A WONDERFUL
PRESENTATION AND FOR YOUR TIME
TODAY.
ALL RIGHT, WE ARE GOING TO MOVE
ONTO THE NEXT TALK.
OUR NEXT SPEAKER IS Dr.
MICHELLE ARKIN.
MICHELLE IS A PROFESSOR OF CHEM
TREE AT THE UNIVERSITY OF
CALIFORNIA SAN FRANCISCO AND
ADJUNCT PROFESSOR AT THE BUCK
INSTITUTE OF RESEARCH.
CHEMICAL OF PROTEIN INTERACTION
NETWORKS AND OTHER CHALLENGING
TARGETS IN DISEASES SUCH AS
ALZHEIMER'S AND CANCER.
MICHELLE ALSO CO DIRECTS THE
UCSF SMALL MOLECULE AND
EDITORIAL BOARD MEMBER OF THE
AGM AND MY PREVIOUS ADVISER SO
I'M REALLY EXCITED TO HAVE HER
WITH US HERE TODAY.
HER TALK IS ENTITLED BIO FISCAL
APPROACHES TO SMALL MOLECULE
DISCOVERY AND VALIDATION.
MICHELLE, THE FLOOR IS YOURS.
>>I LIKE THE DECISIVE BUTTON
PUSH.
LET'S SEE, I HAVE A FANCY --
IT'S FANCY.
THERE WE GO.
I'M NOT SURE IF THIS IS GOING TO
WORK OR NOT BUT I'LL GIVE IT A
TRY.
PLEASURE TO TALK TO YOU TODAY
ABOUT BIO PHYSICAL STUDIES.
SO, WHAT I WANT TO MAKE SURE YOU
UNDERSTAND IS WHAT INFORMATION
DIFFERENT BIO FISCAL CAN
PROVIDE.
IF THERE ARE NEW METHODOLOGIES
YOU'VE HEARD ABOUT THAT I DON'T
TALK ABOUT, PLEASE, FEEL FREE TO
ASK.
IF WE HAVE TIME WE CAN USE
FRAGMENT-BASED DISCOVERY WITH
SAY APPLICATION FOR BIS OWE
PHYSICAL FOR PRIMARY SCREENING.
>>GOOD-BYE.
[LAUGHTER]
>>THAT'S THE SHORTEST TALK I'VE
GIVEN.
IT TAKES PEOPLE A FEW MINUTES
BEFORE THEY BOO ME OFF THE
STAGE.
[LAUGHTER]
SO THE GUIDING PRINCIPLE OF DRUG
DISCOVERY IT'S IMPLICIT IN
EVERYTHING THAT ARE DOING BUT
BIOPHYSICS MAKES IT EXPLICIT SO
THE GOAL OF DRUG DISCOVERY IS TO
FIND A MOLECULE THAT MODULATES
THE FUNCTION OF SOMETHING
USUALLY A PROTEIN BY BINDING TO
A SINGLE CORRECT SITE ON THAT
TARGET.
MOST OF THE AS SAYS DO NOT DO
THAT. THEY SHOW INHIBITION OF A
TARGET OR MODULATION OF A
PHENOTYPE BUT BIS OWE PHYSICAL
METHODS CAN TELL YOU THAT YOU
ARE DOING THAT BY BINDING TO A
SINGLE TARGET AT A SINGLE SITE.
SO THAT IS SUGGESTS WE WANT A
ONE TO ONE WITH THE BINDING SITE
AND THE MOL I'M AND THAT ALLOWS
US TO FIND THE MECHANISM OF HOW
WE'RE MODULATING THAT PROTEIN OR
THAT BIO MOLECULE IS A CO VEIL
APARTMAND THEN IF YOU HAVE AN EE
IS IT BINDING COMPETITIVELY WITH
YOUR SUBSTRATE OR NON
COMPETITIVELY OR UNCOMPETITIVELY
SO UNCOMPETITIVE IS A BACK WATER
BUT A LOT OF NET ABOLISHES
FUNCTION THIS WAY AND WE FIND
COMPOUNDS THAT WORK UM
COMPETITIVELY AND THEY BIND TO
THE ENZYME SUBSTRATE COMPLEX SO
IF YOU HAVE MORE SUBSTRATE YOU
HAVE TIGHTER BINDING OF YOUR
COMPOUND SO THESE ARE EASY
THINGS TO MEASURE AND YOU CAN
LEARN HOW YOUR DRUG IS WORKING.
SO, AS I MENTIONED, MOST
HIGH-THROUGHPUT JONES WHICH IS N
FUNCTION AND VIRTUAL SCREENING
FOCUSES ON VIRTUAL BINDING SO
THAT IS A NATURAL NEXT STEP IS
TO MEASURE REAL BINDING WITH
THOSE COMPOUNDS YOU'VE
IDENTIFIED VIRTUALLY.
SO, BIO PHYSICAL ASSAYS ALLOW
YOU, DEPENDING ON THE ASSAY, TO
MEASURE AFFINITY AND SOMETHING
ABOUT MECHANISM AND FOR THAT
REASON THEY CAN BE USED AT EACH
STAGE OF THE DRUG DISCOVERY
PROCESS AND IN TARGET
IDENTIFICATION, HIT EYE
IDENTIFICATION, WHERE YOU CAN
SCREEN UP TO TENS OF THOUSANDS
OF MOLECULES WITH
HIGH-THROUGHPUT VERSIONS OF
THOSE TECHNOLOGIES, HIT TO LEAD
WHERE WE HAVE A SET OF COMPOUNDS
AND REIMPROVE THE AFFINITY OF
THOSE COMPOUNDS WE WANT TO MICK
SURE WE'RE STAYING ON MECHANISM
AND THEN EVEN IN LEAD ON TOM
EYESIZATION WITH PEOPLE FOCUS ON
AFFINITY AND SELECTIVITY BUT
PEOPLE SAY DON'T FORGET ABOUT
EFFECTS REQUIRED BINDING SO IF
YOU CAN BIND LONGER TO YOUR
TARGET, YOU CAN HAVE A BETTER
EFFECT EVEN IF YOU ARE PHARMA CO
KINETICS, WHEN IT GETS CLEARED
IF IT'S BINDING TO YOUR TARGET
IT CAN BE WORKING.
IT'S EVEN USED IN LEAD ON TOM
OPTIMIZIZATION.
YOU HEARD FROM DOUG THAT
TERRIBLE THINGS CAN HAPPEN IN
HIGH-THROUGHPUT SCREENING.
SO, HIT VALIDATION, THE GOAL OF
THE HIT VALIDATION IS SEPARATING
THE WHEAT FROM THE CHAFF.
HE SAID THIS TO YOU, IT'S TO
REMOVE THE 99% OF THINGS YOU ARE
NOT INTERESTED IN.
AND THEN THE NEXT STEP OF
VALIDATION IS TO SEE WHAT 1% OF
THE 1% OF THINGS THAT YOU GOT
ARE INTERESTING.
SO, IF YOU HAVE A LOT OF ASSAY
INTERFERENCE OR A LOT OF
SATURDAY FACTS IN YOUR ASSAY
IT'S HARD TO PULL OUT THOSE THAT
ARE REAL SO WE TRIED TO DEVELOP
OUR ASSAY SO WE HAVE A FEW
COMMON ARTIFACTS AS POSSIBLEMENT
FOR EXAMPLE, IF YOU HAVE
COMPOUND AGGREGATION USING
DETERGENT AND CARRIER PROTEINS
AND IRRELEVANT PROTEINS CAN
REDUCE THE ARTIFACTS THAT YOU
SEE IN THE FIRST PLACE BUT YOU
STILL NEED TO UNDERSTAND THAT
YOU WILL PROBABLY HAVE ARTIFACTS
IN YOUR SCREEN AND THIS CAN BE
DO YOU TO ASSAY INTERFERENCE AND
METRIC INTERFERENCE AND THE
COMPOUND AGGREGATION THAT IS COM
OND AND CHEMICAL REACTIVITY WE
CAN SEE FROM THE STRUCTURE AND
WE'LL HAVE FLAGS ABOUT WHAT
COMPOUNDS COULD BE CO VAL ANT OR
REACTIVE BUT YOU CAN STILL BE
SURPRISED AND IF YOU CAN HAVE
REVERS ABLE BINDING AND YOU CAN
FIGURE OUT IS IT COVALANT BUT IF
YOU CAN PROVE THE POSITIVE, THAT
YOUR COMPOUND IS BINDING
REVERSIBLY ONE TO ONE BINDING AT
A SINGLE CITE IT ELIMINATES THE
ARTIFACTS.
SO WE HAVE A LOT OF DIFFERENT
ASSAYS WE CAN USE AND DEPENDING
ON THE SIZE AND EXPERTISE OF THE
LABORATORIES, YOUR LAB AND THE
LABS AROUND YOU, YOU MAY HAVE
ACCESS TO A LOT OF THESE.
SO, YOU START WITH THINKING,
WHAT DO I WANT TO DEMONSTRATORS
WHAT DO I NOT KNOW AND WHAT
ASSAY CAN I USE?
THESE ARE SOME OF THE PROPERTIES
OF INHIBITOR THAT YOU MAY BE
CONCERNED ABOUT.
THESE ARE VARIOUS METHOD ANOTHER
GEES AND THIS IS MEANT AS A
SLIDE FOR YOU, A TABLE FOR YOU
TO GO BACK TO BUT I'LL GO
THROUGH A FEW OF THE MOST COMMON
ONES.
SO I'LL START WITH LIGHTS
GATHERING AND WITH DIFFERENTIAL
SCANNING.
IT'S A WAY OF IDENTIFYING
PROTEIN AGO AGREE GATORS,
COMPOUNDS AGGREGATE INTO THESE
STRUCTURES THAT ARE SOL YOU ABLE
AND TRANSPARENT.
CAN'T SPIN THEM OUT AND YOU
CAN'T SEE THEM.
ONE THING THAT I REALLY WANT TO
CALL YOUR ATTENTION TO IS IN THE
EARLY DAYS OF THINKING ABOUT
AGGREGATORS, THIS COM SOUND IS
AN AGGREGATORS BUT OTHER DRUGS
ARE AGO AGREE GATORS TOO UNDER
CIRCUMSTANCES.
THE AGGREGATION DEPENDS ON THE
PROTEIN, IT DEPENDS ON THE
BUFFER CONDITIONS AND
CONCENTRATION DEPENDENT ALSO SO
IT'S SOMETHING WE CAN MEASURE.
MEASURE THE CONCENTRATION
DEPENDENCE OF THE FORMATION OF
THESE SOL YOU ABLE ARTICLE THE
COMPOUND WILL AGGREGATE AND IT'S
NOT A BIG DEAL THE ISSUE IS YOU
WANT IT NOT TO AGGREGATE WITH
THE SAME EC50 OR THE SAME
CONCENTRATION AS THE ACTIVITY
YOU ARE FINDING.
YOU WANT TO DEMONSTRATION THE
AGGREGATION IS NOT THE CAUSE OF
YOUR FUNCTIONAL ASSAY.
AND THIS IS WHAT THE DATA LOOKED
LIKE AND AGGREGATE WILL HAVE
THIS LONG CORRELATION TIME AND
THEN WE FIT THIS, YOU JUST GET A
NUMBER FOR CORRELATION TIME AND
THAT Y AXIS TIME.
YOU CAN LOCK AT THAT AS A
FUNCTION OF CONCENTRATION AND
PLOT THAT VERSUS YOUR IC50 AND
JUST MAKE SURE THEY DON'T CROSS
EACH OTHER.
N THEY'RE JUST ROTATING FREELY N
SOLUTION.
AGGREGATION SO IT WILL MEASURE
THE RADIUS, WE DON'T USUALLY
LOCK AT RADIUS WE JUST LOOK AT
THE PRESENCE OF AGRI GATES.
IT TENDS TO BE A STEEP
CONCENTRATION CURVE LIKE MY CELL
IF YOU WORK IN MY C YOU CELLS.
YOU SEE NOTHING AND NOTHING AND
A BIG STEEP AND THEN IT'S HIGH
AND CONSTANT USUALLY.
SO THESE INSTRUMENTS YOU CAN GET
IN 384 FORMAT NOW AND THEY CAN
BE DONE VERY QUICKLY AND THE
TRICK IS, NOT HAVING ANY MY
CELLS OR BIG THINGS IN YOUR
BUFFER IN THE FIRST PLACE SO
USUALLY WHEN YOU ARE DOING A DLS
EXPERIMENT, YOU WOULDN'T HAVE
CARRIER PROTEINS OR DETER GANTZ
AND YOU ARE NEED TO MAKE SURE
YOU HAVE NO DUST SO THAT IS
REALLY THE TRICK TO THIS IS JUST
DUST FREE AND THEN IT'S A
STRAIGHT FORWARD TECHNIQUE.
ONE THING A LOT OF PEOPLE WILL
DO BECAUSE IT YOU DO IT IN 384
FORMAT STRAIGHT FORWARDLY IS THE
PRINCIPLE BEHIND TERMAL DEN
ATURATION, IT'S MORE STABLE THAN
THE UNBOUND AND IF YOU PUT IT IO
A RTCPR SYSTEM AND YOU ADD A DYE
THAT BINDS TO THE FORM OF A PRE
TEEN SO WHEN A PROTEIN STARTS TO
MELT AND HYDRO PHOBIC SURFACES
BIND AND IT GIVES A INCREASE IN
FLUORESCENCE SIGNAL FROM
TEMPERATURE.
FROM THE MIDDLE GRAPH, YOU CAN
SEE THAT WE HAVE A MELTING
TEMPERATURE FOR THIS PROTEIN OF
ABOUT 60° AND AS AS YOU
INCREASE, YOU SEE FLUORESCENCE
IE YOU START TO SEE PROTEIN
DENATURATION AT HIGHER AND
HIGHER TEMPERATURES AND FROM
THIS YOU CAN MUR KD FOR THE
COMPOUND AND YOU CAN
DEMONSTRATION THAT THE COMPOUND
IS BINDING TO THE PROTEIN
USUALLY AGO AGREE GATERS WILL
NOT INCREASE THE STABILITY OF
YOUR PROTEIN.
WHEN YOU ADD YOUR COMPOUND.
THIS MEANS ALL KINDS OF THINGS,
PEOPLE TEND NOT TO WORRY ABOUT
IT.
IT COULD MEAN IT'S BINDING TO A
LOWER -- A LESS STABLE STATE OF
YOUR PROTEIN.
IT COULD MEAN THAT YOU ARE
BINDING TO THE UNFOLDED STATE OF
YOUR PROTEIN SO YOU HAVE TO
THINK ABOUT WHAT IS LIKELY FOR
YOUR SYSTEM.
ANOTHER WAY TO DO THERMAL
DENATURATION LOWER AND IT'S A
TWO CD AS YOU MELT YOUR PROTEIN
AND YOU LOSE YOUR SECONDARY
STRUCTURE AND YOU CAN SEE IT
CLEARLY.
DO I HAVE D SF 2.0.
SO MOSTLY DSF RIGHT NOW, IS DONE
WITH A DYE CALLED CYBER ORANGE
AND IT WORKS FOR A LOT OF
PROTEINS BUT NOT ALL PROTEINS SO
A COLLEAGUE OF MINE HAS A
RESEARCH PROGRAM FOCUSED ON
SCREENING DYE SO WE HAVE A DYE
LIBRARY AND WE SCREEN THEM FOR
SPECIFIC PROTEINS AND SO IF YOU
ARE COMPOUND DOESN'T, IF YOUR
PREIF PROTEINDOESN'T, WE TRY A E
AND WE ARE PLAYING WITH
DIFFERENT THINGS.
CAN YOU FIND DYES THAT BIND TO
DIFFERENT DOMAINS OF A MULTI
DOMAIN PROTEIN OR TO ONE BUT NOT
THE OTHER PARTNER OF A
PROTEIN-PROTEIN INTERACTION SO
THERE ARE OTHER OPTIONS BUT
THERE'S ONE DYE THAT'S VERY
TYPICALLY USED.
AND THE EVOLUTION OF THIS
TECHNIQUE INTO CELLS SOMETHING
YOU MAY HAVE HEARD OF OR DO
YOURSELF CALLED SETSA AND IT'S
THE SAME THAT YOU CAN MEASURE
BINDING TO YOUR PRE TEEN BUT
ALSO IN A CELL, BY MEASURING HOW
THERMALLY STABLE THE PROTEIN IS
IN THE CELL.
AND IT WAS ORIGINALLY DONE WITH
WESTERN BLOTTING SO YOU HAVE A
PARTICULAR PROTEIN OF INTEREST
AND DIFFERENT TEMPERATURES, PLUS
AND MINUS THE COMPOUND, AND YOU
SEE THAT AT ABOUT 46° YOU START
TO LOSE THE PROTEIN IN THE
ABSENCE OF COMPOUND AND THE
PROTEIN IS STILL STABLE WHEN YOU
HEAT THE WHOLE CELL UP AND EVEN
UP TO 56°.
SO THERE'S A LARGE SHIFT IN THE
THERMAL STABILITY OF THE PROTEIN
IN CELL LIE SATES WHEN YOU HEAT
UP THE CELL AND THEN CAN YOU DO
THIS AS A FUNCTION OF
CONCENTRATION TO GET AN IDEA FOR
THE BINDING AND KD FOR BINDING
TO THAT PROTEIN.
THERE ARE MANY REASONS THAT THAT
MIGHT NOT WORK.
BUT IT SEEMS TO WORK PRETTY
OFTEN.
IT'S REMARKABLY USEFUL
TECHNIQUES AND THE LATEST
INCARNATION OF THIS TECHNOLOGY
IS TO USE MASS SPECTORMETRY SO
NOW IF YOU HAVE A CELL THAT
YOU'VE LABELED SAY WITH LIGHT
MEDIA, SO SAY YOU DO A
EXPERIMENT SO YOU HAVE LIGHT
MEDIA, CELLS WITHOUT COMPOUND
AND THEN HEAVY MEDIA CELLS WITH
COMPOUND, YOU MIX THOSE TOGETHER
AND YOU CAN SEE THE PROTEINS
HAVE SLIGHTLY DIFFERENT MASSES
BECAUSE THEY'RE GREN IN
DIFFERENT MEDIA AND YOU CAN SEE
AS YOU HEAT UP THE MEDIA WHICH
PRE TEENS YOU NO LONGER DETECT
BECAUSE THEY'VE DENATURED AND
WHICH PROTEINS YOU ARE STILL
DETECTING BECAUSE THEY'RE STILL
IN SOLUTION AND IF THAT SHIFTS
OVER, THE CONCENTRATION OVER THE
TEMPERATURE RANGE THAT YOU ARE
LOOKING, THEN YOU CAN SAY THAT
YOU'VE STABILIZED A PROTEIN OR
SET OF PROTEINS WITH THE
COMPOUND.
SO, ANOTHER TECHNIQUE THAT IS
FAIRLY HIGH-THROUGHPUT THAT
PEOPLE WILL USE IN HIT
VALIDATION, IS CALLED MICRO
SCALE THERMO OR MST.
THE PRINCIPLE HERE IS THAT THIS
IS KIND OF A COOKIE TECHNIQUE.
SO THE IDEA IS THAT PROTEINS
WILL MOVE IN A THERMAL GRADIENT
SO YOU KNOW, THINGS WILL MOVE
AND THEY MOVE HOW QUICK WILL HE
THEY MOVE IN SOLUTION IS BASED
ON THEIR SIZE, SHAPE AND CHARGE.
SO THE WAY THIS TECHNOLOGY
WORKS, YOU HAVE AN ICE OWE
TROPIC SOLUTION SO YOU JUST HAVE
YOUR PROTEIN AND SOLUTION AND
THEN AN IR AND LIGHT COMES ON
AND IR LASER COMES ON AND THAT
SCATTERS THE PROTEINS AWAY FROM
THE LIGHT BUT AWAY FROM THE
WARMTH.
AND THEN YOU TURN THE LASER OFF
AND PROTEINS MOVE BACK SO IF THE
PROTEINS ARE LABELED WITH A
FLORA FLOOR OR LABELED WITH A
GREEN FLORA FLOOR, THEN YOU CAN
DETECT THEM MOVING BACK INTO THE
GRADIENT AND SO HERE THEY ARE,
AND THEY'RE GOING AWAY AND
THEY'RE STARTING TO COME BACK
YOU CAN SEE HOW MUCH THEY MOVE
AND HOW LOW THEY COME BACK AS A
CHANGES TO THEIR SIZE, HYDRO
DYNAMIC RADIUS AND CHARGE SO IF
YOU ADD A SMALL MOLECULE, IT
WORKS WELL FOR PROTEIN-PROTEIN
INTERACTIONS AND YOU CHANGE
THOSE THINGS, AND YOU CAN DETECT
THAT AS A FUNCTION OF
CONCENTRATION OF A COMPOUND.
SO THIS IS THE LITTLE PIECE THAT
YOU FIT INTO A DOSE RESPONSE.
TO GET A BINDING CURVE.
SO THOSE ARE TECHNIQUES USED
COMMONLY IN HIGH-THROUGHPUT AND
THEY CAN BE DONE FOR HUNDREDS OF
COMPOUNDS EASILY.
ANOTHER TECHNIQUE THAT IS USED
AT SEVERAL STAGES IN DRUG
DISCOVERY, SMALL MOLECULE
DISCOVER ROW IS SURFACE PLASMA
RIS INANCE.
IT'S A FLEXIBILITY BIO PHYSICAL
FORMAT.
IT'S CALLED A LABEL FREE FORMAT
WHICH IS JUST COMPLETELY NOT
TRUE BECAUSE THE ENTIRE
INSTRUMENT IS THE LABEL.
THE THE PROTEIN IS CLEARLY WHY
WRACK FOR THEM.
I DO USE A LOT OF SPR.
THE PROTEIN IS IMMOBILIZED ON TO
A SURFACE AND THAT'S THE PRO AND
CON OF THE WHOLE TECHNIQUE.
YOU HAVE TO BE VERY CAREFUL
ABOUT HOW YOUR IMMOBILIZING YOUR
PROTEIN AND HOW DENSELY IT'S
IMMOBILIZED AND SO FOURTH BUT
YOU IMMOBILIZE YOUR PROTEIN ON
THIS SURFACE AND YOU FLOW --
THEY'RE CALLED LIGENS AND THEN
YOU FLOW AND ANNA LIGHT OR A
BINDING ELEMENT IN GREN OVER
THAT IMMOBILIZED PROTEIN.
WHAT SPR MEASURES IS REFRACK
TIVE INDEX IN THE DUFFER CLOSE
TO THIS SURFACE.
IT CHANGES THE REFLECTION OF A
LIGHT, LATER ON THE OPPOSITE
FACE.
IT DEPENDS ON REFRACTIVE INDEX
AND THE REAL MAGIC OF SPR FOR
SMALL MOLECULE DISCOVERY, THE
CHANGE IS PROPORTIONAL TO THE
MASS OF MATERIALS ON THE
SURFACE.
MOR OR LESS PROPORTIONAL TO MASS
WHICH MEANS IF YOU KNOW HOW MUCH
MASS YOU'VE IMMOBILIZED AND THE
MOLECULAR WEIGHT OF YOUR PROTEIN
I KNOW HOW MUCH MOLECULES YOU
CAN BIND IF IT'S ONE TO ONE.
SO THE Y AXIS TELLS YOU THE
NUMBER OF MOLECULES THAT ARE
BINDING TO THE SURFACE AND FROM
THAT WEIGHT YOU CAN TELL WHAT
THE STOICHIOMETRY.
THIS IS A REAL-TIME MEASUREMENT
SO SECONDS, NO MATERIALS AND
THEN YOU INJECT YOUR SAMPLE IF
IT DOESN'T BIND YOU HAVE NO
DEFLECTION.
IF IT BINDS, YOU SEE IT THE
MOLECULE BINDING TO THE
IMMOBILIZED PRE TEEN WITH A
CHARACTERISTIC CONCENTRATION
DEPEND ANT AND THEN YOU REACH
EQUILIBRIUM WHERE AS MUCH
COMPOUND WILL BOUN BIND AND A
DISASSOCIATION PHASE WHERE WHICH
INJECT BUFFER AND WATCH THE
MOLECULE COME OFF.
SO THIS IS NOT USUALLY WHAT SPR
DATA LOOKS LIKE IF YOU LOOK ON
THE WEB, IT'S WILL BE A SLOWER
ON RATE AND OFF RATE BUT FOR
SMALL MOLECULE DISCOVERY WHEN
YOU ARE OFTEN FINDING ONE 10
MICRO MOLAR LIGGENS IT SHOULD BE
FAST.
MOLECULE SHOULD COME ON AND OFF
FAST.
YOU CAN MEASURE THE BINDING
STOICHIOMETRY WITH MEASURING THE
MAXIMUM DISPLACEMENT WHERE
ADDING MORE MOLECULE HAS NO MORE
EFFECT ON THE SIGNAL.
YOU CAN MEASURE BINDING AFFINITY
IN TWO DIFFERENT WAYS.
THE YOU CAN MEASURE THE OFF RATE
DIVIDED BY THE ON RATE AND FIT
THAT GLOBAL LOW AT MULTIPLE
CONCENTRATIONS THAT WILL GIVE
YOU THE KD AND YOU CAN ALSO DO
MULTIPLE CONCENTRATIONS AND THE
MEASURE THE MAXIMUM SIGNAL AT
EQUILIBRIUM AS A FUNCTION OF
CONCENTRATION AND THAT GIVES YOU
A BINDING ISO THEY WERE AND
THEY'RE MUCH IN SINK.
SOME OF THE ADVANTAGES TO THIS
TECHNIQUE IS THAT YOU GET
REAL-TIME DETECTION OF BINDING
AND CONNETICS AND YOU CAN MRSTIK
ONLY TREE AND IT'S ALL ABOUT THE
QUALITY OF THE CHIP SURFACE.
I CALL THE CONVENTION SO YOU SEE
FINDING AND YOU ALSO SEE AGO
CREE GAYS, ALREGAYS -- YOU GET K
METRIC BINDING SO IS THE SAME
COMPOUND.
IT'S RAPID ON AND OFF WHERE IT
BINDS NOT SO SUPER
STOICHIOMETRICALLY WITH
CONNETICS TO JUMP TOO.
REMEMBER THE CAC SO IF YOU ARE
BELOW THE CAC IT LOOKS NORMAL
BUT ABOVE THE CRITICAL
AGGREGATION CONCENTRATION YOU
WILL GET HIGH ST STICK ONLY TRED
THIS TELLS YOU WHY YOU SHOULD
ALWAYS USE DETERGENT IN YOUR
BIOCHEMICAL AS SAYS.
-- THIS IS ONE WAY TO AVOID
SEEING ARTIFACTS AND AVOID
THINKING THAT REAL BINDERS ARE
ARTIFACTS BECAUSE YOU ARE SEEING
AGO AGREE GAYS THAT ISN'T
ENDAGO AGREE.
WE HAVE REACH EQUAL RA LIBERAL
AND WE HAVE A FAST OFF-RIGHT SO
WE CAN'T MENTION THE BINDING
CONNETICS AND WE CAN MEASURE THE
BINDING AFFINITY BASED ON THE
DOSE DEPENDENT CURVE AND WE
REACH SATURATION SO EACH COLOR
IS A DIFFERENT DOSE OF COMPOUND
AND IT'S DONE IN DUPLICATES AND
YOU REACH THE TOP, THE LIGHT
BLUE AND DARK BLUE ARE DIFFERENT
BECAUSE THEY REACH SATURATION.
WE HAVE COMPOUNDS THAT COME OUT
OF THIS SCREEN.
WE REPURCHASED THE HITS AND THE
IC50s WERE SIMILAR.
THE MECHANISMS OF ACTION WERE
SIMILAR BUT WHEN WE LOOK AT THE
KDs, WE SEE A BIG DIFFERENCE
BETWEEN THE BINDING AFFINITY
HERE AND THE BINDING IC50.
SO WHAT DO WE BELIEVE?
SO, THE SPC SAYS 88 MICRO MOLAR
ASSAY TO MICRO MOLAR WHERE THIS
IS MICRO MOLAR AND FOR MICRO
MOLAR MUCH CLOSER TO EACH OTHER.
WE ALSO USE DEN OMAR
TECHNOLOGIES BUT THIS COMPOUND
DIDN'T SHOW ANY SHIFT IN THE NMR
SO NO EVIDENCE FOR PRE TEEN TO
THE NMR WHERE THIS COMPOUND
SHOWS EVIDENCE OF BINDING BY NMR
SO WE HAVE TIGER WO TWO BIO PHYL
ASSAYS, THAT SUGGEST BINDING
SIMILAR TO THE CI50 BUT IT
DOESN'T HAVE CELL BASED ACTIVITY
AND WE DON'T HAVE ANY EVIDENCE
IT'S ACTUALLY THROUGH BINDING TO
THE TARGET.
SO YOU NEED TO PUT ALL OF THIS
DATA TOGETHER WHEN YOU MAKE
DECISIONS ABOUT WHAT TO FOLLOW.
SO HERE IS WHAT THE DOSE
RESPONSE LOOKS LIK LOCKS LIKE, L
THIS DATA OUT AND WE RESPONSE RU
AS COMPOUND CONCENTRATION AND
THIS GIVES US A BINDING ICE OWE
THEY WTHERM AND IT LOOKS LIKE A0
CURVE.
AND FROM THIS WE KNOW THAT THE
BMAX IS ONE EQUIVALENT SO HERE
IS A RULE OF THUMB, PROTEINS,
ABOUT 30 TO 50 TIP LIKE AND
SMALL MOLECULES ABOUT 300 TO 500
TYPICALLY SOUR LOOKING FOR A
1/100 RATIO AND YOU IMMOBILIZE A
THOUSAND RUs YOU SHOULD HAVE A
MAX RU OF ABOUT 10 OF YOUR SMALL
MOLECULE BINDING BECAUSE IT'S
100 THE MASS.
SO IN TOTAL WHAT YOU ARE LOOKING
FOR IN A HIT VALIDATION PACKAGE,
IS DEMONSTRATING REPRODUCIBILITY
SO YOU REPURCHASE AND YOU STILL
GET GOOD ACTIVITY AND THE
INHIBITION MECHANISM.
SO I DON'T THINK, I DON'T TALK
ABOUT MIKAELA KINETICS BUT I
WILL BE HAPPY TO DURING A BREAK.
AND BINDING DATA SO SPR GIVES
YOU THIS IF IT WORKS.
I DO TALK ABOUT IT.
YOU IT TALK ABOUT SOMETHING ELSE
BECAUSE I DO TALK ABOUT ENZYME
CONNETICS.
SO ENZYME CONNETICS, OFTEN YOU
WILL GET A MIXED MECHANISM WHICH
IS KIND OF A DRAG BECAUSE IT'S A
FAIRMENT OF WORK TO DO THESE SO
THERE ARE THREE WAYS TO
REPRESENT DATA SO WHAT MIKAELA
SMITTEN DATA MEASURES IS THE
EFFECT OF INHIBITION AS A
FUNCTION OF TIME USUALLY SO WE
MEASURE THE INITIAL VOLUME OS
TEE OF THE ENZYME REACTION AS A
FUNCTION OF YOUR INHIBITOR AND
YOUR STUB STRAIGHT.
SO HERE WE HAVE A LOT OF WAYS TO
FIT THIS DATA.
CONCENTRATION OF SUBSTRATE AND
VERSUS CONCENTRATION OF
COMPOUNDS SO YOU SEE THAT YOU
INHIBIT AS YOU ADD THE COMPOUNDS
YOU INHIBIT THE ASSAY.
THIS IS THE MODERN WAY TO FIT
THE DATA BECAUSE YOU ARE NOT DEG
ANY MANIPULATION OF THE DATA YOU
ARE FITTING THE DATA AS YOU
MEASURE IT AND YOU FIT THESE NON
LINEAR CURVES USING PRISM OR
SOMETHING.
THE OLD WAY IS A LIVER WEAVER
BURKE PLOT.
PEOPLE DON'T LIKE DO TO THIS
ANYMORE BECAUSE IT EMPHASIZES
CERTAIN ASPECTS OF THE CURVE
MORE THAN OTHERS BUT IT IS
REALLY NICE AS A QUICK VISUAL
SAYING OK, IF IT'S PARALLEL LIKE
THIS THIS IS AN UNCOMPETITIVE
MECHANISM IF IT HITS THE X AXIS
IT'S ONE AND Y AXIS IT'S ANOTHER
SO YOU CAN GET A QUICK IDEA OF
MECHANISM FROM LOOKING AT A LINE
WEAVER BURKE PLOT BUT WHAT IS
PRACTICAL IS TO JUST MEASURE
YOUR IC50 CURVE FOR YOUR SMALL
MOLECULE OTHERS A FUNCTION OF
SUBSTRATE CONCENTRATION USING
THE IC50 OF THE MOLECULE SO
HERE, I HAVE AN UNCOMPETITIVE,
IT'S THE SAME COMPOUND, AN
UNCOMPETITIVE MECHANISM OF
ACTION AND I KNOW THAT BECAUSE
IF I HAVE A LOW CONCENTRATION 6F
BE STRAIGHT I HAVE A WEAK IC50
AND
A HIGH
CONCENTRATION I HAVE A POTENT
IC50 SO IT GETS MORE POTENT AS
YOU ADD SUBSTRATE AND THAT IS
UNCOMPETITIVE, IT BINDS TO THE
ENZYME SUBSTRATE COMPLEX.
TYPICALLY, YOU WILL SEE
COMPETITIVE INHIBITION WHICH
WOULD MEAN THE MORE SUBSTRATE
YOU ADD THE WEAKER YOUR COMPOUND
IS SO YOU GET A LEFT -- A
RIGHT-WORD SHIFT IN THAT GRAPH.
IT GETS WEAKER.
THAT'S A PRACTICAL WAY TO
EVALUATE THE PHARMACOLOGY OF
YOUR COMPOUND EVEN IF YOU HAVE
SOME MIXED MECHANISMS BECAUSE
IT'S HARD TO MEASURE THESE
ACCURATELY.
FOR THIS SAME EXAMPLE, THE SAME
PROJECT, WE CONDUCTED IT WAS
UNCOMPETITIVE AND NOW SPR
VERIFIES THAT AND WE HAVE THESE
TWO INHIBITORS, THEY BIND TO
DIFFERENT CONFIRMATIONS AND
ENZYME CONNETICS CAN'T MEASURE
THE NO SUBSTRATE BOUNDS STATE
BECAUSE YOU HAVE TO HAVE SOME
SUBSTRATE TO DO IT AND SO IN A
WAY, SPR GIVES YOU MORE
INFORMATION.
SO HERE IS AN UNCOMPETITIVE
COMPOUND SO THIS UNCOMPETITIVE
COMPOUND DOESN'T BIND.
IT BINDS TO THE SUBSTRATE BOUND
COMPLEX WHICH IS CONSISTENT WITH
AN UNCOMPETITIVE MECHANISM AND
SOMETHING ELSE YOU CAN'T REALLY
DO BY ASSAY, IT BINDS BETTER TO
THE PRODUCT.
IS HE THE WAY THIS COMPOUND
WORKS IS IT INHIBITS PRODUCT
RELEASE SO IT BINDS TO THE
PRODUCT BOUND ENZYME COMPLEX.
THIS COMPOUND IS NOT NON
COMPETITIVE AND IT DOESN'T
REALLY CARE VERY MUCH WHETHER
THERE'S NUCLEOTIDE OR NO
NUCLEOTIDE IT BINDS WESTBOUND TO
BETTER -- ITDOESN'T BOU BIND TON
THEY'RE SUBSTRATE IT'S NO
SUBSTRATE ENZYME.
SO SPR VERIFIES THAT YOU
DETERMINE BY CONNETICS SO THERE
ARE MANY INSTRUMENTS FOR SPR AND
RELATED TECHNOLOGIES, BLI IS A
RELATED TECHNOLOGY AND THERE ARE
MANY DIFFERENT FORMATS NOW AND
MANY OF THESE FORMATS RELY ON A
FLOW FORMAT.
BIACORE, SENSIQ AND THERE ARE
DIFFERENT INSTRUMENTS AND THE
NOISE THING ABOUT A FLOW BASED
IS IT MEASURES ACCURATELY.
THE INSTEAD OF FLOWING MATERIAL
OVER A SURFACE, YOU PUT YOUR PRE
TEEN SURFACE IS ON A TIP OF A
FIBER OPTIC AND YOU DIP IT INTO
YOUR SOLUTIONS IT RELIES ON
DIFFUSION FOR BINDING AND
UNBINDING SO THE MATH IS
DIFFERENT AND JUST IN MY
OBSERVATION IT'S NOT AS GOOD AS
MEASURING OFF RATES.
SOME OF THE KEY ELEMENTS FOR
ASSAY DEVELOPMENT.
THE I AM MOBILIZATION STRATEGY
AND THE SURFACE CHEMISTRY.
IF YOU CAN USE A LABEL PROTEIN
THEN YOU HAVE HAVE MORE ROW
PRODUCING ABLE ENZYME, MORE
REPRODUCE ABLE SURFACES AND YOU
CAN ALSO DO RANDOM IMMUNE
COUPLING OR RANDOM LYSINE
COUPLING AND RANDOM LYSINE
COUPLING, IF YOU ARE DOING
KINASES, VERY TRICKY IT MAKES ME
THINK YOU CAN HAVE THIS FOR
ANIEN TIME THAT HAS LIESINGS IN
THE SITE.
IF YOU MOBILIZE AUCOI KINASE, YD
TO BECAUSE IT'S REACTIVE IT WILL
STICK TO YOUR CHIP AND IT WON'T
STICK TO YOUR PROTEIN SO IT'S
SOMETHING TRICKY YOU HAVE TO
KEEP IN MIND.
HOW AM I DOING FOR TIME?
THE OTHER THING IS YEAH PROTEIN
STABILITY, ACTIVITY
CONFIRMATIONAL, FLEXIBILITY ARE
VERY IMPORTANT SO SOME PRE TEENS
ARE REALLY UNSTABLE OBJECT THE
CHIP UNLESS THEY'RE DENSE AND
THEY'RE CLOSE TO EACH OTHER AND
THE OPPOSITE CAN ALSO BE TRUE.
THEY AGGREGATE ON THE SURFACE IS
THE SURFACE DENSITY HAS TO BE
SLOW SO THESE ARE THINGS YOU
NEED TO CONSIDER.
PROTEIN STABILITY OVER TIME IS
VERY IMPORTANT AND SURFACE
ACTIVITY BECAUSE WE'RE GOING TO
USE, WE'RE GOING TO ASSUME THE
MASS OF THE PROTEIN ON THE
SURFACE IS ACTIVE UNLESS PROVEN
OTHERWISE AND SO YOU ARE STRIKE
ONLY TREE WILL BE STRONG IF IT'S
EFFECTIVE SO IF YOU HAVE A
POSITIVE CONTROL OR SUBSTRATE
THAT YOU CAN MEASURE THE BINDING
AND YOU SAY I'M SATURATING MY
BINDING AT 80% OF WHAT I EXPECT
SO MY SURFACE 180% ACTIVE IT'S
FINE JUST KEEP THAT IN MIND WHEN
YOU ANALYZE YOUR DATA AND THAT
POSITIVE CONTROL YOU INJECT IT T
AND SOME USE INTERNAL AUTO MAKE
GOING ON FOR SEVERAL DAYS.
SO YOUR PROTEIN SURFACE
STABILITY TEE IS IMPORTANT AND
YOU CAN MULTI PLEX.
MOST OF THESE MODERN INSTRUMENTS
HAVE -- THEY DIFFER IN HOW
THEY'RE DESIGNED BUT THIS IS AN
OLD CHIP AND IT HAS FIVE SPOTS
PER CHANNEL AND SO EACH OF THESE
IS A CHANNEL AND SO THAT'S THE
MICRO FLUID I CAN CHANNEL AND
THE PLACE THE PROTEIN IS
IMMOBILIZED IS THIS GREN SPOT ON
THESE MICRO FLUIDIC CHANNELS AND
EACH TAKES ONE COMPOUND AT A
TIME SO ONE AT A TIME AND EACH
ANNA LIGHT CAN GO OVER ALL FIVE
SURFACES AT THE SAME TIME SO FOR
EXAMPLE, ONE THAT HAS YOUR
PROTEIN OF INTEREST, ONE THAT
HAS YOUR PROTEIN OF INTEREST
WITH THE ACTIVE SITE BLOCKED OR
A DIFFERENT DENSITY OR SOMETHING
ELSE AND IT CAN BE A RELEVANT
PROTEIN OR ANNA LOGS OF YOUR
PROTEINS THAT YOU DON'T WANT TO
HIT.
AND REQUIRE DOING A LIPID KINASE
PROJECT RIGHT NOW WITH NCATS AND
WE PIT THROW ISOFORMS ON THE
CHIP AT THE SAME TIME.
SO THESE ARE ALL THINGS THAT ARE
EASY TO DO AND YOU BASICALLY GET
THIS DATA ALL AT THE SAME TIME.
SO IT'S A VERY DATA-RICH
TECHNIQUE AND WHEEL COME BACK TO
THAT IN LEAD OPTIMIZIZATION.
I WANT TO TALK ABOUT NMR WHICH A
VERY FLEXIBLE METHODOLOGY THAT
THERE ARE A LOT OF WAYS TO USE
IT IN SMALL MOLECULE DISCOVERY
AND I DON'T KNOW ABOUT YOU GUYS
BUT I HAVE A HARD TIME
CONVINCING ANYONE TO DO IT.
MAYBE I'LL CONVINCE YOU OR YOU
WILL HAVE MORE BALANCE AS TO
TAKE BACK TO YOUR LAB TO
CONVINCE PEOPLE TO DO IT BECAUSE
THERE ARE SO MANY NMR
EXPERIENCES YOU CAN DO.
YOU CAN DO SMALL MOLECULE
DETECTED NMR.
DOESN'T USE A LOT OF PROTEIN,
VERY FAST.
IT'S PROTEIN NMR BASED
TECHNIQUES.
YOU CAN DO PROTEIN NMR WHICH CAN
BE A LITTLE MORE COSTLY AND IT
TAKES MORE PROTEIN BUT YOU CAN
GET BINDING SITE INFORMATION SO
IT'S INFORMATION RICH TECHNIQUE
AND THEN THERE ARE OTHER KINDS
OF FANCY METHODOLOGIES AND I'LL
SEND YOU TO THIS REVIEW.
FROM 2008 BUT IT IS STILL
COMPREHENSIVE AND NOT A LOT OF
BRAND NEW TECHNIQUES AND WAYS OF
LOOKING AT THE EVIDENCE OF
BINDING WHERE THE BINDING SITE
IS BETWEEN THE MOLECULE AND THE
PRE TEEN AND SO THESE ARE FANCY
TECHNIQUES BUT SOME ARE
ROUTINELY USED SO I'LL TELL YOU
ABOUT THOSE.
SO THE MOST COMMON METHODOLOGIES
TAKE ADVANTAGE OF THE
INTERACTION BETWEEN THE SMALL
MOLECULES WHICH HAS A SET OF
CHARACTERISTICS RATES OF DECAY,
RATES OF SPIN DOUGH K SPIN DECA.
THE PROTEIN HAS DIFFERENT ONCE.
PROTEIN IS BIG, IT MOVES SLOWLY,
IT'S LAZY AND IT MOVES SLOWLY
AND ROW LAXES QUICKLY AND SMALL
MOLECULE IS THE OPPOSITE.
THEY SPIN QUICKLY THROUGH SPACE
AND THEY'RE SMALL AND THEY HAVE
SHARP PEAKS.
SO WHEN YOU SEE A PROTEIN, A
SMALL MOLECULE NMR IT LOOKS LIKE
THIS.
SHARP PEAKS AND NICE SPLITTINGS
AND THE PRE TEEN HAS THESE BIG
BLOBY PEAKS.
IF YOU BIND -- SO, IF YOU IT IT
TOUCHES THE PROTEIN IT ADOPTS
THE PROPERTIES OF THE PROTEIN.
IT'S LIKE THE NMR VERSION OF A
FLORENCE IF YOU ARE FAMILIAR
WITH THAT TECHNIQUE SO YOU HAVE
SOMETHING SMALL BOUND TO
SOMETHING BIG IT TAKES ON THE
MOLECULAR PROPERTIES OF THE BIG
THING.
AND SO WE CAN SEE THAT AND HERE
IS AN EXAMPLE IF YOU HAVE THE
SMALL MOLECULE BOUND TO A
PROTEIN SO THE BIG BLOBBY SPEAK
IS PROTEASOME TEEN AND YOU SEE
HOW BROADER THESE SMALL MOLECULE
PEEKS WHEN THERE IS NO PROTEIN
BOUND BECAUSE SOME OF THAT
MOLECULE IS BINDING TO THE
PROTEIN AND TAKING ON THE
CHARACTERISTICS, THE SPIN
CHARACTERISTICS OF THE PROTEIN
AND IF YOU ADD PROTEIN, AND YOU
DON'T BIND, YOU DON'T SEE ANY
SHIFTS.
THIS IS A SATURATION TRANSFER
DIFFERENCE EXPERIMENT.
OR WALK LOR EXPERIMENTS, IN ONE
TECHNIQUE, YOU RADIATE THE
PROTEIN AND THEY RADIATION IS
TRANSFERRED TO THE SMALL
MOLECULE.
IT CAN TRANSFER IT'S MAG
NICATION TO THE SMALL MOLECULE
AND WHEN YOU DO THAT TECHNIQUE,
YOU CAN SEE ALL KINDS OF
INTERESTING THINGS, FOR EXAMPLE,
YOU CAN DO IT IN SUCH A WAY THAT
YOU SEE THOSE PEAKS THAT ARE
MOST BROAD END OR MOST EFFECTED
ARE THOSE THAT ARE MOST BOUND TO
THE PROTEIN.
YOU CAN GET A GENERAL IDEA WHICH
PARTS ARE BURDEN H BURIED AND SY
BURIED SO ADVANTAGE TO NMR IS
EVEN JUST PLAIN OLD PROTEIN IF
YOU DO PROTON YOU CAN SEE THE
SHIFTS AND THESE LINE
BROADENNINGS AND THEY'RE FAST
AND YOU ZOOS A SMALL
CONCENTRATION OF PROTEIN AND A
HIGH STICK ONLY TREE OF TOM
POUND SO IT LOOKS LIKE THE WAY
YOU DO AN ASSAY WHERE YOU HAVE
ABOUT 100 TIMES MORE MOLECULE
THAN PROTEIN AND IF YOU DO IT
IT'S THE SAME THING HERE AND
THIS ASSAY RELIES ON FAST
CONNETICS THAT THEY BIND AND
ABSORB AND COME OFF IN THE NEXT
COMPOUND BINDS.
BECAUSE YOU RELY ON THIS
AMPLIFICATION OF SIGNAL IT'S
ONLY GO AHEAD WHEN YOU HAVE FAST
ON AND OFF RATES.
SO IT'S THE BEST IN EARLY STAGE
DISCOVERY BEFORE YOUR COMPOUNDS
BIND TIGHTLY SO IT'S A VERY NICE
TECHNIQUE FOR VALIDATING
SCREENING HITS AND IT'S RATIO OF
COMPOUND TO PROTEIN AND IT'S
WHAT YOU GET OUT OF HTS.
IT'S NOT A 384 WORLD TECHNIQUE
IT'S STILL INDIVIDUAL COMPOUNDS,
ONE WAY YOU GET AROUND THE
SLOWNESS IS IF YOU HAVE
COMPOUNDS THAT HAVE REALLY
DISTINCT NMR SPECTRA YOU CAN
MULTI PLEX THE COMPOUNDS SO YOU
CAN HAVE 10 COMPOUNDS IN A TUBE
AT A TIME.
SO PROTEIN NMR DETECTION IS A
VERY POWERFUL TECHNIQUE IF YOU
ARE PROTEIN IS ASSIGNED
ESPECIALLY IF THE BACKBONE OF
YOUR PROTEIN IS ASSIGNED SO YOU
SEE THIS ONE IS A PROTON
NITROGEN AND YOU CAN DO LARGER
PROTEINS AND IT'S CALLED AN HMQC
BUT YOU SEE 2D AXIS EACH DOT
HERE IS ONE IN THIS EXPERIMENT.
IT WOULD BE A MENTHOL GROUP SO
IT'S ONE AM ID BOND SO YOU HAVEA
SINGLE PEAK ACID AND THESE
BACKBONE NITROGEN BONDS ARE
SENSITIVE TO ENVIRONMENT SO IF
THEY'RE INSIDE THE PROTEIN THEY
HAVE CHARACTERISTICS SHIFTS IF
THEY'RE OUTSIDE THE PROTEIN,
THEY HAVE DIFFERENT SHIFTS AND
THEY'RE SENSITIVE TO BUFFER AND
PH BE CAREFUL, SALT BE CAREFUL
BUT COMPOUND BINDING SO WE DID A
SCREEN ON THIS SMALL PROTEIN IN
GRAY AND THIS IS MAKES
INTERACTIONS SHOWN IN GROWN SO
WE WERE LOOKING FOR COMPOUNDS
THAT WOULD COMPETE WITH THIS
OTHER PROTEIN SUGGESTING THAT
THEY WOULD SHOW SHIFTS ALONG
THIS BINDING SITE BUT ALL THE
COMPOUNDS SHOWED SHIFTS ON THESE
AROUND THIS BINDING SITE SO
BACKBONE THAT CORRESPOND SO
THERE'S A POCKET NEXT TO THE
MAIN BINDING SITE AND
INTERESTINGLY, THESE STABILIZE
SO THEY HELD IT INTO A BINDING
COM FIRMATION AND SO THAT IS WHY
I'M SHOWING IT TO YOU HERE
INSTEAD OF PUBLISHING IT.
YOU CAN SHOW CLEARLY WELCOME
WHERE THEIRUSING THIS TECHNIQUEE
A SHIFT IN THIS RESIDUE IN
PARTICULAR.
UPON BINDING.
EVEN IF YOU DO HAVE THE SPECTRUM
ASSIGNED, SO YOU SEE ALL THESE
PEAKS AND YOU DID KNOW WHICH
AMINO ACID THEY CORRESPOND TO
YOU CAN GET USEFUL INFORMATION
SO YOU CAN SAY WELL 10 OF MY
MOLECULES BIND IN THE SAME PLACE
OR THE SAME PLACE AS THE
SUBSTRATE OR THE PROTEIN OR
SOMETHING SUE CAN STILL GET
USEFUL INFORMATION.
SO THE FIRST FRAGMENT WAS SAR BY
NMR AND IT USED THIS HSQC
PROTEIN TECHNIQUE BECAUSE IT'S
SENSITIVE TO THESE ENVIROMENTAL
CHANGES AND SO THE WAY THIS
WORKED, SAY YOU HAVE A PROTEIN
SURFACE, A BINDING SITE WHICH
TWO SUB POCKETS IN IT AND BY SAR
BY NMR YOU COULD FIND SHIFTS IN
THE BINDING POCKET WHEN A
MOLECULE BOUND THEN YOU OPTIMIZE
FOR BETTER AND BETTER SHIFTS,
LARGER SHIFTS, MAYBE MORE
SHIFTS.
AND THEN YOU SCREEN FOR A SECOND
SITE IN THE PRESENCE OF THIS
OPTIMIZED FIRST SITE FRAGMENT
AND OPTIMIZE AND THAT FRAGMENT
AND YOU HAVE TO LINK THESE TWO
FRAGMENTS IN THE SECOND STEPS
AND IT'S DRUG DISCOVER ROW BUT
IT'S POWERFUL TECHNIQUES FOR
DOING THAT.
ONE OF THE KEY POINTS THERE IS
THAT INTERESTINGMENT BASED DRUG
DISCOVERY USE SMALLER LIBRARIES
INSTEAD OF USING 100,000 OR A
MILLION IT USED A THOUSAND TO
10,000 MOLECULES AND BECAUSE
THOSE LIBRARIES SIZES ARE
SMALLER THEY'RE IN THE SAME
REGIME THAT CAN YOU DO .
PHYSICAL TECHNIQUES FOR PRIMARY
SCREENING AND ALSO BECAUSE
FRAGMENT THOSE LITTLE MOLECULES
BOUBIND WEAKLY AND ASSAYS CAN BE
PROBLEMATIC YOU HAVE A LOT OF
FALSE NEGATIVES AND FALSE
POSITIVES WHEREAS THESE BINDING
ASSAYS ARE IN A CONCENTRATION
REGIME AND MEASURING BINDING
DIRECTLY AND THEY TEND TO BE
MUCH LESSER OR PRONE.
SO A FEW MORE TECHNIQUES, HIGHER
INFORMATION CONTENT LOWER
THROUGH PUT TECHNIQUES.
IT'S COME ALONG IN SPEED PHOTO
AFFINITY LABELING IS A TECHNIQUE
THAT YOU MAY SEE PEOPLE USE.
THE IDEAS THAT YOU TAKE YOUR
MOLECULE AND YOU ADD SOMETHING
TO IT THAT WILL BIND TO YOUR
SMALL MOLECULE, OR YOUR PROTEIN
AND THE MORE ROW ACTIVE IT IS,
THE COLLIER HE IS IT BINDS IN
THE BINDING SITE AND THERE'S
SEVERAL DIFFERENT REACTIVE
PROBES THAW MIGHT USE, SOME
REQUIRE LIGHT, PHOTO A FIN TOE
LABELING AND OTHERS DO REQUIRE
LATE THEY'RE JUST ROW ACTIVE AND
FROM THAT YOU CAN GET AN IDEA
WHEN YOU THEN CO CHOP UP THE
PROTEIN YOU CAN FIND OUT WHERE
THE MOLECULE IS BINDING BASED ON
WHAT IS ATTACHED TO AND THIS
GIVES YOU A MODEL FOR HOW THE
COMPOUND BINDS.
IT'S NOT USED THAT OFTEN BUT IT
CAN BE POWERFUL.
ANOTHER TECHNIQUE CAN BE
POWERFUL AND I DON'T KNOW HOW
OFTEN IT'S USED IN INDUSTRY
BECAUSE IT'S FAIRLY SLOW.
LOW THROUGH PUT AND IT USES A
LOT OF MATERIAL.
IT GIVES YOU VERY NICE DATA.
THE PRINCIPLES OF ICE OWE NORMAL
CALIORMETRY IT BINDS HEAT.
YOU GET ENERGY BY BINDING.
IT BINDS BECAUSE IT GIVES YOU
ENERGY AND SO WHEN YOU ADD A
COMPOUND SO HERE WE PUT THIS IN
A CAREFULLY TEMPERATURE
CONTROLLED AND HERE IS THE
PROTEIN AND WE ADD COMPOUND AND
AS WE ADD THE COMPOUND IT BIND
TO THE PROTEIN AND HEATS UP AND
WE MEASURE THE HEAT AND THE HEAT
GOES AWAY REALLY QUICKLY AS IT
RE' CALIBRATION.
WE GET HEAT AND IT GOES SO AND
SO UNTIL WE ADD COMPOUND AND WE
GET NO MORE HEAT BECAUSE THE COM
UNDER HAS SATURATED THE BINDING
SITE.
AND FROM THIS CURVE, YOU CAN GET
AFFINITY INFORMATION FROM THE
EC50.
YOU CAN GET STRIKE ONLY TREE
INFORMATION BECAUSE IT'S DONE
QUANTITIES OF COMPOUND AND
PROTEIN AND YOU GET THE STICK
ONLY TREE AND WHETHER IT'SEN
TROPIC DRIVEN SO IF YOU HAVE A
BIG HEAT THEN IT'S BECAUSE YOU
ARE GETTING A LOT OF ENTHALPY
AND IF YOU DON'T GET A HIGH
SIGNAL, MOST THE BINDING ENERGY
IS COMING FROM CHANGES IN
ENTROPY.
AND IT'S HIGH-THROUGHPUT
TECHNIQUE A VERY STANDARD
TECHNIQUE WHEN YOU CAN DO IT BY
SOAKING YOU CAN ACTUALLY USE IT
FOR PRIMARY SCREEN AND PEOPLE DO
THAT FOR FRAGMENT BASED SCREENS
AND THEY'LL HAVE POOLS OF 10
MOLECULES AND A SET OF 90 OR 160
CRYSTALS AND SOAK THE SO MANY
POUNDS INTO THE CRYSTALS AND SEE
IF THERE'S DENSITY THERE THAT
THEY CAN ASSIGN TO THE SMALL
MOLECULE FRAGMENTS AND
TECHNOLOGY THAT'S COMING ALONG
FOR SMALL MOLECULE DISCOVERY IS
CRYO EM AND I THINK THIS IS
GOING TO BE AN IMPORTANT
TECHNOLOGY FOR LEAD O
OPTIMIZATION.
IT'S NOT HIGH ENOUGH RESOLUTION
TO SEE HYDROGEN BONDS BUT YOU
JUST MODEL THEM ANYWAY, LET'S
PHASE IT.
AND LOW RESOLUTION IS REALLY
WHAT YOU WANT KNOW WHERE IT'S A
BOUND AND HOW CAN IT MAKE THE
BOUND BETTER AND IT'S UP TO THAT
TASK.
SO WE TALKED ABOUT BINDING
VALIDATION, MECHANISM OF ACTION
AND I MENTIONED OFF RATE
SELECTION.
AND SO JUST THE POINT IS THAT
ALL THESE METHODS NEED TO BE
BALANCED BY SECONDARY SAYS AND
THAT IS THE GOAL.
WHATEVER YOU KNOW, THERE'S
SOMETHING YOU DO KNOW SO USE A
VARIETY OF TECHNIQUES TO ANSWER
ALL THE QUESTIONS THAT YOU HAVE
DO IT EARLY AND DO IT OFTEN,
DON'T WASTE YOU OR YOUR CHEMIST
TIME ON ARTIFACTS.
AND WITH THAT I'LL TURN IT OVER
TO YOU.
[APPLAUSE]
>>THANK YOU SO MUCH FOR AN
AWESOME TALK.
QUESTIONS FOR MICHELLE?
GO AHEAD.
>>THANK YOU FOR THE NICE TALK.
I HAVE A QUESTION ABOUT THE DSF
ASSAYS.
WE SEE IT TWO PICKS INSTEAD OF
ONE AND PEM THINK IT'S A MULTI
DOMAIN SO THAT DO YOU THINK
ABOUT THAT?
>>IF YOU HAVE A MULTI DOMAIN
PROTEIN YOU WILL OFTEN SEE TWO
PEAKS BECAUSE THEY MELT
INDEPENDENTLY.
IF YOU HAVE A SINGLE DOMAIN
PROTEIN AND YOU ARE GETTING TWO
PEAKS, I'M NOT SURE.
IF YOU HAVE SOMETHING SLOWLY
ININTEREST CONVERTING IT'S
SIMILAR BUT THAT'S UNUSUAL.
THANK YOU.
>>WHAT DO YOU RECOMMEND FOR
PROTEINS THAT ARE MEMBRANE BOUND
LIKE GCPR THAT ARE HARD TO
PURIFY AND SOL YOU SIZE?
>>WHAT DO I RECOMMEND FOR
GCPRs?
PEOPLE HAVE BEEN WORKING ON SPR
FOR GCPR FOR A LONG TIME AND I
DON'T KNOW THE LATEST.
IT'S BEEN DIFFICULT.
YEAH, USUALLY THE PEOPLE THAT I
WORK WITH THEY SOL BUELL EYES
THEM IN DETERGENT AND NANO DISKS
AND SPINNING SOLID STATE NMR IS
SOMETHING TO CONSIDER AND LET ME
THINK WHAT ELSE.
YOU MAY BE ABLE TO GET
INFORMATION TOO FROM MASS
SPECTORMETRY FROM HD EXCHANGE OR
SOMETHING THAT IS DOU DENATURIND
YOU CHOP UP THE PROTEIN BUT YOU
SEE THE ECHO OF WHAT WAS BOUND
FROM THAT CROSS LINKING OR HD
EXCHANGE OR PROTECTION FROM
DIGESTION OR SOMETHING.
I HAVE TO THINK ABOUT THAT MORE.
ONE MORE OVER HERE.
>>I JUST WANTED TO ADD, WORE
TRYING TO DEVELOP BRETT BASED
PROBES ON GCPRs SO IT'S
SOMETHING WE'RE TRYING TO DO
WITHIN A PEPTIDE BASE PROJECT SO
WE HAVE A GCPR AND IS EXPRESSING
NANO LUKE AND THEN THE PEPTIDE
HAS AN ACCEPTOR.
>>SO THAT OBJECT INHIBITION
ANDAL CONTINUE TIVE COMPETITION
ASSAY.
>>YES.
YES.
>>NOT DIRECT BINDING.
>>YES.
CORRECT.
>>THAT'S USEFUL.
>>ANYMORE QUESTIONS FOR
MICHELLE?
ALL RIGHT, I DON'T SEE ANYMORE
HANDS.
THANK YOU SO MUCH, MICHELLE.
AGAIN.
[APPLAUSE]
WE ARE GOING TO TAKE A SHORT
BREAK AND WE'LL RECONVENE AT
11:00.
SEE YOU ALL AT 11:00.
>>HIS TALK TODAY IS ENTITLED
"LEAD SELECTION AND
OPTIMIZIZATION BY MEDICINE
CHEMISTRY.
WELCOME.
>>THANK YOU.
I'LL TRYING TO FIGURE OUT AND
AND I WAS TRYING TO BEGIN THIS
TALK BY ASKING A CHEMIST AND WE
SAW A WONDERFUL TALKS AND ASSAYS
AND HOW THEY'RE AND FOR SMALL
MOLECULES AND WE USE INFORMATION
FROM THESE ASSAYS AND TO EMILY
GENERATE STRUCTURE OF AND I
UNDERSTAND AND WHAT THESE ARE
AND HOW THEY UTILIZE TO MAKE AND
HERE IS A A PICTURE AND I WAS
AND I DIDN'T HAVE FOR AT LEAST A
YEAR AND WHAT I WANT TO
ENCOURAGE CHEMIST IS TO
ESSENTIALLY NOT GO DOWN THIS AND
WHERE YOU ARE HAVING A
CONVERSATION WITH THEM AND
POSITIONING YOURSELF FOR SUCCESS
AND DEVISING AND MAKING A
HOLIDAY AND HOW MODERATE WHICH
WAS DO WE SEE AN EFFECT IN THE
DISEASE.
SO I'M GOING TO ACTUALLY
EXEMPLIFY THESE CONCEPTS BY
THROW CASE STUDIES THE FIRST
DEALING WITH KINASE INHIBITORS
THIS IS CAR SOON STRUCTURE FOR
KINASE AND IN THIS KINASE,
THERE'S ATP, THE COFACTOR OF THE
KINASE WHICH IS THERE'S
STRUCTURE WHERE YOU NEED LOG
WHICH HAS A NUMBER OF BETA
SHEETS AND THEN THERE'S ANOTHER
DOMAIN WHICH HAS A BUNCH OF
THOSE CONNECTED BY THIS NARROW
THREAD OF PEPTIDES WHICH IS
CALLED THE HINGE REGION AND ALL
KINASE IS ESSENTIALLY ATP WHICH
IS THE COFACTOR AND TRANSFER IT
TO ANOTHER PROTEIN LIPID SO THE
PORTION BINDS TO THE HINGE
REGION AND MOST KINASE
INHIBITORS TROY TO COPY THIS
CONCEPT SO THEY TRY TO BIND TO
THE SAME REGION OF THE KINASE.
THE PROBLEM IS WE HAVE ABOUT 500
PLUS KINASES AND IN YOU HAVE
DESIGNING A ATP YOU WILL HAVE CO
ACTIVITY AGAINST ALL THESE
KINASES BUT THE FORTUNATE THING
IS THAT ALTHOUGH THIS DOMAIN OF
THE KINASE IS OUR CONSERVE THE
DOMAIN NEARBY AND NOT CON SIDE
AND YOU CAN EXPLOIT THAT
DIFFERENCE TO MAKE COMPOUNDS
WHICH ARE SELECTED AND HERE I'M
SHOWING YOU TWO VERY FAMOUS
KINASE INHAD BEEN TOURS AND YOU
CAN SEE THIS PINK PORTION WHICH
IS THE REGION BY KINDS TO THE
REGION AND THERE ARE OTHER
PORTIONS OF THE COMPOUND THAT
YOU CAN SEE HOW THEY'RE
POSITIONED WITHIN THE KINASE AND
THE CONFIRMATIONS ARE DIFFERENT
IN THE WAY THEY'RE POSITION AND
THAT WHAT BUILDS THE
SELECTIVITY.
IT'S SHOWN HERE AND THE FIRST
STUDY I'LL TALK ABOUT ACTUALLY
BEGAN IN 2005 AND 2008 SO IT'S
SLIGHTLY DATED BUT I THINK THAT
THE CONCEPTS THAT I LEARNED ARE
STILL APPLICABLE TODAY AND AND
SO THE FIRST CASE STUDY I'LL
SHOWCASE WHERE WE HAD RECEPTORS
AND THERE WAS A LOT OF LISTURE
OUT THERE WHICH MADE SENSE TO GO
AFTER THIS TARGET AND JUST TO
QUICKLY GO THROUGH THE SIGNALING
PATHWAY THIS WAS A KINASE IT'S
ACTUALLY A DIMER THERE'S AN
ALPHA SUBUNIT WHEN THE BINDS TO
THE EXTRA SER YOU LAR DOMAIN AND
THE DIMER COMES TOGETHER AND DUE
TO THE CONFORMATIONAL CHANGES
DUE TO THE BINDING EVENT, WHAT
HAPPENS IN THE DOMAIN IS THAT
ONE BETA UNIT RECRUITS ATM AND
PHOSPHORYLATION THE BETA UNIT
NEARBY SO THAT LEADS TO
CONFIRMATION CHANGE AND
RECRUITMENT OF SEVERAL DIFFERENT
PLAYERS WHICH ULTIMATELY STARTS
SIGNALING CASCADES WHICH
ULTIMATELY ACTUALLY LEAD TO THE
TURNING ON OF TRANSCRIPTION
FACTORS WHICH LEAD TO THE
PRODUCTION OF PROTEINS WHICH
LEAD TO PROLIFERATION,
MIGRATION, HALLMARK PHENOTYPES
OF CANCER.
NOW, WHILE A LOT OF DATA WITH
RESPECT TO SILENCING KNOCKING
OUT THE TARGET INDICATED IT
WOULD HAVE AN EFFECT ON CELL
PROLIFERATION AND THE MOST
IMPORTANT DATA WAS THAT PATIENTS
WHO WERE BEING TREATED FOR A
BREAST CANCER WERE BEING TREATED
WITH HERCEPTIN AND THEY WERE
DEVELOPING A RESISTANCE AND WHAT
WAS FOUND IN THE CLINIC WAS THAT
RESISTANCE WAS DRIVEN BY THIS
SIGNALING ACCESS AND ONE PROBLEM
WE REALIZED WAS THAT THE INSULIN
GROWTH FACTOR ONE REP SCEPTER
HAS THE INSULIN RECEPTORS AND IF
WE HAVE COMPOUNDS WE MIGHT HAVE
A PROBLEM WITH INSULIN SIGNALING
AND GLUCOSE HOMEOSTASIS, TIM AND
NATHAN WENT THROUGH A NUMBER OF
PLATFORMS WHICH CAN BE UTILIZED
TO DESIGN KINASE INHIBITORS.
YOU CAN LOOK AT AN ATP AND FOSS
FIR LAYING OF SUBSTRATE AND
MONITOR ATP CONCENTRATIONS WHICH
IS THE COFACTOR AND HAVE
CELLULAR ASSAYS IN THIS CASE
ITSELF AND YOU CAN HAVE ASSAYS
EXPLAINED JUST NOW BY MICHELLE
AND YOU HAVE ALL THESE DIFFERENT
THINGS THAT YOU CAN DO
DOWNSTREAM ASSAYS WHERE YOU LOCK
AT PHOSPHORYLATION OF DOWNSTREAM
SUBSTRATES AS WELL AS THE
TRANSCRIPTION AND PROLIFERATION
OF CELLS.
IF YOU HAVE A PLATFORM, YOU CAN
HAVE THE SAME PLATFORM AND
EXPAND IT TO OTHER KINASE WHICH
WILL ALLOW YOU TO THEN FIND OUT
HOW SELECTIVE YOUR COMPOUNDS
ARE.
SO, LET ME QUICKLY DESCRIBE YOU
THE FIRST SAY WHICH WAS THE
WORKFORCE ASSAY WE USED AND THIS
ARTIFICIAL PEPTIDE SUBSTRATE
THAT'S BIO TIN HERE SO WHEN YOU
DO THE PHOSPHORYLATION YOU CAN
ADD AN ANTI BODY A EUROPEAN CRIP
TATE AND IF YOU ADD ANOTHER
REALITY WHICH HAS AN ACCEPT OR,
YOU CAN HAVE A LIGHT TRANSFER
AND THEN THIS EMITS LIGHT AT A
DIFFERENT FREQUENCY SO BY
MEASURING THE FREQUENCY THAT
THESE LIGHTS, YOU CAN
ESSENTIALLY FIND OUT HOW MUCH
PHOSPHORYLATED PEPTIDE YOU HAVE
AND WHAT YOU WANT TO SEE IS BY
THE ADDITION OF COMPOUND YOU
SHOULD SEE A DOSE DEPENDENT
REDUCTION OF THE SIGNAL WHICH
TELLS YOU HOW MUCH YOU HAVE OF
THE SIGNAL YOU HAVE PEPTIDE YOU
HAVE.
I'LL BE SHOWING IC50 IN THE
NUMBER WHICH IS ESSENTIALLY THE
CONCENTRATION AT I WITH YOU SEE
50% INHIBITION OF THE SIGNAL AND
NOW, AS YOU HAVE ALREADY
REALIZED, THIS IS THE KINASE
DOMAIN IS INEXTRA CELLULAR SO WE
HAD ANOTHER ASSAY WHICH ALLOWED
US TO MEASURE FOSS FOLLOW
RELATED INCELLS SO IN THIS CASE,
WE WOULD ACTUALLY TAKE CELLS
WHICH WERE OVER EXPRESSING THE
KINASE OF INTEREST AND ADD THE
COMPOUNDS AND THIS PLATE
ACTUALLY HAD AN ANTI BODY COATED
ON THE SURFACE WHICH WAS AN ANTI
BODY SO THAT WOULD THEN CAP
AFTER AND WE ADD ANOTHER
ANTIBODY WHICH RECOGNIZES THE
FOSS FOR RELATED KINASE AND THIS
IS CHARGED WITH A CRIP TATE YOU
ADD RE AGENTS THAT LEAD IT OUT
AND THAT GENERATES A LIGHT
SIGNAL WHICH CAN BE MONITORED SO
THIS LIGHT SIGNAL IS ESSENTIALLY
REFLECTIVE OF HOW MUCH KINASE
THAT YOU HAVE AND IF YOU ADD
COMPOUND, WHAT I WANT TO SEE IS
THE REDUCTION OF THE SIGNAL SO
THAT TELLS YOU THAT IF YOU HAVE
A COMPOUND AND YOU ARE
INHIBITING THAT SIGNALS WHICH IS
WHAT YOU WANT.
SO WE NEED A STARTING WE HAD
PRONOUNCED CELLS AND THE PROFILE
WHICH WE WANTED WAS ACTUALLY OF
GROWTH INHIBITION BUT NOT OF A
CELL.
AND WE HAVE SOME OTHER KINASES
AND HERE THE DATA IS SHOWN IN
LOG PIC50 WHICH IS THE NEGATIVE
LOG SO OF IC50 SO IF YOU HAVE A
NUMBER OF SIX IT'S ONE MICRO
MOLAR IF IT'S NINE IT'S ONE NANO
MOLAR AND BEFORE YOU START ANY
PROGRAM FOR MEDICINAL CHEMIST WE
CAN LOOK AT THE PRIOR ART SO WE
WERE LATE IN THE FIELD AND THESE
COMPOUNDS IN IN LITERATURE SO WE
COULD ADD THESE TO OUR ASSAYS
AND MEASURE HOW WE'RE RANKING,
WHAT IS THE RANK ORDER WITH
RESPECT TO THESE COMPOUNDS.
ARE WE IMPROVING OR ARE WE
BEHIND WHAT'S DO WE NEED TO BE?
SO THIS IS TO HAVE SI SYNTHESIS
WHICH CAN BE DIVERSIFIED IN THE
END.
I DON'T WANT TO GO THROUGH THE
WHOLE SYNTHESIS BUT THIS IS FOR
THE CHEMIST AND WE DISCOVERED
CHEM TREE WHERE YOU TAKE THIS
AND YOU ADD ASSAYS, YOU HAVE AN
INTERNAL CYCLIZATION REACTION
AND YOU COULD OPEN IT UP IN THIS
PORTION OF THE MOLECULE AND
ULTIMATELY YOU COULD MAKE THIS
SAYINGEL AND GENERATE STRUCTURE
DIVERSITY AND THIS BOTTOM
PORTION OF THE MOL YOU MOLECULE.
IT EVALUATES IN THE ASSAY AND
SEE WHAT MOVEMENT YOU SEE IN THE
ACTIVITY WITH RESPECT TO THOSE
CHANGES AND SO IN YELLOW THE
ASSAYS I DESCRIBED AND THE PANEL
WE ALSO HAD ASSAYS WHICH ALLOWED
US TO MEASURE THE EFFECT IN CELL
PROLIFERATION AND WE HAD A
PROTEIN SHIFT ASSAY WHERE WE
WANTED TO SEE AND BIND TO GO THE
PROTEIN AND WE WANTED EFFICACY
IN PD EXPERIMENTS AND WE WERE
ALSO MONITORING ACTUALLY GROUP
IN THE LEVELS BECAUSE OF THE
INSULIN RECEPTORS ACTIVITY AND
WE HAVE A HOST OF ASSAYS WHICH
ALLOWED US TO ESSENTIALLY
MEASURE THE DRUG ABILITY OF THE
COMPOUND AND IT'S CALL CAN I
DOSE IT IN A RODENT?
CAN I SEE SOME EXPOSURE.
DOES IT REACH THE TUMOR WHICH IS
THE TARGET OF INTEREST.
FOR THE INTEREST OF TIME, I'LL
HAVE ONLY TIME TO JUST TALK
ABOUT THESE STARRED ITEMS TODAY.
SO, LET'S LOOK AT A DATA TABLE.
AND SEE WHAT HAPPENS WHEN WE DIG
THIS COMPOUND WHICH WAS A
STARTING POINT AND JUST MODIFY
THIS SMALL PORTION R1 AND WHAT
I'M SHOWING YOU HERE IS THE
ASSAY, IC50 WITH RESPECT TO IGFR
WHICH IS A TARGET OF INTEREST
AND YOU HAVE AURORA B AND
JUNCTION ONE WHICH WERE THE CO
ACTIVITIES IN THE STARTING
COMPOUND AND I HAVE THE CELLULAR
IC15 THE PHOSPHORYLATED ASSAY
AND WHAT YOU CAN SEE IS THAT BY
CHANGING THIS GROUP, WE'RE
SLOWLY ERODING AWAY THE AURORA B
ACTIVITY IN FACT, IT'S
COMPLETELY, WE HAVE COMPLETELY
REMOVED THE ACTIVITY ON THE
OTHER HAND, THIS BUYS US MORE
POTENCY WITH RESPECT TO THE IGFR
NUMBERS HERE WE HAVE SMALLER
NUMBERS HERE.
I WANT TO EMPHASIZE THAT
SOMETIMES ONE TO ONE
CORRELATIONS DON'T EXIST BUT YOU
CAN STILL SEE TRENDS AND IT'S
FINE.
THE OTHER THING WE ALSO REALIZE
IS THAT WE CAN ACTUALLY HANG A
BUNCH OF WATER GROUPS THAT TOLD
US THIS WAS PROBABLY OUT WITH
THE SOLVENT FRONT SO WE HAVE
FLEXIBILITY.
HOWEVER, THE POINT I WANT TO
EMPHASIZE IS THAT ROBUST ASSAYS
ALLOW US TO A ARRIVE AT DATA
LIKE THIS.
SO, QUICKLY, I WANT TO SHOW YOU
SOME IN VIVO DATA AS WELL AND SO
WHEN A COMPOUND IS DOSED, BY THE
IV DOSE, YOU OFTEN SEE A CURVE
LIKE THIS AND THIS IS BECAUSE
PRPREDOMINANTLY IT REACHES THE
LIVER WHERE THIS ARE THESE
WONDERFUL ENZYMES WHICH ACTUALLY
WILL DO THESE TRANSFORMATIONS
WHERE THEY TAKE A HYDROGEN AND
CONVERT IT TO HYDROXY AND THESE
ARE CALLED P450 IT'S GOOD THAT
THEY'RE THERE BECAUSE THEY'VE
HELD PROTECT YOU FROM ZENO
BIOTICS BUT WE HAVE TO REACH OUR
TARGET OF INTEREST.
THIS IS THE FIRST PASS AND OTHER
ENZYMES WATER AND YOU CAN
ESSENTIALLY ELIMINATE THESE
COMPOUNDS AND FOR THE CHEMIST IN
THE AUDIENCE, THERE'S A NOISE
MECHANISM FOR YOU TO THINK ABOUT
HOW THESE WORK.
SO ANYWAY, IF WE GO BACK TO THE
IV, DOSING CURVE, WE SEE IS A
CURVE WHICH LOOKS LIKE THIS AND
YOU CAN USE PARAMETERS LIKE
UNDER THE COVER AND THE
CONCENTRATION OF THE LAST TIME
POINT TO COMPARE AND CONTRAST
COMPOUNDS AND IF YOU DOSE A
COMPOUND ORDERLY, YOU SEE AN AB
PORTION FACE WHEN IT'S BEING
ABSORBED INTO THE LIVER AND YOU
CAN ALSO SEE ELIMINATION PHASE
YOU CAN USE PARAMETER LIKE THE
MAXIMUM CONCENTRATION OR THE
CURVE DIVIDED BY THE DOSE YOU
CAN USE IT TO COMPARE AND
CONTRAST AND EXPOSURE YOU ARE
GETTING THE DIFFERENT AND IN THE
VERSE RELATIONSHIP AND FROM THE
CURVE SO IT WAS DATA SO SEE WHAT
HAPPENS WHEN YOU OUTPUT THIS
COMPOUND AND MEETING AND WHAT
HAPPENS WHEN YOU MAKE VERY SMALL
CHANGES TO THIS PORTION OF THE
MOLECULE AND IF YOU DID THE MOL
YOU'LL, WHERE R1 IS HYDROGEN AND
SO WHAT WE REALIZE IS THAT OUR
CLEARANCE WAS HIGH AND IF YOU
MAKE AND YOU SEE AN IMPROVEMENT
AND CONCENTRATION AT EIGHT HOURS
AND YOU SEE IMPROVEMENT YOU
HONOR THOSE AND R2 AND SAME AND
PAM AR TERES BUT UNFORTUNATELY
WE'RE LOSING ACTIVITY AND
ATTENDING THE DATA LIKE THIS AND
CHEMIST AND YOU CAN USE THESE
RESULTS AND THEN DICTATE WHICH
YOU WANT TO KEEP AND ZENO GRAPH
MODEL AND WHAT WE REALIZE WAS
THIS HAD A NARROW IN THE CERTAIN
AND WE SENT BACK AND SAID WAIT A
SECOND, WE STILL HAVE THE JUNK
ACTIVITY AND PAPER WE SHOULD
DIAL IT OUT AND I'M CHANGING AND
THIS COMPOUND AND HAD THIS FROM
NITROGEN AND IT AND INTEREST AND
IT HAD GREAT CELLULAR ACTIVITY
AND THE JUNK ACTIVITY AND WE'LL
BE FOUND IS THAT THIS PARTICULAR
GROUP AND I LOST SELECTIVITY
AGAINST THE JUNK KINASE SO
ROBUST REPRODUCIBILITY ASSAYS
ALLOW YOU TO MAKE THESE SUBTLE
CONCLUSIONS WITH ROW STRICT TO
THE RELATIONSHIP.
THIS WAS ACTUALLY WAS A
CANDIDATE AND WE DECIDE TODAY
LOOK AT THE KINASE SELECTIVITY
AGAINST A BROADER PANEL AN 4% OF
PATIENTS HAVE A REARRANGEMENT
WITH PROTEIN AGAIN THE ML4 AND
WE WERE EVALUATING THE BROAD
ASSAY PLATFORM SO WE THOUGHT IT
WAS GREAT AND WE PUSHED THIS
ACTIVITY AND WENT TO THE
MANAGEMENT AND THIS IS A GREAT
CANDIDATE.
THE FUNNY THING IS THE PROOF OF
CONCEPT FOR CANCER IS STILL OUT
THERE AND THE COMPOUNDS FOR
CLINIC FOR CANCER HAVE NOT SEEN
THE LIGHT OF DAY.
BUT, ALK INHIBITORS HAVE.
UNFORTUNATELY WE WERE TOO
SUCCESSFUL IN MULTIPLE PROGRAMS
SO THE MANAGEMENT SAID WE CAN
JUST TAKE ONE COMPOUND, FORWARD
IT AND THEY TOOK THE CANDIDATE
WHICH WAS A DRUG BUT THIS WAS
SHELVED AND WHEN I JOINED NCATS
IT GOT APPROVED AND FOUR HAVE
BEEN APPROVED SO THIS IS MY I
COULD HAVE BEEN THERE.
SO YOU KNOW, SO IN THE END, YOU
HAVE A REPORT CARD OF THE
COMPOUND AND ALL THE ACTIVITIES
AND TALK TO YOUR TEAMMATES WHO
GENERATE THIS DATA AND HAVE A
GRASP OF HOW THIS DATA HAS BEEN
GENERATED AND WHAT IT MEANS AND
SHIFT GEARS TO THE NEXT KINASE
HIN HIB TO BE PROJECT WHERE WE
HAD A COMPOUND FOR DUCHENE
MUSCULAR DYSTROPHY SO IS IT
EFFECTS 1/5,000 BOYS AND I'M
SHOWING YOU THIS CARTOON WHERE
I'M SHOWING YOU THIS SARCOLAMA.
IN THE UNITS OF MUSCLE CELLS
THEY ACTUALLY ALIGN AND FORM
THESE TUBES AND SO THE PLASMA
MEMBRANE WHICH IS CALLED IS
ALMOST LIKE A CONTINUOUS SORT OF
STRUCTURE WHERE WE HAVE ALL
THESE DIFFERENT STRUCTURE
PROTEINS, ALL THESE DIFFERENT
STRUCTURE PROTEINS WHICH REALLY
GIVE A LOT OF STRUCTURE AND
RIGIDITY TO THE MUSCLE AND
STRENGTH AND WHAT HAPPENS IN DUE
LANE, IN THIS PRE TEEN, THIS
STRUCTURE OF PROTEIN IS ABSENT
AND THAT MAKES THIS THE MUSCLES
EXTREMELY WEAK IN THESE YOUNG
MEN AND WHAT OUR COLLABORATORS
THOUGHT, WELL, WHAT IF WE UP
REGULATED THE PRODUCTION OF
ANOTHER STRUCTURE PROTEIN AND
MAYBE WE CAN COM PEN TATE FOR
THE LOSS AND THE ASSAYS WAS
DESIGNED IN THE WAY THAT WE
LOOKED AT THAT ALPHA WHICH IS A
STRUCTURAL PROTEIN AND WE TOOK
ONE OF THE ALLELES OUT FROM THE
ALPHA 7 INTRA GRIN PROTEIN AND
AND IF THAT PROTEIN IS
EXPRESSED, YOU SEE AN EXPRESSION
OF THIS REPORTER AND YOU CAN ADD
THE SUBSTRATE AND GET A SIGNAL
SO THE LIGHT SIGNAL ESSENTIALLY
REFLECTS HOW MUCH PROTEIN THAT
YOU HAVE AND WHEN WE DID
REPURPOSE AND SCREEN WE
DISCOVERED THIS COMPOUND THE
CDK2 INHIBITOR WHICH WAS ABLE TO
UP REGULATE THE PROTECTION OF
THIS PROTEIN AND THIS PICTURE OF
THE -- WHAT IT IS, I'M SORRY,
MIO TUBES, YOU CAN SEE IN THE
MIO TUBES THERE'S A GRO GREEN GL
TELLING YOU THIS COMPOUND IS
ABLE TO UP REGULATE THE PROCESS
OF THE PROTEIN.
THAT WAS GREAT SO THEN THE
QUESTION IS, WELL, IT'S A CDK2
INHIBITOR KNOWN IN LITERATURE SO
THAT PROBABLY IS OUR TARGET BUZZ
IT'S A PHENOTYPIC ASSAY SO WE
LET LET'S FIND OUT THE KINASE
THIS COMPOUND IS ENGAGING WITH
IN THESE MIO TUBES AND SO I HAVE
TO INTRODUCE YOU TO THIS
TECHNOLOGY PLATFORM HERE AND
WHERE YOU HAVE THESE PROBLEMS
THAT LOOK LIKE ATP AND WHILE
TENDS HERE YOU HAVE A CARBON
HE'LL GROUP WHICH ACTUALLY IS
REACTIVE AND IF YOU ARE PAYING
ATTENTION, TO THE TOP BEFORE,
THERE ARE CONSERVED LICE EVENS
IN KINASE AT THE OUTER PARK ET
AND THESE CONSERVE ACTUALLY CAN
REACT WITH PROBLEMS LIKE THIS
AND THIS PROBE IS CHARGED WITH
BIO TIN SO WHEN THIS PROBE
INTERACTS WITH KINASES IN CELLS,
AND THIS THE LIE SEEN IS
ATTACHED TO THIS AND THEN CAN
YOU ADD A PRO TEE ACE AND CHOP
OFF THE KINASE AND YOU HAVE A
UNIQUE PEPTIDE SIGNATURE FROM
EACH FROM BINDING SO WHEN YOU DO
IT, YOU WILL ACTUALLY LOSE THAT
SIGNAL FROM THE LCMS EXPERIMENT.
SO, LET'S SEE WHAT HAPPENS WHERE
IT'S SHOWING THE REGULATION OF
THE ALPHABET TA PROTEIN SO I
DON'T WANT TO GO THROUGH THIS
BUT I WANT TO SHOW YOU HOW THE
DATA LOOKS SO YOU HAVE ALL THESE
DIFFERENT KINASES AND THESE PEP
SIDES AND WE HAVE THREE
DIFFERENT CONCENTRATIONS SO
GREEN IS WE DON'T SEE A BINDING
EVENT AND RED WE SEE A GOOD
BINDING EVENT AND IF WE FOCUS ON
THE KINASE WHERE WE SHOW
ACTIVITY OR BINDING OF THIS
PROBE WE SEE CDK2 AND BINDING IN
ALL THREE DIFFERENT
CONCENTRATIONS AND WE SEE CDK4,
5 WHICH IS EXPECTED AND WE
DISCOVER SOME OTHER KINASES
WHERE WE SEE MUCH BETTER
BINDINGS THAN CDK2 WHICH IS THE
ORIGINAL TARGET.
THAT WAS VERY SURPRISING.
WE SAID WELL MAYBE, THE ACTIVITY
OF THIS COMPOUND AGAINST THESE
KINASES IS ALREADY KNOWN AND
SURE ENOUGH, REACTION BIOLOGY
WHICH IS A PLATFORM WHICH
UTILIZES THIS SORT OF TECHNOLOGY
WHERE THEY HAVE ARTIFICIAL
SUBSTRATES WHICH IS FOSS FOR
LATED BY A RADIOACTIVE PHOSPHATE
AND PUBLISH THIS WONDERFUL PAPER
WHERE THEY TOOK COMPOUNDS AND
THEY DID PROFILING AND PUBLISHED
THIS LITERATURE AND WHEN WE
LOOKED AT OUR COMPOUNDS, SU9516
WE SAW THAT AS PREDICTED, IT WAS
A GREAT INHIBITOR OF THIS
FUNCTIONAL ACTIVITY AGAINST CDK5
BUT IT WAS DEAD AGAINST THE TWO
KINASES THAT WE HAD DISCOVERED
IN THE MIO TUBE EXPERIMENT SO
THAT TELLS YOU THAT DEPENDING ON
THE PLATFORM YOU HAVE, YOU CAN
HAVE VERY DIFFERENT RESULTS AND
OBVIOUSLY THE QUESTION IS, WHAT
IS THE TRUTH?
THE BIOCHEMICAL OR THIS ONE AND
SO, YOU KNOW, JUST WANT TO HIT
HOME THIS CONCEPT WHERE YOU TAKE
CELLS OF COMPOUNDS WITHOUT
COMPOUNDS AND TAKE THE LICE IT'S
A AND INCUBATE WITH THESE BEADS
AND THEY WILL BIND TO THE KINASE
AND PULL THEM OUT AND YOU CAN DO
IT AGAIN AND IF YOU HAVE
COMPETING COMPOUNDS YOU WON'T
SEE THAT BINDING SO THIS IS A
GREAT PAPER AND CAN YOU READ AND
HERE TOO, THEY FOUND DIFFERENCES
BETWEEN BIOCHEMICAL AND CELLULAR
TARGETING ENGAGEMENT.
ALL RIGHT.
LAST CASE STUDY, I'LL DISCUSS A
RARE DISEASE AND IT'S GOUSHER
DISEASE, THIS IS A RARE
RECESSIVE DISORDER AND DISEASE
AND BECAUSE OF THE LACK OF
ACTIVITY BECAUSE OF THIS ENZYME
YOU SEE ACCUMULATIONS OF THESE
SUBSTRATES WITHIN THE LICE OWE
SOME THAT'S WHY IT'S THE
DISORDER AND THERE ARE THREE
FORMS OF THIS DISEASE I'LL FOCUS
ON EXTRA THE TYPE ONE AND IT HAS
ESSENTIALLY MOSTLY MAN FESS
INDICATIONS IN THE VISCERA AND
THEY HAVE LARGE SPLEENS AND
LIVES AND THEN THERE ARE TWO
UNFORTUNATE TYPES WHICH HAVE A
PATHIC COMPONENT AND ONE OF THE
GENOTYPES RENDERS IT UNSTABLE.
IF YOU HAVE THE MOST COMMON
SUBTYPE OF THIS HYDRO LAYS, IF
IT MAKES IT TO THE LICE OSEM IT
HAS CATALYTIC ACTIVITY BUT THE
PORTING DOESN'T FOLD PROPERLY SO
IT'S TAGGED FOR DEGRADATION SO
THE PROBLEM IS YOU HAVE LESS
PROTEIN.
SO, THE QUESTION IS HOW DO YOU
THEN FIND COMPOUNDS WHICH CAN BE
THERAPEUTIC AND OUR GOAL WAS TO
ACTUALLY DEVELOP SMALL GROUPS
AND THIS WAS ALSO A CONCEPT
EXPLAINED IN THE PREVIOUS TALK,
YOU CAN SOMETIMES FIND COMPOUNDS
WHICH BIND TO PROTEINS AND MAKE
THEM STABLE AND PREVENT THEM
FROM DEGRADATION SO THAT WAS OUR
GOAL.
THE QUESTION WAS LET'S DESIGN .
CHEMICAL ASSAY.
WE CAN OBVIOUSLY HAVE RECOMMON
PROTEIN AND HERE IS WHAT THE
PROTEIN DOES, IT FORMS A PART OF
THE MEMBRANE AND IT ACTUALLY
INTERACTS WITH CO FACTORS, NOT
CO FACTORS, OTHER PROTEINS WHICH
HELP ACTUALLY ORIENT THE
SUBSTRATE IN A PROPER WAY SO IT
CAN CHOP IT.
THE PROBLEM WITH THE N37S IT WAS
HARD TO MAKE IT BECAUSE IT WAS
UNSTABLE SO WE WENT TO A
CLINICIAN AND BECAUSE THE
PATIENTS HAVE ENLARGED SPLEEN,
SHE HAD SPLEEN SAMPLES FROM THE
PATIENTS IN HER FREEZER SO WE
TOOK THOSE SPLEENS AND PUT THEM
IN A GRINDER AND WE HAD A SOUP
THAT HAD THIS ENZYME AND THAT
ENZYME PRESENT IN THE SPLEEN,
WAS ABLE TO ACTUALLY HAD CAT A
LAD I CAN FUNCTION AND THE
QUESTION IS WHAT DO WE HAVE FOR
SUBSTRATE?
WE HAVE THE TYPICAL ACTUAL
SUBSTRATE AND MONITORING THE
HYDROLYSIS OF THIS IS DIFFICULT
SO WE HAD SUBSTRATES WHERE WE
HAD A GROUP COULD FLORES AT THE
RED REGION OF THE SPECTRA AND I
JUST WANTED TO SHOW YOU WHAT
HAPPENS WHEN WE DO A SCREENING
WITH THESE DIFFERENT SOURCES AND
DIFFERENT SUBSTRATES AND SO HERE
IS WHAT THE RESULTS OF A
SCREENING OF A DECK OF 558,000
COMPOUNDS SO IF WE USE THE WILD
TYPE PROTEIN WE DISCOVERED A
NUMBER OF INHIBITORS AND FEW
ACTIVATORS ON THE OTHER HAND,
WHEN WE TOOK THE SAME DECK AND
SCREENED AGAINST THE N370S IN
THE SPLEEN FROM PATIENTS WE
DISCOVERED MORE ACTIVATORS AND
THEN LESS INHIBITORS AND THIS
SCREENING WAS DONE WITH THIS
BLUE SUBSTRATE SO IT TELLS YOU,
DEPENDING ON THE SOURCE OF THE
ENZYME AND THE SUBSTRATE CHOICE
YOU HAVE, THE SUBSTRATE CHOICE
I'LL TALK ABOUT IT IN THE NEXT
SLIDE, YOU CAN HAVE VERY
DIFFERENT RESULTS AND THE WAY WE
RATIONALIZED THIS OUTCOME WAS
WHEN WE TAKE THE ENZYME WE
ACTUALLY HAVE TO ADD DETERGENT
TO MAKE IT ACTIVE AND WE THINK
THAT THAT ACTIVATES THE ENZYME
AND PREVENTS YOU FROM
DISCOVERING ACTIVATORS AND LET
ME SHOW YOU QUICKLY FOR TYPES WE
DISCOVERED AND SHOW YOU WHAT
HAPPENS WHEN WE TAKE JUST THE
N370S MUTANT, THE COURSE COULD
BE THE REKUM BAN RE-- AND THE DS
SHOWN IN LIGHT AND IN BOLD IS
THE SPLEEN DATA.
HERE IS THE COMPOUND WHICH SHOWS
INHIBITION OF THESE TWO
SUBSTRATES ONLY WHEN YOU USE THE
SPLEEN.
THIS COMPOUND SHO SHOWS ACTIVITY
WHEN YOU USE THE PROTEIN.
THIS IS GREAT FROM CHEMISTS
BECAUSE NO MATTER WHAT YOU DO IT
WORKS.
IF YOU REMEMBER, THE DISEASE IS
DUE TO TO DEFICIENCY OF THE PRO.
WE WANT A COUPLE POUND THAT
ACTIVATES THE PROTEIN OR AS I
MENTIONED, INCREASES THE
CONCENTRATION OF THE PROTEIN AND
SO THIS WAS A COMPOUND WE
DISCOVERED WHERE WE SAW
ACTIVATION WITH RESPECT TO THE
HYDROLYSIS OF THIS ARTIFICIAL
SUBSTRATE AND WE SAW ACTIVATION
ONLY WITH THE BLUE SUBSTRATE.
AND IN FACT, THIS ASSAY WAS THEN
UTILIZED TO EXECUTE A MEDICINAL
CHEM TREE COMPOUND WITH THE
SPLEEN AND THE BLUE SUBSTRATE
AND WE DID, WHY WANT TO GO
THROUGH THIS DATA, WE PUBLISHED
THIS A LONG TIME BACK BUT WE HAD
A COMPOUND ML198 WHICH WAS MUCH
MORE POTENT THAN THE STARTING
HIT THAT WE HAD AND THEN WE WENT
ON TO PUBLISH THIS DATA AND THEN
I'LL SHOW YOU FURTHER
CHARACTERIZATION WE DID TO THIS
COMPOUND.
I WANT TO SHOW YOU THESE
COMPOUNDS BECAUSE I'M GOING TO
TALK ABOUT THIS AGAIN.
WE MADE THESE COMPOUNDS WHERE WE
REPLACED THE RING WITH FOUND
THEY HAD NOT THAT GREAT POTENCY
BUT THEY WERE IMPORTANT LATER
ON.
SO WE TOOK THIS COMPOUND AND
ACTUALLY SHOWED THAT NOT ONLY IS
THE COMPOUND ABLE TO ACTUALLY
INCREASE THE CONCENTRATION OF
PATIENT DERIVED MACROPHAGES.
SO MACROPHAGES ARE THE CELLS
WHERE THE DISEASE ACTUALLY
HAPPENS AND WHERE WE SEE VERY
LOW QUANTITIES OF IN GROWN AND
WHEN YOU ADD THE COMPOUND YOU
CAN SEE THE GREEN GOES TO THE
RED THAT'S WHY YOU HAVE THIS
YELLOW SIGNAL SO WITH THE
COMPOUND INCUBATION WE SEE A
PROFOUND INCREASE IN THE
CONCENTRATION OF THE ENZYME IN
THE LICE OWE SOMEWHERE WE WANT
IT TO GO.
THERE'S AN OWESSIZATION OF ASSON
PARKINSON'S DISEASE, WE TOOK
SHELLS FROM PARKINSON PATIENTS
AND FIBER BLAST AND GENERATE
IPSCs AND DIFFERENTIATE THEM
INTO NEURONS AND WE WERE ABLE TO
SHOW THIS COMPOUND IS ABLE TO
CLEAR AGO AGREE GATES FROM THESE
NEURONS AND I CAN TALK ABOUT
THIS IF YOU GUYS ARE INTERESTED.
ESSENTIALLY WHAT I'M SHOWING
YOU.
THESE ASSAYS ARE DIFFICULT TO DO
AND YOU HAVE TO AGE THE NEURONS
FOR SEVERAL DAYS TO SEE THE
PHONE OWE TYPE AND WHAT OUR
BIOLOGY COLLEAGUES WERE ABLE TO
SHOW THIS COMPOUND ACTUALLY IS
ABLE TO DO THIS PROFOUND
REDUCTION OF AGO AGREE GATES.
IT WAS LICENSED TO ACCOMPANY AND
THEY MODIFIED THIS TO THIS
CLINICAL COMPOUND WHICH WENT TO
THE CLINIC AND I'M LOOKING TO
SEE WHAT HAPPENS WITH THIS CHEMO
TYPE.
SORRY, I HAVE TO ADVERTISE THIS.
SO THIS IS AN ARTICLE I ACTUALLY
WROTE WITH POSTDOCTORAL TRAINEES
IN MY GROUP.
WE WENT THROUGH SIMILAR CASE
STUDIES TO SHOWCASE THAT THE
ASSAY IS IMPORTANT IN DEFINING
THE BIOLOGY OF YOUR COMPOUNDS
AND LASTLY, I JUST WANT TO HIT
HOME THE SAME MESSAGE THAT YOU
KNOW, ASSAYS CAN BE REALLY
IMPORTANT AND WORK WITH YOUR
BIOLOGIST TO UNDERSTAND THEM.
THEY CAN MAKE OR BREAK A
CAMPAIGN AND OBVIOUSLY, I HAVE
TO THANK A NUMBER OF PEOPLE WHO
ARE WORKING THESE PROJECTS AND
THANK YOU FOR YOUR TIME.
>>THANK YOU SO MUCH, SAM, FOR A
WONDERFUL TALK AS USUAL.
WE HAVE QUESTIONS HERE.
>>THANK YOU FOR A WONDERFUL AND
JUST REFERENCES WOULD THAT
FOLLOW THE SAME PRINCIPLES THE
CONTRACTOR, THE CONCEPT WHERE
YOU LOOK AT THINGS LIKE, YOU
KNOW, WHERE YOU HAVE A KINASE
THAT PHOSPHORYLATION A BUNCH OF
DIFFERENT KINASES TO SEE HOW
THEY'RE IMPACTED WITH THE DRUG.
SIMILARLY, WOULD THAT APPLY TO
THE POST TRANSLATIONAL
MODIFICATION AS WELL OR IS IT
JUST FOR PHOSPHORYLATION.
>>IF THERE'S BIOLOGY THAT LOOKS
AT DOWNSTREAM EVENTS, I WOULD
ALWAYS PREFER THAT WE MONITOR
THAT SO WHEN YOU ARE ESSENTIALLY
OPTIMIZING COMPOUNDS WHICH ARE
DIRECTLY BINDING WITH THE
TARGET, THE DOWNSTREAM EVENTS
TAKE PLACE AS WELL.
SOMETIMES IT CAN BE DIFFICULT
BECAUSE DESIGNING ASSAYS FOR
DOWNSTREAM AT LEAST TARGET
ENGAGEMENT IF THE COMPOUND IS
MAKING IS ENGAGING WITH THE
TARGET BECAUSE YOU CAN DO ASSAYS
IN HIGH-THROUGHPUT WE HAVE FOLKS
HERE WHO IT COULD BE LIMITEDDED
WHAT IS AVAILABLE TO YOU AND
YEAH.
>>DO YOU LIKE AT WHAT WAS
DRIVING THAT?
>>THAT'S A GREAT QUESTION, THIS
IS SOMETHING THAT PEOPLE OFTEN
ASKED ME BECAUSE I SHOWED
DIFFERENT ENZYME SOURCES AND
JUST WHAT IS THE STRUCTURAL
BASIS AND IN THE KINASE ASSAY,
WE, I CAN SPECULATE THAT PERHAPS
IN THIS THE CELLULAR ENVIRONMENT
IT'S MORE PHYSIOLOGICALLY
RELEVANT AND IT'S PROBABLY
INTERACTING.
YEAH -- I WOULD BELIEVE THE CELL
BASE YEAH.
THEY HAVE MARK HERE WHO WAS
HEADING THE BIOLOGY THERE AND HE
CAN TELL YOU WHAT HE BELIEVES.
YEAH, I WOULD BELIEVE THAT.
[OFF MIC]
>>YES, THAT IS TRUE.
YOU ARE RIGHT.
I DON'T THINK -- THAT'S WHY I
HAVE TO WORK WITH PEOPLE LIKE
YOU.
THE QUESTION WAS HERE, NATHAN
SAID, THAT THE ATP CONCENTRATION
AND THE BIOCHEMICAL ASSAY COULD
BE IMPORTANT AND MAYBE THAT WAS
ONE OF THE REASONS WHY WE DIDN'T
SEE ACTIVITY AGAINST THE TWO
OTHER BIOLOGICAL TARGETS.
THAT IS WHY YOU HAVE TO WORK
WITH FOLKS LIKE YOU.
WHO WILL READ THE PORT CAL.
[OFF MIC]
YES.
[OFF MIC]
YES.
SO THIS IS A VERY IMPORTANT
POINT.
SOMETIMES THE FOLKS WHO ACTUALLY
DEVELOP THESE PLATFORMS, THEY
WILL LOOK AT SPECIFIC SEQUENCES
OF PEPTIDES OF THE SUBSTRATE AND
DESIGN THE PEPTIDE SEQUENCE.
IF YOU HAVE THAT IT'S MUCH MORE
RELEVANT THAN SUBSTRATE.
SOMETIMES YOU GO WITH WHAT YOU
HAVE.
[OFF MIC]
>>YES.
SO THAT'S A VERY -- YOU PICKED
THAT UP.
SO, EVEN WITHOUT THE DIMER, YOU
CAN ADD IN A SUBSTRATE AND IT
FOSS FOR LATES AND IT DOES THE
AUTO PHOSPHORYLATION.
WONDERFUL.
THANK YOU SO MUCH AGAIN, SAM.
[APPLAUSE]
ALL RIGHT.
OUR NEXT SPEAKER IS Dr. SOFIE
A SENIOR SCIENTIST AT PFIZER
WITH WHERE SHE SUPPOSE SCREENING
PLATFORM AND HIT DISCOVERY
WITHIN THE PRIMARY PHARMACOLOGY
GROUP.
HER TALK TODAY IS ENTITLED
BUILDING THE EFFICIENT DISCOVERY
OF ACTIONABLE CHEMICAL MATTER
FROM DNA-EN CODED LIBRARIES.
THANK YOU SO MUCH, SOPHIA.
>>THANK YOU FOR THE KIND
INTRODUCTION AND THANK YOU FOR
GIVING ME THE OPPORTUNITY TO BE
HERE AT THIS WORKSHOP.
AS THE TITLE INDICATES, MY TALK
BEFORE WAS ON THE UNCODED
LIBRARY SCREENING AND THE WORK
FLOW THAT THE PFIZER GROUP
IMPLEMENTED IN THE LAST COUPLE
OF YEARS TO ENSURE THE
ACTIONABLE CHEMICAL MATTER AT
THE END OF THE SCREENING.
NOW BEFORE I START, HOW MANY OF
YOU ARE FAMILIAR WITH THE
SCREENING, RAISE YOUR HANDS?
A FEW OF YOU, THAT'S NOT BAD.
SO FOR PEOPLE WHO ARE NOT
FAMILIAR WITH IT DON'T WORRY
BECAUSE I'M GOING TO START MY
TALK WITH A BRIEF INTRODUCTION
TO THE SCREENING.
AND AFTER THAT, I'LL SHOW YOU
HOW WE AT PFIZER VALIDATE OUR
PROTEIN REAGENTS BEFORE THE
SELECTION AND LASTLY I'LL TALK
ABOUT THE WORK FLOW THAT WE
IMPLEMENTED TO ENSURE THAT WE
HAVE HIGH HEAT CONFIRMATION
RATES WHEN WE SYNTHESIZE THE
COMPOUNDS OF DNA AND ENSURE THE
COMPOUNDS HAVE GOOD DRUG-LIKE
PROPERTIES AND HOPEFULLY IN THE
LAST COUPLE OF MINUTES I'LL TALK
ABOUT CELL BASED SCREENING AS
WELL AND MEMBRANE PROTEINS.
LET ME START WITH A BRIEF
INTRODUCTION.
SO, THE DNA-ENCODED LIBRARY
SCREENING IS A RELATIVELY NEW
TECHNOLOGY.
IT WAS DEVELOPED IN THE LAST
20-25 YEARS BUT IT REALLY CAUGHT
ON IN THE LAST DECADE OR SO.
SO, THOSE ARE A COLLECTION OF
COMPOUNDS THAT ARE LINKED TO A
DNA TAG AND SO, EVERY MOLECULE
IN YOUR LIBRARY WHICH WE CALL
WAR HEADS HAS A UNIQUE DNA TAG
WHO SEQUENCES DESCRI DESCRIBES E
BUILDING BLOCKS IN THE LIBRARY
SO FOR EXAMPLE THE BLUE BUILDING
BLOCKS IS ENCODED BY THE BLUE
SEQUENCE AND THE RED BY THE RED
SEQUENCE AND SO ON.
NOW, MY TALK WILL FOCUS ON AND
SCREENING AND NOT SYNTHESIS BUT
STILL I WANTED TO SHOW YOU HOW
THESE COMPOUNDS ARE MADE BECAUSE
I THINK IT'S QUITE WE REFER TO
THIS A METHODOLOGY SO WE START
WITH A DNA HAT PIECE KICKED TO A
LINKER AND WE SPLIT THAT AND
PLACE IT INTO A MORE PLATE AND
LET'S SAY A PLATE.
NOW, AFTER THAT, WE DO OUR FIRST
LIGATION OF THE DNA TAG.
SO, AT THIS STEP, EVERY SINGLE
WELL WE RECEIVED A UNIQUE DNA
SEQUENCE SO AFTER YOUR LIGATION,
YOU END UP WITH 96 UNIQUE DNA
SEQUENCES.
SO WHEN YOU LIGATE YOUR FIRST
DNA TAG, YOU ADD YOUR FIRST
BUILDING BLOCK AND AGAIN, EACH
WELL WE RECEIVED THE FIRST
BUILDING BLOCKS SO AFTER YOUR
SYNTHESIS, YOU WIND UP WITH 96
DIFFERENT COMPOUNDS WITH ITS OWN
UNIQUE DNA SEQUENCES.
SO AFTER THIS SYNTHESIS, YOU
POOL ALL YOUR TOGETHER INTO A
TUBE AND MIX THEM EFFICIENTLY
AND REDISTRIBUTE THEM.
EVERY SINGLE WELL IN YOUR PLATE
WILL HAVE ALL THE COMPOUNDS THAT
YOU JUST SYNTHESIZED IN THE
FIRST ROUND.
AND THEN YOU REPEAT THIS CYCLE A
COUPLE MORE TIMES.
SO AGAIN, YOU ARE GOING TO ADD
YOUR SECOND DNA TAG, YOUR
BUILDING BUILDING BLOCK AND
EVERY SINGLE WILL RECEIVE ITS
UNIQUE BLOCKS AND SO YOU REPEAT
THIS TWO OR THREE TIMES
DEPENDING ON THE COMPOUNDS THAT
YOU WANT TO SYNTHESIZE SO FOR
EXAMPLE, AT THE END HERE, YOU
WILL HAVE A COMPOUND WITH THROW
DIFFERENT BUILDING BLOCKS AND
IT'S OWN UNIQUE DNA TAG.
NOW JUST SOMETHING TO KEEP IN
MIND, SINCE WE'RE DOING ORGAN
SYNTHESIS IN THE PRESENCE OF DNA
WE HAVE TO HAVE WATER PRESENT SO
IT'S POSSIBLE THAT UNDER THESE
CONDITIONS, NOT EVERY CHEMICAL
REACTION WE PROCEED AS EXPECTED,
AND SOME OF YOUR BUILDING BLOCKS
JUST WON'T BE INCORPORATED.
SO, THE NICE THING ABOUT THE
SPLIT AND PULL TECHNOLOGY THAT
JUST BY USING A SINGLE PLATE WE
COULD MAKE A MILLION COMPOUNDS
AND WE REALLY NEEDED JUST 96 FOR
THAT AND NOT ONE MILLION
DIFFERENT VIALS.
NOW, AGAIN, I'M NOT GOING TO
TALK ABOUT DELL SYNTHESIS AND
GREAT PUBLICATIONS OUT THERE
THAT YOU COULD JUST READ UP ON.
THE LIBRARIES ARE POISE TOGETHER
AND THIS IS WHAT WE USE FOR
SCREENING AND SO WE INCUBATE THE
PROTEIN AND WHEN YOU REACH
BINDING EQUITY BIOME YOU CAN
JUST USE THE MAGNET AND PULL
EVERYTHING DOWN, YOU PULL DOWN
YOUR BEADS, PROTEIN, BIO
COMPOUNDS AND REMOVE THE UNBOUND
COMPOUNDS.
AFTER THAT, YOU TAKE YOUR BEADS
AND YOUR PROTEINS THROUGH A
COUPLE OF WASH CYCLES TO REMOVE
THE BINDE BINDERS SO WE RUN A CE
OF SELECTIONS WHICH MEANS THAT
WE TAKE THESE SPECIFIC ALLUDED
COMPOUNDS FROM ROUND ONE AND WE
TAKE THEM INTO ROUND TWO AS
INPUT COMPOUNDS.
AND RUNNING A COUPLE ROUNDS OF
SELECTION IS NECESSARY BECAUSE
YOU WANT TO REACH YOUR SPECIFIC
LIG ANTS ENOUGH AND REUSE YOUR
BACKGROUND SO YOU CAN DOSE YOUR
COMPOUNDS ON A SEQUENCING CHIP.
NOW, THE SELECTION TAKES A
COUPLE OF HOURS BUT WE DON'T
HAVE A SCIENTIST CHAINED TO THE
BENCH AND RUNNING THIS FOR HOURS
AND HOURS.
AT THIS POINT, EVERYTHING IS
AUTOMATED SO YOU LITERALLY JUST
TAKE YOUR PLATE, PIPE IT IN YOUR
REACH AND THE REST IS TAKEN CARE
OF BY AN INSTRUMENT.
THE ADVANTAGE OF -- THEY HAVE
SEVERAL ADVANTAGES COMPARED TO
TRADITIONAL HDS FOR EXAMPLE WE
REQUIRE LESS PROTEIN REAGENT
COMPARED TO HDS IN OUR CASE, WE
USUALLY ASK FOR TWO OR THROW
MILL A GRAMS OF PROTEIN AND THAT
IS ENOUGH FOR REGION VALIDATION
AND FOR SCREENING AND FOR HIT
VALIDATION AND FOR THE
OCCASIONAL MESS UPS AND ASSAY
REPEATS.
NOW, ALSO DELL LIBRARIES DON'T
HAVE ANY BIAS AGAINST THEM.
AND WHAT I MEAN BY THAT, IS FOR
EXAMPLE, IF YOUR COMPANY HAS
BEEN HEAVILY WORKED IN
NEUROSCIENCE SPACE, YOU PROBABLY
HAVE A CHEMICAL MATTER WHICH HAS
BIAS AGAINST COMPOUNDS THAT HAVE
SPECIFIC PHYSICAL CHEMICAL
PROPERTIES THAT ALLOWS THEM TO
CROSS THE BROAD BAIN BARRIERS
AND YOUR CHEMICAL METHOD DOESN'T
COVER ALL THE OTHER CHEMICAL
SPACE HOWEVER THIS IS NOT TRUE
FOR DEL SO THEY DON'T HAVE BIAS
AGAINST THEM AND THEY COVER A
WIDE CHEMICAL SPACE.
NOW, DEL VALIDATION AND
SCREENING ITSELF IS RELATIVELY
FAST AND WE CAN RUN THESE IN
JUST A COUPLE OF WEEKS.
AND ALSO, WE CAN PENALIZE
MULTIPLE CONDITIONS BUT I'LL GET
BACK TO THAT A LITTLE BIT LATER.
BEFORE I GET INTO THE DETAILS,
OF SCREENING AND THESE ANALYZES,
LET ME JUST WALK YOU THROUGH
WHAT IT TAKES TO BE A HIT IN DEL
SELECTION AND WHAT IS EXPECTED
OUTCOME THAT YOU WOULD SEE BASED
ON THE BIO CHEM TREES THAT IS
GOING ON IN YOUR BINDING
REACTION.
IN BOTH CASES, THE CONCENTRATION
OF EITHER THE LIG ANT OR THE
PROTEIN HAS TO EXCEED THE KD OF
THE INTERACTION.
SO, IN TRADITIONAL HDS, YOU HAVE
YOUR ACCESS AND SO YOU WORK WITH
LIGANDS AND YOU LOW
CONCENTRATION OF PROTEIN.
SO YOU CAN EXPRESSION IT AND
THIS IS SWITCHED.
WE HAVE A HIGH CONCENTRATION OF
PROTEIN, USUALLY IN THE AND LOW
CONCENTRATION OF COMPOUNDS SO IF
YOU WANT TO HAVE THE BOUNDS YOU
COULD USE THE REST TO DO THAT.
NOW, IN PRACTICE, WE HAVE ONE
MILLION COPIES PAIR COMPOUNDS IN
OUR LIBRARY.
IF YOU WANT TO PUT THAT INTO
CONCENTRATION, THAT IS IN THE
MOLAR IMAGINE 10 TO THE MINUS 12
AND 10 TO THE MINUS 15.
SO WE HAVE A SMALL AMOUNT OF
COMPOUNDS IN OUR ASSAYS.
YOU CAN SEE IN DEL THE BINDING
IS DRIVEN BY PROTEIN
CONCENTRATION AND HDS THE
BINDING IS DRIVEN BY LIGAND
CONCENTRATION AND WE UTILIZE
THIS DURING THE SELECTION SO WE
ACTUALLY HAVE SAMPLES WHICH HAVE
HIGH PRE TEEN CONCENTRATION
AROUND TWO, TWO AND A HALF MACRO
MOLAR AND WE HAVE CONDITIONS
WHERE WE HAVE LOW PROTEIN
CONCENTRATIONS AND LET'S SAY .5
OR MICRO MORAL AND THIS GIVES US
AN IDEA OF THE RELATIVE AFFINITY
OF THE COMPOUNDS SO IF YOU HAVE
A COMPOUND THAT BINDS TO BOTH
SAMPLES THAN THE COMPOUND HAS
HIGHER AFFINITY.
IN A TYPICAL DELL SCREENING WE
OBSERVE BETWEEN 200 AND 500 FULL
ENRICHMENT PAIR ROUND OF
SELECTION SO THAT MEANS WE HAVE
TO RUN.
>>Alex: SELECTION TO REACH OUM
BOUNDS AND DRIVE THE BACKGROUND
DOWN SO HERE ON THE BOTTOM RIGHT
THERE'S JUST A SELECTION
STIMULATION AND ARE YOU SEE THE
ROUNDS OF DEL SELECTION FOR
COMPOUNDS WITH VARIOUS
AFFINITIES AND YOU CAN SEE IS
THAT THE COMPOUND THAT IS A
KNOWN BINDER SO IT HAS 100 MICRO
MOLARS IT'S WASHED OUT THE
SOLUTION AFTER THE SECOND ROUND
OF SELECTION SO AT THIS POINT,
WHAT YOU HAVE ARE COMPOUNDS WITH
10 MICRO MOLAR OR LOWER.
NOW, ESSENTIALLY, YOU COULD RUN
ENOUGH ROUNDS OF DEL SELECTION
TO REMOVE ALL OF YOUR LOWER
AFFINITY COMPOUNDS AND THE
REMAININGS ARE JUST THE HIGH
AFFINITY COMPOUNDS, HOWEVER IN
PRACTICE, WHAT WE DO IS WE LOWER
THE TOTAL NUMBER OF COMPOUNDS
JUST ENOUGH SO THAT WE CAN LOAD
THEM ONTO THE SEQUENCING CHIP
AND SO WE USUALLY RUN TWO OR
THREE ROUNDS OF SELECTION AND ON
UNDER THESE CONDITIONS, OUR HITS
HAVE EITHER SINGLE DIGIT MICRO
MOLAR AFFINITY SO NO MOLAR
AFFINITY SO THIS IS JUST AFTER
OVERVIEW OF THE VALIDATION AND
SCREENING PROCESS AND WE START
WITH A PRO SELECTION VALIDATION
AND AND WE RESYNTHESIZE ON DNA
TO CONFIRM THOSE HITS AND IF WE
CONFIRM THEY MAKE THEM OF DNA AS
WELL AND THEY'RE PUT INTO SOME
KIND OF BIOCHEMICAL AS SAY.
IT PROVED TO BE IMPORTANT IN OUR
WORK FLOW AND I WILL TALK ABOUT
THAT AS WELL A LITTLE BIT LATER.
AND LET ME START BY TELLING YOU
HOW WE VALIDATE OUR TARGETS AT
PFIZER.
EVERY SUCCESSFUL HEAT ID
PLATFORM DEPENDS ON THE REGIONS
AND THIS IS TRUE FOR DAL SINCE I
AS I MENTIONED, BINDING IS
DRIVEN BY PROTEIN CONCENTRATION
AND SO YOU REALLY WANT THE
MAJORITY OF YOUR PROTEINS TO BE
WELL BEHAVED.
SO, WITH THIS IN MIND, WE SET UP
A WORK FLOW BEFORE WE TAKE THEM
INTO THE SCREENING AND
ESSENTIALLY, WHAT WE DO OR TRY
TO DO IS TO QUANTIFY THE ACTIVE
SIDES OF THE PROTEIN AND BEFORE
I AM MOBILIZIMMOBILIZE, THE ACTN
BE LOST FOR SEVERAL REASONS, FOR
EXAMPLE, BECAUSE OF LOSS OF
TRANSITION OF FREEDOM IN THE
SPACE AND BECAUSE YOUR PROTEIN
REGION IS NOT JUST COMFORTABLE
WITH THE DEL REGIONS LIKE HIGH
SOLUBLE OR DNA.
THIS GRAPH I'M SHOWING YOU HERE
IS ONE OF THE OUR EARLIER TIMES
TO HAVE DATE A TARGET AND THIS
IS A FLUORESCENCE POLARIZATION
OF A COMPOUND BINDING TO THE
PROTEIN AND THIS IS PROTEIN IN
SOLUTIONS SO WITHOUT I AM
MOBILIZATION AND THIS IS HERE
WITH I AM MOBILIZATION AND NOW,
YOU CAN SEE THAT THE
PHARMACOLOGY MORE OR LESS REMAIN
THE SAME SO WE HAVE THE SAME
IC50 VALUE HOWEVER IF YOU LOOK
AT THE SIGNAL WINDOW IT'S
SIGNIFICANTLY DECREASES FOR THE
IMMOBILIZED PROTEIN.
WE'RE NOT SURE WHETHER THIS WAS
AN ARTIFACT OF THE ASSAYS OR
WHETHER MOST OF OUR PROTEINS
JUST DEMATURES AFTER
MOBILIZATION AND IT COULDN'T
BIND TO THE COMPOUND.
SO IT WAS CLEAR THAT THIS MATTER
IS NOT THE WAY FORWARD TO
VALIDATE OUR REGIONS FOR DEL
SINCE THIS IS NOT QUANTITIVE.
THE WORK WENT AHEAD AND SET UP A
WORKING FLOW AND IT'S CALLED
BELLY.
AND THE WORK FLOW IS A VERY
SIMILAR TO THE ACTUAL DEL WORK
FLOW SO YOU TAKE YOUR RECEPTORS
AND YOU IMMOBILIZE IT ON
AFFINITY BEADS AND AFTER I AM
MOBILIZATION YOU -- YOU SEPARATE
THE UNBOUND LIGAND AND THEN YOU
HAVE YOUR BOUNDING ON THE BEAT
FRACTION AND YOU USE BOTH OF
THESE FRACTION AND SOME KIND OF
DETECTION METHOD
THAT MEANS IT
DIDN'T BIND TO RECEPTOR.
HOWEVER, IF IT ENDED UP IN
THE BEAD OR FRACTION, THAT
INDICATE BINDING TO YOUR
PROTEIN. SO HERE ON THE TOP
LEFT, THIS IS A GRAPH USING
THE SAME PROTEIN
FLUORESCENCE LIGAND WE USED
BEFORE. WE USE ONE SINGLE
CONCENTRATION OF PROTEIN,
AND WE TITRATED IT WITH THE
TRACER. AFTER THAT WE
MEASURE FLUORESCENCE IN BOTH
THE ELUOTE AND SUPERNATANT
CONDITION. IN THESE
CONDITIONS THE BINDING
SHOULD BE STOIC METRIC AND
THE FLUORESCENT TRACER
NICELY SHOWED UP IN THE
ELUOTE FRACTION AS EXPECTED
AND THAT WAS INFLECTION
POINT OF 500 NANOMOLAR WHICH
IS CONCENTRATION OF PROTEIN
WE PUT INTO THE ASSAY. SO
THIS IS A VERY NICE DATA, WE
WERE HAPPY TO SEE THIS.
HOWEVER IF YOU THINK ABOUT
IT FOR MOST OF YOUR TARGET
FOR MOST OF YOUR PROJECT,
YOU ARE NOT GOING TO HAVE A
FLUORESCENT LIGAND. IT WOULD
TAKE JUST TOO MUCH TIME AND
EFFORT TO SYNTHESIZE
FLUORESCENT LIGAND. TO
ADDRESS THIS, THE GROUP
QUESTIONED WHETHER WE COULD
USE MASS SPEC AS DETECTION
METHOD TOWARD THIS BALI WORK
FLOW. SO WE REPEATED THE
ASSAY USING THE SAME
PROTEIN, BUT THE STANDARD
LIGAND DIDN'T VICE PRESIDENT
LIGAND ON IT, SEPARATED AND
SEPARATED THE COMPOUND USING
MASS SPMASS SPEC. YOU CAN SEE
ACCUMULATION OF COMPOUND
WITH INFLECTION POINT AROUND
750 NANOMOLAR. THE
INFLECTION POINT BETWEEN THE
TWO EXPERIMENT SHIFT AD
LITTLE BIT SIMPLY BECAUSE WE
PUT MORE PROTEIN INTO THE
BALI MS EXPERIMENT. SO THIS
BALI MS BECAME THE CORNER E
STONE OF REGION VALIDATION
AT PFIZER, IT IS VERY QUICK
TO RUN, IF YOUR PROTEIN IS
MAL BEHAVED WE CAN VALIDATE
WITHIN A WEEK OR TWO. SO YOU
VALIDATED YOUR REAGENT, YOU
RAN YOUR SCREEN AND AT THE
END YOU HAVE NO HITS. OR YOU
HAVE HITS WITH POOR PHYSICAL
CHEMICAL PROPERTIES OR YOU
MAKE YOUR HIT OF DNA AND
THEY JUST CONFIRM. SO WHAT
DO YOU DO NEXT? TO ADDRESS
ALL OF THESE QUESTIONS, THE
PFIZER GROUP SET UP A WORK
FLOW, BUT BEFORE I GET INTO
THE DETAILS LET ME SHOW YOU
HOW WIAL VISUALIZE AND
ANALYZE THE DATA SINCE I'M
GOING TO BE SHOWING THESE
PLOT BY PLOT. SO THIS IS A
3D PLOT WE CALL THIS THE
CUBE. AND ON THE ACCESS OF
THE CUBE YOU HAVE YOUR
BUILDING BLOCKS YOUR
MONOMERS. SO THIS MEANS THAT
EVERY SINGLE COMPOUND IN
YOUR LIBRARY, WILL HAVE A
UNIQUE PHYSICAL ADDRESS IN
THE 3D SPAC SPACE. SO THESE
SPHERES YOU SEE HERE ARE
COMPOUNDS. AND THEY ARE
SIZED BY THE READING COUNT
WE GOT OUT OF NEXT
GENERATION SEQUENCING. NOW
WHEN MEDICINAL CHEMIST LOOKS
AT THIS DATA THEY LOOK FOR
FEATURES LIKE LINE FEATURE
OR A PLANE FEATURE. SO FOR
EXAMPLE THIS LINE FEATURE IS
MEDICINAL CHEMIST IS
INTERESTED IN. THESE
FEATURES CAN OCCUR FOR
VARIOUS REASON. IT CAN BE IN
THIS CASE IT CAN BE STR
AROUND THIS PINK BUILDING
BLOCK OR IT ALSO THE
POSSIBLE THE BUILDING BLOCK
THE PINK BUILDING BLOCK IS
NOT IN THE BINDING POCKET SO
IT DOES NOT CONTRIBUTE TO
BINDING. OR THE ASSERTION
IS MAYBE BECAUSE OF FAIR
CHEMISTRY OR TRUNCATIONS AND
I WILL MENTION THAT A LITTLE
BIT LATER AS WELL. SO AS I
SAID AT THE BEGINNING, AT
THE END OF OUR SCREEN IF YOU
WANT TO END UP WITH
COMPOUNDS WITH GOOD PHYSICAL
CHEMICAL PROPERTIES, DRUG
LIKE PROPERTIES SOMETHING
MEDICINAL CHEMIST CAN
CONVERT TO FURTHER DEVELOP
AND DO ASSAY ALL AROUND. NOW
A LOT OF THINGS THAT
ATTRIBUTES TO THESE -- A LOT
OF PROPERTIES THAT
ATTRIBUTES TO THIS,
MOLECULAR WEIGHT, HYDROGEN
COUNTS, AUTOMATIC RINGS C
LOG D, SO ON. SO WE
ESTABLISHED AN MPO SCORING
SYSTEM TO GUIDE OUR HIT
SELECTION DURING ANALYSIS.
THIS SCORING SYSTEM RELIES
ON MOLECULAR WEIGHT M
HYDROGEN BOND, AUTOMATIC
RING COUNTS, AND USE
VISUALIZATION TOOLS TO HELP
THE SELECTION. SO THIS IS
JUST AN EXAMPLE AT THE
BOTTOM. SO THIS LINE
FEATURE, WAS A HIT IN ONE OF
OUR SELECTION. AND YOU CAN
SEE THAT THE BIGGEST
SPHERES, THE ONES THAT HAVE
THE MOST COUNTS, THEY ARE
ACTUALLY COLORED RED AND
ORANGE AND THAT MEANS THAT
THEY HAVE A LOW MPO SCORE,
SO THEY HAVE VERY POOR
PHYSICAL CHEMICAL
PROPERTIES. HOWEVER, ONCE WE
APPLY MPO SCORE BASE
FILTERING WE REMAIN THESE
GREENISH COLOR SPHERES,
THESE ARE COMPOUNDS THAT
HAVE BETTER PHYSICAL
CHEMICAL PROPERTIES
SOMETHING YOU WANTS TO WORK
WITH T AT THE END. NOW, I
SHOWED THIS FILTERING JUST
FOR DEMONSTRATION PURPOSES,
IN REALITY WE DON'T USE THE
MPO SCORING TO FILTER OUT
DATA, WE JUST USE IT AS A
TOOL TO GUIDE HIT
SELECTIONS. OKAY. SO AT THE
BEGINNING WHEN THE GROUP
JUST STARTED A COUPLE OF
YEARS AGO, THE -- OUR
SCREENING PROCESS CONSISTED
OF DOING THE SELECTION,
ANALYZE THE DATA, CALLING
THE HITS, THEN RIGHT AWAY
SYNTHESIZE THE COMPOUNDS OF
DNA AND PUT THEM INTO SOME
KIND OF FUNCTIONALIZED
BINDING ASSAY. WHAT WE SAW
IS THAT AFTER ALL THAT
EFFORT AND RESOURCES THAT WE
PUT INTO MAKING THE
COMPOUNDS OF DNA, MOST OF
THEM DIDN'T CONFIRM. AND
THEY REALLY HAD A -- WE HAD
A LOW CONFIRMATION RATE. SO
NOW HOW IS THIS POSSIBLE
THAT BALI INDICATED THE
COMPOUND IS A HIT AND MAKE
IT OF DNA AND IT DOESN'T
CONFIRM? REMEMBER AT THE
BEGINNING I SAID EVERY DNA
TAG WAS ONE COMPOUND. BUT I
LIED. IN REALITY, WHAT
HAPPENS IS THAT DNA LIGATION
OCCURS WITH HIGH FIDELITY AS
YOU WOULD EXPECT IT.
HOWEVER, THIS IS NOT TRUE
FOR CHEMICAL SYNTHESIS.
ESPECIALLY UNDER CONDITIONS
THAT WE USE, TO PREVENT DNA
DAMAGE. SO FOR EXAMPLE IN
THREE CYCLE CHEMISTRY, IF
THIS IS YOUR TARGET
COMPOUND, IT IS POSSIBLE
SOMETHING GOES BAD DURING
CYCLE 1, AND SAY THE NYTRIL
GROUP IS NOT REDUCED TO
AMINE OR FIRST BLOCK IS NOT
INCORPORATED.
FOR THIS ONE FINAL COMPOUND,
POTENTIALLY YOU COULD GET ALL OF
THESE CYCLE DUCTS YOU CAN SEE ON
THE SCREEN, AND IT FORMED ALL
THE CENTER DUCTS HAVE THE SAME
DNA TAG. SO THE ONE DNA CODE
EQUALS ONE SMALL MOLECULE
STATEMENT IS NOT TRUE. AND THIS
FAILED CHEMISTRY AND TRUNCATED
STRUCTURES NEEDS TO BE TAKEN
ACCOUNT DURING DATA ANALYSIS.
BUT EXPERIENCE MEDICINAL
SCIENTIST CAN ACTUALLY DO THAT.
OKAY. SO NOW WE KNOW THAT WE
HAVE PROBABLY ALL THESE TYPE OF
REACTION MIXTURE, SO WE HAD TO
SET UP A WORK FLOW TO ADDRESS
OUR LOW CONFIRMATION RATE. AND
I'M GOING TO USE THIS COMPOUND
BOND AS AN EXAMPLE. SO FROM THIS
SLIDE FEATURE COMPOUND ONE WAS
CALLED AS A HIT BECAUSE OF GOOD
PHYSICAL CHEMICAL PROPERTIES
THAT IT HAS. HOWEVER, WE MADE
THEM OF DNA AND IT JUST DIDN'T
CONFIRM. SO THE GROUP WENT AND
RESYNTHESIZED THIS COMPOUND ON
THE DNA HEADPIECE,S USING THE
EXACT SAME CHEMICAL RULE THAT
WAS USED DURING THE ORIGINAL
SYNTHESIS. SO AFTER
RESYNTHESIZING THIS, THEY
ANALYZED THE COMPOUND MIXTURE,
AND THIS IS JUST THE HPLC TRACE
FOR THAT MIXTURE. AND
SURPRISINGLY COMPOUND ONE WASN'T
EVEN THERE. INSTEAD, WE HAD TWO
MAJOR CYCLE DUCTS WHERE THE
FIRST BUILDING BLOCK WASN'T
INCORPORATED, OR THE SECOND
BUILDING BLOCK WAS INCORPORATED
TWICE. SO WE ENDED UP WITH THIS
COMPOUND 2 AND COMPOUND 3, WHICH
HAD REVERSE CHEMICAL PHYSICAL
PROPERTY -- WORSE PROPERTIES
THAN COMPOUND ONE. THIS IS
GREAT, WE KNOW WHAT WAS IN OUR
ORIGINAL SELECTION BUT THIS
STILL DOESN'T GIVE THE ANSWER
WHICH COMPOUND BOUND TO PROTEIN.
SO WE WENT AHEAD AND UTILIZED
THIS BALI MS PET PLATFORM FOR
DNA CONFIRMATION AS WELL. SO AS
BEFORE, WE IMMOBILIZE OUR
RECEPTOR UNDER BEADS THEN
INCUBATED WITH THE OWN DNA
COMPOUND MIXTURE. AFTER
REACHING REWILLIE YUM WE
SEPARATE SUPERNATANT FROM BEADS
AND INJECT INTO LCMS. DURING
INJECTION YOU USE INTERNAL
SENDER SO THAT ALLOWS US TO
QUANTIFY TO NORMALIZE OUR DATA
AND ACCESS THE DATA
QUANTITATIVELY. SO BASED ON THE
LCMS DATA, WE SAW A FIVE FOLD
ENRICHMENT FOR COMPOUND 2 AND
SURFACE 3, 4, INRICHMENT FOR
COMPOUND 3 SO IT IS HIGHLY
LIKELY THE ORIGINAL WAS THE SUM
OF BOTH OF THESE CONTRIBUTIONS.
SO NOW, INSTEAD OF GOING
STRAIGHT TO OFF DNA SYNTHESIS,
FIRST WE RESYNTHESIZED OUR DEL
HITS ON DNA TO GET THIS HIT TREE
THEN ANALYZE THAT MIXTURE USING
OUR BALI MS PLATFORM. THIS THOSE
COMPOUNDS -- IF THOSE COMPOUNDS
CONFIRM WE MAKE THEM OF DNA AS
WELL AND PUT THEM INTO A
FUNCTIONAL ASSAY. SO WITH THIS
APPROACH WE GOT HIGHER
CONFIRMATION RATE AND THIS ALSO
ALLOWED US TO FOLLOW-UP ON MORE
CHEMICAL SERIES AND MAKE FEWER
OF DNA COMPOUNDS SO ESSENTIALLY
SAVING THESE SOURCES FOR THE
PROAPOLOGETIC. NOW THAT YOU KNOW
ALL ABOUT DEL, I WANTED TO SHOW
YOU AN EXAMPLE OF SELECTION
PLAN. AS I MENTIONED BEFORE, WE
CAN PENALIZE CONDITIONS DURING
DEL SELECTION. SO THAT MEANS
THAT YOU CAN HAVE YOUR FULL
PROTEIN FOR EXAMPLE IN ONE
CONDITION AND IN OTHER CONDITION
MAYBE YOU HAVE THE PROTEIN IN
COMPLEX OR MAYBE YOU HAVE THE
MUTANT PROTEIN. BUT IT'S ALSO
POSSIBLE YOU WANT TO ADD
SUPPLEMENTS LIKE CHAPERONE OR
CO-FACTOR OR SOME SUBSTRATE AS
WELL TO SEE COMPETITION WITH THE
SUBSTRATE. I MENTIONED BEFORE
THAT WE USUALLY RUN TWO
CONCENTRATIONS, HIGHER AND LOWER
CONCENTRATION, TO GET THE IDEA
OF THE RELATIVE AFFINITY OF THE
COMPOUNDS. I DIDN'T MENTION
REALLY THIS, BUT WE TRY TO ALSO
INCLUDE A POSITIVE CONTROL IN
OUR ASSAY. SO IF THE PROTEIN OF
INTEREST HAS SOME KIND OF
CHEMICAL MATTER, THAT BINDS TO
THE PROTEIN WITH RELATIVELY HIGH
AFFINITY, WE CAN MAKE THAT
COMPOUND ON THE DNA SPIKE IT
INTO THE DNA DEL POOL, AND THEN
USE IT DURING SELECTION. SO
THAT'S HIGH COMPOUND WILL SERVE
AS POSITIVE CONTROL, AND WE CAN
MONITOR IT DURING SELECTION AND
SEE IF IT WAS ENRICHED. SO FOR
EXAMPLE, IF AT THE END OF THE
DAY YOU DON'T GET ANY HIT OUT OF
YOUR DEL SELECTION BUT YOUR
POSITIVE CONTROL WAS ENRICHED,
YOU CAN BE SURE THAT THE DEL
SELECTION WENT WELL JUST
SOMETHING ELSE HAPPENING THAT
DIDN'T GIVE YOU ANY HITS. THIS
SMALL TABLE IS TO GIVE YOU AN
IDEA OF THE SELECTION PLAN, SO
FOR EXAMPLE IN SAMPLE ONE AND
TWO, WE HAVE HIGH PROTEIN
CONCENTRATION, FOR SAMPLE TWO WE
ADDED A NON-BINDER AS WELL TO
IDENTIFY DEL COMPOUNDS THAT
COULD COMPETE WITH THIS
NON-BINDER. SO ESSENTIALLY BIND
TO THE SAME BINDING POCKET. AND
THEN IN FOUR SAMPLES 3 AND 4 WE
HAVE THE LOW PROTEIN
CONCENTRATION AND AGAIN FOR 4 WE
ADDED A NON-BINDER. FOR SAMPLE 5
THAT'S BASICALLY YOUR NEGATIVE
CONTROL, YOU DON'T ADD ANY
PROTEIN AND YOU JUST WORK WITH
THE BEADS TO IDENTIFY BINDERS
THAT ARE BEING CAPTURED BY
MAGNETIC BEADS. SO I THINK I
HAVE SOME TIME LEFT TO MENTION
CELL BASED SCREENING AND THUS
SCREENING FOR MEMBRANE PROTEINS
SO WHY ARE WE INTERESTED IN
MEMBRANE PROTEIN?
BECAUSE THEY PERFORM A MYRIAD OF
BIOLOGICAL FUNCTIONS AND THEY
ARE IMPORTANT DRUG TARGETS. SO
THIS GRAPH IS SHOWS THE FD
APPROVED SMALL MOLECULAR DRUGS
TARGETING MAJOR PROTEIN
FAMILIES. IT IS FROM 2017 SO
NUMBERS MIGHT BE A LITTLE BIT
OFF BUT YOU CAN SO E THAT MORE
THAN 50% OF THE DRUGS ARE
TARGETING MEMBRANE PROTEINS.
UNFORTUNATELY, RUNNING THE DEL
SCREEN FOR MEMBRANE PROTEINS IS
VERY CHALLENGING. SO WHY IS
THAT? YOU BASICALLY HAVE -- YOU
HAVE A COUPLE OF CHOICES OF HOW
YOU RUN THIS SELECTION. IF YOU
ARE LUCKY AND YOU CAN FORTIFY
YOUR PROTEIN AND STABLE ENOUGH
YOU CAN TRY TO DO THAT. HOWEVER,
UNFORTUNATELY MOST OF THE TIME
IT IS NOT GOING TO HAPPEN. YOU
WILL HAVE PA HARD TIME
FORTIFYING THE PROTEIN, KEEPING
IT IN SOLUTION AND FOLDED. SO
ANOTHER OPTION WOULD BE RUNNING
IT ON USING CELLS. WELL YOU CAN
MENTION THAT IF YOU WANT TO USE
CELL FOR DEL SELECTION YOUR
BACKGROUND WOULD BE MUCH HIGHER
DUE TO NON-SPECIFIC BINDING BUT
THIS IS SOMETHING YOU CAN
ADDRESS WITH THE RIGHT NEGATIVE
CONTROL, FOR EXAMPLE USING THE
PARENTAL CELL LINE. HOWEVER, THE
REAL CHALLENGE HERE COMES FROM
THE FACT THAT AS I SAID, BALI --
BINDING IN DEL IS DRIVEN BY THE
PROTEIN CONCENTRATION. WHICH
MEANS THAT CELL BASED SELECTION
WE WOULD NEED CELLS THAT
EXPRESSES THE RECEPTOR OF
INTEREST IN VERY HIGH
CONCENTRATCONCENTRATION. SO THEA
COUPLE OF PAPERS OUT THERE I
WANTED TO MENTION QUICKLY. FOR
EXAMPLE, DUKE UNIVERSITY IN
COLLABORATION WITH LEXICON, THEY
WORKED WITH THE BETA ADRENERGIC
RECEPTOR, THEY FORTIFY AND ON
MAGNETE SURFACE AND KEPT THE
RECEPTOR IN A SOLUTION. ASTRA
ZENECA USED A SIMILAR METHOD FOR
THEIR COMPANY BUT IN THIS CASE
THEY INTRODUCED POINT MUTATIONS
TO STABILIZE THE PROTEIN IN AN
ANTIBODY STABLE CONFIRMATION.
AND AGAIN, THEY USE (INAUDIBLE)
TO KEEP PROTEIN IN SOLUTION. AS
I SAID BEFORE THIS IS A POSSIBLE
ROUTE BUT IT IS VERY LIKELY THAT
IT IS LIKELY YOU WON'T BE ABLE
TO EXPRESS AND FORTIFY YOUR
PROTEIN AND ALSO ALL OF THESE
DATA CHARTS THAT THE DIFFERENT
GROUPS USE, THEIR COMPATIBILITY
WAS NEVER ACTUALLY STUDIED WITH
DEL SO WE HAVE NO INFORMATION
ABOUT THEM. THERE ARE A COUPLE
OF PUBLICATIONS THAT USE SLIDE
CELLS AS WELL. SO FOR EXAMPLE
HERE, THE GROUP EXPRESSED FOLATE
RECEPTOR IN CELLS AND FOLATE HAD
A DNA TAG ATTACHED TO IT. SO
THIS DNA TAG ESSENTIALLY
HYBRIDIZES WITH THE DEL TAG
ITSELF AND ESSENTIALLY GUIDING
THE COMPOUNDS TO THE RECEPTOR
AND INITIATING THE BINDING. SO
IN THIS CASE THEY DIDN'T NEED TO
EXPRESS THE PROTEIN AT HIGH
CONCENTRATION. AND THERE WAS A
NICE ELEGANT WORK FROM GSK
WORKING WITH NK 3 RECEPTOR,
WHERE THEY TRANSDUCED HEP 293
CELL WITHBACTER VIRUS AND THEY
WERE ABLE TO GET 500 RECEPTORS
PER CELL, VERY HIGH
CONCENTRATION AND THEN RAN THE
DEL SELECTION ON THESE SLIVE
CELLS. THE GSK PAPER SUGGESTS TO
HAVE AT LEAST 100,000 COPIES PER
CELL, BUT THE MORE YOU HAVE THE
BETTER CHANCE YOU ARE GOING TO
GET FOR DEL SELECTION. AND THIS
METHOD IS ACTUALLY SOMETHING
THAT NOW WE ARE EXPLORING AT
PFIZER AS WELL AND TRYING TO
LEARN MORE ABOUT IT. NOW, JUST A
QUICK SUMMARY. DEL SCREENS ARE
RELATIVELY LOW COST ONCE YOU
HAVE ESTABLISH YOUR LIBRARY AND
YOUR PLATFORM AND CAN BE
COMPLETED IN JUST A COUPLE OF
MONTHS AS WELL. IT IS USUALLY
RESULTS IN MINIMAL OFF DNA
SYNTHESIS SO TEN COMPOUNDS OR
SO. MAKE SURE THAT YOU VALIDATE
YOUR REAGENTS BEFORE DEL
SCREENING, THAT SHOULD BE THE
CORNERSTONE OF YOUR WORK FLOW.
AND USING MPO SCORING AND ON DNA
RESYNTHESIS YOU CAN INCREASE
CONFIRMATION THE HIT
CONFIRMATION RATE AND ENSURE YOU
HAVE A GOOD DRUG LIKE PROPERTY
AT THE END OF IT. NOW, DEL IS
REALLY A TEAM WORK AND I WANT TO
ACKNOWLEDGE EVERYBODY AT PFIZER
AND ALSO COLLABORATORS AT HIT
GEN. AS I SAID DURING THE DEL
SELECTION BIOLOGICAL CHEMISTS
HAVE TO WORK TOGETHER TO ENSURE
SUCCESSFUL COMPANY. THANK YOU
FOR YOUR ATTENTION AND HAPPY TO
ANSWER ANY QUESTIONS.
[APPLAUSE]
>>THANK YOU SO MUCH SOFIA FOR A
WONDERFUL TALK. QUESTIONS FOR
SOPHIA?
>>DO YOU MIND APPROACHING THE
MIC OVER THERE?
(OFF MIC)
>>THAT WAS REALLY EXCELLENT
TALK AND EXPLAINED A LOT ABOUT
DEL LIBRARIES WHICH ARE REALLY
INTERESTING. ONE THING THAT I
HAVE NEVER QUITE UNDERSTOOD IS
HOW AFTER AMPLIFICATION AFTER
THE FIRST ROUND YOU RESYNTHESIZE
YOUR COMPOUND LIBRARY ALONG WITH
THE PCR PRODUCT. CAN YOU DISCUSS
THAT?
>>I'M NOT SURE I UNDERSTAND
YOUR QUESTION. SO AFTER YOU DID
YOUR ROUND OF DEL SELECTIONS?
>>YES.
>>OKAY. SO AFTER THAT YOU
BASICALLY AMPLIFY YOUR DNA TAGS
DURING PCR. AND THEN YOU HAVE TO
TAKE IT INTO NEXT GENERATION
SEQUENCING. SO THERE WERE A
COUPLE OF STEPS INVOLVED HERE SO
AFTER PCR AMPLIFICATION YOU HAVE
TO ADD SOME KIND OF ADAPTER THAT
WILL ALLOW YOUR DNA TAG TO
ATTACH TO THE SEQUENCING CHIP AS
WELL. BUT AT THAT -- AT THIS
POINT YOU ARE LOOKING AT THIS
LIKE DNA SEQUENCE. YOU KIND OF
-- YOU FORGET ABOUT THIS BEING
COMPOUNDS, YOU ARE GOING TO JUST
AMPLIFY YOUR SEQUENCE USING PCR
AND WORK WITH THAT LATER ON.
>>THEN SO THE SECOND ROUND OF
SELECTION, YOU REPEAT THE 96
WELL PLATE COMBINATORIAL
CHEMISTRY?
>>NO. SO IN THE SECOND -- AFTER
THE FIRST ROUND OF SELECTION,
YOU ADD TO YOUR COMPOUNDS. YOU
HAVE SOME YOU ADD THEN YOU TAKE
THE EXACT SAME COMPOUNDS AND
TAKE INTO YOUR SECOND ROUND OF
SELECTION AS AN INPUT.
>>OKAY. THANKS.
>>YEP.
>>THANK YOU FOR YOUR TALK. SO I
WANT TO KNOW MORE ABOUT BUILD
SCREENING FOR THE DNA BINDING
PROTEINS. SO SOME OF THE
PROTEINS COULD HAVE DNA BINDING
POCKET OR WHATEVER YOU CALL, SO
DO YOU THINK THE CHANCE OF
GETTING MORE OFF TARGET OR I
WANT TO KNOW MORE ABOUT THAT.
THANK YOU.
>>THAT IS ACTUALLY REALLY GOOD
QUESTION. WE HAD A COUPLE OF
TARGETS WHICH WERE EITHER DNA OR
RNA BINDING PROTEIN. IN THAT
CASE WE WERE LUCKY, SO WE DID
SEE BINDING TO DNA TAG BUT THAT
BINDING WAS NON-SPECIFIC. SO
THAT MEANS IT WASN'T ENRICHED
DURING SELECTION AND IT IS JUST
RESULTING IN HIGH BACKGROUND
WHICH WE COULD GET RID OF DURING
THE DATA ANALYSIS. HOWEVER, FOR
EXAMPLE, HITGEN OUR
COLLABORATOR, THEY HAVEN'T
ESTABLISHED WORK FLOW, WELL THEY
ACTUALLY ADD -- SO IN TERMS OF
RNA BINDING PROTEINS YOUR
PROBLEM IS THAT THE DNA TAG HAS
A SINGLE CHAIN REGION AS WELL.
AND HITGEN CAME UP WITH AN
APPROACH WHERE THEY HYDROLYZE
THE SINGLE CHAIN INTO SMALL RNA
PIECES AND THEN RUN THE DEL
SELECTION ON THAT, SO THEY WERE
ABLE TO REDUCE THE BACKGROUND.
BUT AS I SAID IN OUR CASE, WE
NEVER -- WE DIDN'T HAVE PROBLEM
BECAUSE BINDING WAS ALWAYS
NON-SPECIFIC SO YES IT INCREASED
OUR BACKGROUND BUT WE COULD GET
RID OF THAT DURING DATA
ANALYSIS.
>>ACTUALLY HAVE TWO QUESTIONS.
SO THE FIRST ONE, SO (INAU --
>>GOT YOU. SORRY ABOUT THAT. I
HAVE TWO QUESTIONS. FIRST WHEN
YOU POOL YOUR LIBRARIES TOGETHER
LIKE YOU WORRY ABOUT THE SIZE
DIFFERENCE OF THE LIBRARIES? SAY
LIKE YOU POOL A LIBRARY THAT HAS
BEADS OF MOLECULES AND YOU PUT
IT TOGETHER WITH A LIBRARY THAT
ONLY HAS MILLIONS OF MOLECULE,
DO YOU WORRY THAT THE LARGE
LIBRARY WILL BE DILUTED IN THIS
WAY?
>>THAT IS A GOOD POINT. AND
THEN WHEN YOU POOL THEM TOGETHER
FOR SELECTION, WE PULL IT IN A
WAY THAT AT THE END EVERY SINGLE
COMPOUND WE HAVE COPY, SO THAT
IS NOT SOMETHING YOU HAVE TO
WORRY ABOUT BECAUSE THAT IS A
GOOD POINT.
>>MY SECOND QUESTION IS SO FOR
THE CELL BASED DEL ASSAY, SO CAN
YOU ADD SOME COMMENTS ON LIKE
POSSIBLE NO TARGET CONTROL LIKE
YOU JUST USE THE SAME CELL LINE
THAT DOESN'T EXPRESS THE PROTEIN
OR YOU HAVE LIKE OTHER -- LIKE
NO TARGET CONTROLS?
>>YEAH, SO AS I SAID WE ARE
JUST GETTING TO THIS SPACE AND
WE ARE LEARNING ABOUT IT. BUT
RIGHT NOW OUR STRATEGY IS TO USE
THE PARENTAL CELL LINE OR THE
CELL LINE THAT WAS TRANSDUCED
NEGATIVE CONTROL VIRUS OR
SOMETHING LIKE THAT.
>>GOT YOU. THANKS.
>>SO GPCR PROJECTS THAT
INTERESTING DO THEY TALK ABOUT
WHETHER THEY HAVE TO CALL
AMPLIFY OR CO-TRANSFECT IN THE G
ALPHA COMPONENT WITH THE ACTUAL
RECEPTOR ITSELF?
>>YES, SO IN THESE PAPERS, THEY
DIDN'T DO THAT. BUT THAT IS
ACTUALLY AN OPTION AS WELL THAT
WE ARE EXPLORING. SO THAT IS A
VERY GOOD POINT. YES.
>>ALL RIGHT. THANK YOU VERY
MUCH, SOPHIA, AGAIN. SO COUPLE
OF QUICK ANNOUNCEMENTS BEFORE WE
GO TO LUNCH, ONE THING I WANT TO
REMIND YOU ALL THAT WE ACTUALLY
HAVE TOPIC FOCUSED WORKSHOP ON
DEL TWO DAYS OF VIDEOS YOU CAN
WATCH ONLINE, ON DEMAND NIH
VIDEOCAST WEBSITE, WE'LL SHARE
ALL THESE IF THAT IS SOMETHING
OF INTEREST AND YOU WANT TO DIVE
MORE INTO DNA LIBRARIES. SO WE
ARE -- WE ARE GOING TO TAKE AN
HOUR BREAK NOW. WE WILL
RECONVENE AT 1:30. WE HAVE
EXCELLENT TALKS THIS AFTERNOON.
SO COME BACK, WE WILL HAVE TALKS
ON HTS DATA ANALYSIS. DRUG
SCREENING USING 3D TISSUE
MODELS. COUPLE OF MORE EXCITING
TALKS AWAITING YOU THIS
AFTERNOON. WE WILL SEE YOU AT
1:30. THANK YOU.
WELCOME BACK,
EVERYONE. OUR NEXT SPEAKER IS
DR. THOMAS CHUNG. DR. CHUNG IS
THE DIRECTOR OF TRANSLATIONAL
PROGRAMS OUTREACH AT THE PREBYS
CENTER FOR CHEMICAL GENOMICS AT
SANFORD PREBYS MEDICAL DISCOVERY
INSTITUTE SVP IN LA JOLLA,
CALIFORNIA. DR. CHUNG IS A LONG
STANDING EDITORIAL BOARD MEMBER
OF THE AGM AND HAS MULTIPLE
RECOGNIZED CHAPTERS IN THE AGM
SO WE ARE EXCITED TO HAVE HIM
HERE TODAY. HIS TALK IS
ENTITLED BASIC ASSAYS
STATISTICS, DATA ANALYSIS AND
RULES OF THUMB. TC, THE FLOOR IS
YOURS.
[APPLAUSE]
>>I HAVE HAD A LONG HISTORY
WITH THE SOAS AND THE AGM SO IT
IS A PLEASURE. TODAY I'M TRYING
TO TALK BASIC FOUNDATIONS,
ADEQUATELY COVERED DEVELOPMENT
AND VARIOUS THINGS. WHAT I'M
HERE TO GIVE YOU IS RULES OF
THUMB, WHAT I'M PRESENTING IS
REALLY A DILUTED STATISTICS
CLASS, IT IS REALLY A REAL
STATISTICIAN PROBABLY CRUCIFY
ME. REALLY I WANT TO HELP
EVERYONE THINK HOW WHAT YOU MEAN
BY ASSAY ROBUSTNESS. AND WHAT
VALIDATION MEANS IN TERMS OF
ROBUSTNESS OF ASSAY. SO YOU HAVE
HEARD SEVERAL TIMES THERE'S
ASSAYS AND THE ASSAYS ARE
EMPLOYED IN HTS HEALTHCARE YOU
HAVE ONE CHANCE TO TEST THAT
COMPOUND SO YOU WANT TO BE AT
LEAST SURE THAT THE ASSAY ITSELF
WASN'T -- WAS BEEHIVING W BEHAVO
THEY HAVE TO BE RIGOROUSLY
VALIDATED THESE ASSAYS FOR
VIABILITY AS WELL AS
PHARMACOLOGICAL EVENTS NOT JUST
IN THE DISEASE STATE BUT
ACTUALLY IN TERMS OF THE
PHENOMENOLOGICAL AFFECT WHEN
COMPOUND RICH REACHES THE TARGEU
HEARD FROM NATHAN AND TIM ABOUT
VERIFYING REAGENTS STABILITY FOR
STORAGE AS WELL AS DURING THE
ASSAY. AND THINGS LIKE ROBUST
FREEZE THAWING AND -- TRY THIS.
THAT'S GOOD. FREEZE THAWING AND
LONG TERM TEMPERATURES. WITH
THESE ASSAYS WHEN YOU START
DOING BIG CAMPAIGNS, YOU ARE
TRYING TO DO IT IN RAPID
SUCCESSION AND YOU HAVE TO WORK
WITH -- YOU HAVE TO HAVE THE
STOCK SOLUTION MADE TEMPORARY
WORKING STOCKS AND U YOU WANT TO
MAKE SURE THEY ARE USEFUL AND
STABLE FOR THAT. WHAT DO YOU DO
WITH THE LEFT OVERS? SOMETIMES
YOU JUST THROW THEM AWAY BUT
OCCASIONALLY YOU CAN REFREEZE
THEM AND FIND THAT YOU CAN USE
IT FOR FOLLOW UP STUDIES. FOR
ASSAY ROBUSTNESS AND PERFORMANCE
OTHER THINGS TO DO IS YOU WANT
TO IDENTIFY AND ELIMINATE THE
SOURCES OF SYSTEMIC ERRORS SO
YOU HAVE HEARD ABOUT CHECKING
FOR DMS COMPATIBILITY. THE OTHER
IS TO CHECK PHYSICAL UNIFORMITY,
NOW DAYS THE VENDORS ARE GOOD
ABOUT THOSE. IN THE OLD DAYS
PLATES WARPED DEPENDING THE
VENDOR YOU WENT TO. BUT THERE IS
OTHER EFFECTS LIKE IF YOU THINGS
EDGES OF PLACE TEND TO BE HEATED
SOONER THAN THE CENTERS OF THE
PLATE BECAUSE PLASTIC IS VERY
PORE CONDUCTOR OF HEAT.
DEPENDING UPON YOUR LIQUID
HANDLING SYSTEMS YOU CAN SEE
SYSTEMIC CLOG TIPS WILL LEAF A
STRIPING. I WILL SHOW YOU
EXAMPLES LATER. YOU WANT TO
CHECK FOR INNER PLATE AND
INTERDAY VARIATIONS, TRYING TO
MINIMIZE THAT. THERE'S WAYS OF
USING DIFFERENT TYPES OF
STATISTICS FOR THAT. THE OTHER
THING IS YOU WANT TO REALLY
CHECK THAT YOU ARE ON THE LINEAR
RANGE IF ENZYMATIC OR BINDING
ASSAY YOU ARE LINEAR PART OF THE
RESPONSE CURVE WHERE IT IS THE
MOST SENSITIVE RATHER THAN
PLATEAUING WHERE RECENTLY
ASIMILAR TAUPE. SO YOU ARE
TRYING TO DEAL WITH RANDOM
ERRORS AN ELIMINATING THE
SOURCES THINGS YOU CAN CORRECT
FOR. THE REASON FOR THAT IS
RANDOM ERRORS YOU CAN'T DO
ANYTHING ABOUT BUT YOU CAN TREAT
THEM STATISTICALLY. SO TYPICAL
ASSAY IS YO YOU HAVE A PRIMARY
ASSAY THAT MAPS BASICALLY LARGE
NUMBERS OF COMPOUNDS TO
BIOLOGICAL ASSAY, YOU HAVE TO
DEFINE HITS, THRESHOLD. AND
FOLLOWED BY HITS YOU WILL
SECONDARY ASSAYS THESE
ORTHOGONAL ASSAYS, THE HIT
TRIAGE THAT WAS DISCUSSED FROM
SEVERAL SPEAKERS. THIS IS A VERY
TYPICAL FORMAT IF YOU ARE 96
FORMAT YOU HAVE TYPICALLY MOST
PEOPLE USE A FIRST TWO COLUMNS
AS EMPTY OR FIRST AND LAST
COLUMN IS EMPTY. AND THESE ARE
WHERE YOUR CONTROLS ARE PUT, AND
WHEN YOUR CONTROLS ARE PUT, THEY
GIVE YOU THE HIGH SIGNAL AND THE
LOW SIGNAL. AND IN THE PAST
VARIOUS ASSAY PARAMETERS HAVE
BEEN CALCULATED FOR THESE SUCH
AS NORMALIZE INHIBITION, THAT'S
WHAT WE DO, WE ACTUALLY
NORMALIZE IT TO HIGH AND LOW,
TAKE IT AS PERCENTAGE OF THAT,
PUT IT ON THE SAME SCALE TO
COMPARE THINGS. WE USE A Z SCORE
WHICH IS FORMALLY UNITS OF
STANDARD DEVIATION AWAY FROM THE
MEAN, THAT'S SOMETHING IS, AND
YOU USE THAT IN TERMS -- YOU
OFTEN USE THAT WHEN YOU DON'T
HAVE A HIGH AND LOW CONTROL
ABILITY CONTROL AND MORE
COMPLICATED THINGS CAN BE SCORE.
SO THESE ARIST H I CAN ARE.
THESE EXIST BECAUSE BUT MOST
DONE TALK ABOUT THE B SCORE. YOU
DO NPIs AND PEOPLE OFTEN TALK
Z SCORE. THE REASON WHY THE B
SCORES, IT IS HARD CONCEPT AND
TAKES A LOT OF MATHEMATICAL
CALCULATIONS. SO PEOPLE LOOK FOR
SIMPLER THINGS. SO LET ME JUST
GET A LITTLE BIT OF A PRIMER ON
STATISTICS. SO THERE'S
STATISTICS ARE JUST COMPARISONS
AND THERE'S LOT OF USE AND
MISUSE OF THAT. AND I'M NOT SURE
IF YOU STUDY AMERICAN LITERATURE
BUT MARC TWAIN HAS BEEN
DESCRIBED TO SAY THERE'S THREE
KINDS OF LIES. THERE'S LIES,
DATA LIES AND STATISTICS. IF YOU
LOOK FOR THAT, THERE'S ENGLISH
PERSON THAT FIRST TALKED ABOUT
THAT. SO YOU NEED TO HAVE RULES
OF THUMB ABOUT STATISTICS AND
SIMPLE MEASURES FOR THE ASSAY
ROBUSTNESS. FIRST IN TERMS OF
WHAT IS THE STATISTICS? HERE IS
THE FORMULA DEFINITION FROM THIS
BOOK THAT I ALWAYS CITE ON THE
BOTTOM OF ALL MY SLIDES. IT IS A
COLLECTION OF METHODS FROM
PLANNING YOUR EXPERIMENTS TO
OBTAIN AND ORGANIZING YOUR DATA
SO SUMMARIZE RESULTS SO THAT YOU
CAN SAY YOU CAN DRAW CONCLUSIONS
THAT'S WHAT YOU ARE MEASURING IS
DIFFERENT HAHN THE NOISE WITH
THE BACKGROUND OR UNPERTURBED
SYSTEMS. YOU BASICALLY MAKE A
BUNCH OF MEASUREMENTS OR MAKE
BUNCH OF DETERMINATIONS AND EACH
OF THOSE MEASUREMENTS COMPRISE
SOMETHING CALLED POPULATION
ENSEMBLE OF MEASUREMENTS SO
STATISTICS HERE WOULD BE THERE
IS ABOUT A HUNDRED OF US HERE,
WE EACH HAVE A HEIGHT AND IF YOU
POP ON THE TABLE THAT'S YOUR
POPULATION METRIC. CENSUS IS
WHEN YOU HAVE A REALLY LARGE
POPULATION YOU CAN'T
INDIVIDUALLY TEST EVERYONE BUT
YOU DO SAMPLE GROUPS AND BLEND
THEM TOGETHER AND YOU CAN THEN
ASSUME THAT REPRESENTS THE WHOLE
POPULATION. THERE'S WAYS OF
TESTING FOR THAT. (INAUDIBLE) IS
A SUB COLLECTION OF ELEMENTS
FROM THAT POPULATION. AND THE
PARAMETER IS THE ACTUAL
NUMERICAL MEASUREMENT YOU MAKE
TO CHARACTERIZE THAT POPULATION.
A STATISTIC IS NUMERICAL
MEASUREMENT THAT DESCRIBES
CHARACTERISTIC OF THE SAMPLE.
THERE'S TWO KINDS OF DATA, WE
MOSTLY DEAL WITH QUANTITATIVE
DATA FOR INHIBITION OF CERTAIN
VALUE. BUT STATISTICS ON
CATEGORICAL DATA, A DIFFERENT
COLOR OR SOMETHING LARGER OR
LESS THAN SOMETHING ELSE. HERE
ARE EXAMPLES OF MISUSE OF
STATISTICS THE USE OF STATISTICS
IS TO TALK ABOUT QUALITY
CONTROL. IF SOMETHING HAS TIGHT
QUALITY IT HAS VERY LITTLE
VARIANCE, DOESN'T CHANGE DAY TO
DAY. YOU WANT TO BE ABLE TO GIVE
A NUMBER THAT ESTIMATES THAT
STATISTIC THEY HAVE SO SAY HOW
MANY PEOPLE IT AFFECTS WHAT IS
MY ASSURETY OF THAT THING. SO
POPULATION YOU SAY SOMETHING YOU
HAVE TO COUNT THEM AND CHECK ON
TRENDS. BASE STATISTICS WHEN
THEY SET A SPEED LIMIT IT IS NOT
JUST COMPLETELY ARBITRARY THEY
SAY HOW MANY ACCIDENTS AT THIS
SPEED THEY THINK IT IS NOT SAFE
THEY REDUCE IT SO THIS STATISTIC
THERE IS NUMBER OF ACCIDENTS PER
NUMBER OF MILES DRIVEN. SO YOU
HAVE TO ANALYZE. ESPECIALLY IN
OUR INDUSTRY WE HAVE TO BE ABLE
TO RECOGNIZE AND WHETHER STORY
STATISTICS. SO THERE ARE LOTS
OF ABUSES. SOME ABUSE IS LIKE
YOU HAVE SMALL OR BAD SAMPLE AND
IT IS KIND OF LINE, YOU CAN SAY
SEVEN OUT OF TEN DENTISTS.
SOUND LIKE 70% BUT THEY ONLY
ENTER TEN DENTISTS SO THAT IS A
SMALL STATISTIC OR EITHER YET
YOU FIND THEY WERE RESPOND CORED
BY DRUG COMPANIES SO THEIR PAID
SO THAT IS A BIAS STATISTIC. THE
OTHER ONE THAT IS COMMON, THIS
IS REFERS TO SIGNIFICANT FIGURES
AS WELL. THERE IS PRECISE
NUMBERS APPARENTLY PRECISE WHICH
GIVES YOU A FALSE SENSE OF
ACCURACY. LIKE $37,700. IS THAT
REALLY, IS IT REALLY BETTER TO
GIVE THEM ALL THESE ADDITIONAL
VIEW -- UNLESS YOU ARE COUNTING
THAT'S NOT IMPORTANT BECAUSE YOU
TRY TO SAY SOMETHING MORE
IMPORTANT. I HAVE BEEN IN
MEETINGS WITH VERY SERIOUS
PEOPLE WILL ARGUE WHAT IS LIKE
IC 50 OF 3.25 VERSUS 3.21. I ASK
HOW MANY SIGNIFICANT FIGURES
THEY RUN THAT COMPOUND OUT TO,
ATELL ME TWO MILLIGRAMS PLUS OR
MINUS ONE MILLIGRAM. SO THERE IS
ONLY ONE SIGNIFICANT FIGURE IN
THAT ENTIRE THING. SO ALWAYS ASK
THAT. THE ONLY TIME I USE
BALANCES YOU CAN MEASURE THINGS
TO THREE SIGNIFICANT FIGURES,
ACTUALLY FOUR IN TERMS OF
HUNDREDS OF MILLIGRAM. BUT IF
YOU PUT A FINGER PRINT YOU
CHANGE THE WAY SO JUST HAVE TO
DEAL WIT. THE OTHER ONE THAT'S
POPULAR IN JOURNALS IS SOMETHING
LIKE ON THE RIGHT. THIS IS THE
SAME DATA BUT PRESENTED THIS. SO
IF YOU ARE NOT CAREFUL YOU THINK
THAT IS A BIG CHANGE. IT IS A
SMALL PERCENT DIFFERENCE. THIS
IS THE THING TO WORRY ABOUT AND
IF YOU READ JOURNAL ARTICLES YOU
WILL SEE THIS. FOR NOW --
ANALYSIS OF HDS DATA WE DO HAVE
TO PREPROCESS DATA, THAT IS
NORMALIZATION. BUT BEFORE
NORMALIZATION LOOK AT THE RAW
DATA. TO SEE IF THERE IS SOME
BIAS TO THAT. WITH THE KINDS OF
CORRECTIONS THE REASON TO DONOR
MALLIZATION IS TO CHECK PLATE BY
PLATED BUT SOME OF THE STANDARD
CONTROLS IN TERMS OF CORRECTING
FOR SYSTEMATIC ERRORS ARE
EXPERIMENTAL ERRORS TO SEE IF
THERE'S OUTLIERS IN THE CONTROL,
WHETHER THERE'S POSITIONAL
EFFECTS THE WAY YOU CHECK
POSITIONAL AFFECTS IF YOU TAKE
THE PLATE AND READ IT AND FLIP
360 DEGREES AND SEE IF LOW WELLS
TRANSPOSE WITH THE GEOMETRY
VERSUS THE ACTUAL PLATE ITSELF.
THEN YOU WANT TO LOOK AT CONTROL
WELLS AND OVERALL PLATE AND SEE
IF IT DRIFTS. ALL THIS WILL
EFFECT HOW YOU SELECT. RIGHT?
YOU WANT TO GET RID OF THOSE TO
ANALYSIS DISTRIBUTION. SO THE
VARIABILITY REPLICATE CONTROLS
IS USED TO ESTIMATE THE
VARIABILITY. I WANT TO
EMPHASIZE, WHEN YOU ARE STARTING
ASSAY YOU CAN TAKE WHOLE PLATE,
HIGH AND LOW CONTROL AND YOU CAN
CALCULATE THIS VARIABILITY FROM
THAT. WHEN YOU SCREEN YOU ONLY
HAVE CERTAIN WELL THAT ARE
CONTROLS AND YOU SCREEN ACTUAL
COMPOUND. BUT FOR MOST ASSAYS
AND TYPES OF WAY WE SCREEN, IT
TURNS OUT THAT VARIABILITY OF
REPLICATE WITHOUT COMPOUNDS YOU
ARE TESTING, IS OFTEN THE
OVERALL VARIABILITY, OF THOSE
CONTROLS, DO REFLECT THE
VARIABILITY OF YOUR SCREEN. WAS
BECAUSE MOST ARE INACTIVE. 99
ARE HITS AND THE REST ARE
INACTIVE COMPOUNDS. THAT'S WHAT
THE LAST LINE MEANS SO YOU CAN
LOOK AT RANDOM ERRORS VARIABLE
AGAINST ALL COMPOUNDS IN A PLATE
BECAUSE MOST OF THE COMPOUNDS
AREN'T ACTIVE AND INACTIVE
COMPOUNDS JUST REFLECT BY RANDOM
ERROR SO THAT SHOULD BE
DISTRIBUTED OR GALSION. SO THIS
IS AN EXAMPLE OF 1530 WELL PLATE
I HIGHLIGHT BASICALLY IF YOU
LOOK AT MOST 1536 WELL PLACE,
LIKE IN OUR LAB WE TEND TO PUT
MORE HIGHER CONTROLS THAN LOWER
CONTROLS BECAUSE THERE IS MORE
VARIANTS IN THE HIGH CONTROLS,
HIGH SIGNAL CONTROLS. SO WE DO
THAT. IN OUR CASE, MANY OF OUR
DISPENSERS THEY WORK IN A
CERTAIN FASHION SO YOU HAVE A
CLOGGED TIP YOU CAN SEE SPREAD
QUICKLY. SO THESE ARE OBVIOUS
THINGS YOU CAN ELIMINATE THOSE
DATA FROM ANALYSIS OR TRY TO
CORRECT THEM. WERE AVERAGE
CORRECTION ALGORITHM. ONE OF THE
REASON I ACTUALLY SHOW THIS
PARTICULAR EXAMPLE IS ONE WAY TO
PLOT TO MAKE SURE ASSAY IS
WORKING WELL IS LOOK AT
DISTRIBUTION OF CURRENT
DEFLECTION, IF FLAT LIKE THIS,
PROBABLY A GOOD PLATE TESTIMONY
OTHER THING, IF THERE'S AN AGE
EFFECT ESPECIALLY 1536, THESE
PLACE TEND TO HEAT UP FASTER.
MOST CONSEQUENCE IS ON CELL
BASED ASSAYS WHERE THEY DRY OUT
FASTER. SO ONE PRACTICAL ASPECT
WHEN SCREENERS ARE APPROACHED BY
THEY WANT A NINE DAY ASSAY YOU
HAVE TO TELL THEM PRACTICALLY
YOU CAN'T DO MORE THAN A 5A
ASSAY WITHOUT HEROIC MEASURES.
THAT'S LIMITATION. AND THE
LIMITATION IS ADDRESSED PROBABLY
BY GOING TO 3D FOUR WELL PLACE.
384 DOES WORK IN MANY FORMATS.
THIS TABLE I PUT TOGETHER MAKES
A POINT THAT NATHAN MIGHT HAVE
SHOWED TO BUT 96 WELLS AND 36
WELLS, THE BIG SURPRISE THING IS
MOST 1536 WELLS TEND TO BE LONG
TALL COLUMNS AND 384 -- 96 WELLS
TEND PLAID FLAT WIDE DISCS OR
BARRELS SO WHAT IT MEANS IS IF
YOU HAVE SOMETHING THAT IS
EVAPORATES IN TERMS OF OR TRYING
TO GET AREA EXCHANGE, THE
SURFACE AREA OF O MENISCUS IS
SMALLER RELATIVE TO CONTAIN FOR
1536. SO 384 AIR RATES WELL,
1536 STRUGGLES THEY DON'T GROW
WELL HOWEVER THE VOLUME OF THE
THAT SURFACE AREA VOLUME CONTEXT
IN A 1536 IS HIGHER MORE SURFACE
CONTACT IN THE BAR SO THAT HAS
COUPLE OF EFFECTS. IF YOU DO SO
LIKE WASH ASSAY THAT RELY ON
ADHERENCE OF YOUR TARGET TO
WELLS, 1536 SCALES BETTER. IT
ALSO MEANS ANY TYPE OF STICKY
COMPOUNDS ARE EXACERBATED IN
TERMS OF DEPLETION WITH 1536
PLATE. THAT'S ONE PRACTICAL
ASPECT. SO THIS IS HOW YOU LOOK
AT SYSTEMATIC BASED ERRORS LOOK
AT THINGS LIKE IF YOU PLOT
THINGS BY COLUMNS, OR BY ROWS
YOU CAN SEE IF A COLUMN IS
ALWAYS HIGH VERSUS OTHER COLUMNS
SO THIS IS A COLUMN THAT MIGHT
BE EDGE EFFECT SO YOU WANT TO DO
THAT. YOU WANT TO SEE IF -- SO
SAME THING HERE THIS SYSTEMATIC
DRIFT EACH TIME OR HERE YOU ARE
SEEING WITH INCUBATION VERY
PATTERN DRIFT SO EVERY COLUMN IS
INCREASING IN TIME, YOU CAN PLOT
THIS WAY AND THIS GIVES YOU
CLEARER IDEAS IF THERE'S
SYSTEMATIC ERRORS. THE WAY YOU
DO THAT IS WAY YOU CORRECT IS
REDESIGN THE SAY, YOU GET RID OF
THE OUTSIDE WELLS OR IF YOU
CAN'T GET RID OF THEM YOU CAN
PUT SMOOTHING ALGORITHMS AND
TAKE THE AVERAGE SO WHEN YOU ARE
DONE WITH THAT, YOU ARE LEFT
WITH RANDOM ERROR. I'M GOING TO
JUMP HERE TO THIS THING WHICH
HAS BEEN TAUGHT TO ME BUT IT IS
A LITTLE COUNTER INTUITIVE. I
WILL WALK -- THIS IS A
DISTRIBUTION OF ALL THESE PEOPLE
ABOUT I PRESIDENT HOW MANY
PEOPLE -- 200 DIGITS SO TAKE
THIS SOCIAL SECURITY NUMBER OF
ANYONE RANDOMLY AND LOOK AT FOUR
OF THE THEM. RIGHT? FROM 0 TO #E
DISTRIBUTION IS IF YOU TAKE 200,
IT SHOULD BE ABOUT THERE'S NINE
RANDOM NUMBERS, EQUALLY
POSSIBLE. SO THEY DID THIS AND
THEN THIS IS THE DISTRIBUTION
YOU GET. IT IS RELATIVELY FLAT.
YOU CAN FORMALLY CALCULATE THE
MEAN OF THIS, THE MEAN IS FOUR
AND A HALF BECAUSE ONE TO -- IT
IS 0 TO 9. SO THAT MAKES SENSE.
THE SIGNIFICANT FIGURES
REPRESENTS JUST COUNTING
STATISTICS WHEN YOU DO IT. WHAT
IS INTERESTING IS IF YOU FAKE
NOT THE ACTUAL DISTRIBUTION
WHICH IS FLAT, BUT CALCULATE THE
MEANS OF THE DISTRIBUTION -- IF
YOU TAKE ALL THESE SAMPLES AND
CALIFORNIA COW LATE THE MEAN OF
FOUR SIGNATURES, AND PLOT, SO
HERE IS THE MEAN OF FOUR DIGITS
AND LOUT THAT NUMBER, THE MEAN
EVERY FOUR AND PLOT THE
DISTRIBUTION OF THE MEANS YOU
SEE HOW IT LOOKS SORT OF LIKE A
NORMAL DISTRIBUTION. IF YOU
ACTUALLY INCREASE THAT NUMBER OF
SAMPLES YOU DO TO CALCULATE THE
MEAN YOU MAKE THAT N VALUE
LARGER IT BECOMES MORE GALSION.
SO WHY IS THIS IMPORTANT? IT IS
COUNTER INTUITIVE THAT THIS
SHOULD BE BUT YOU CAN DO IT MANY
THINGS. SHOW YOU IN THE NEXT
SLIDE. THE MEAN STAYS THE SAME
FROM THE FIRST DISTRIBUTION --
FROM THE POPULATION DISTRIBUTION
TO MEAN SAMPLE DISTRIBUTION THAT
IS CONSTANT. WHAT IS INTERESTING
IS THIS -- AS YOU GO TO
POPULATION DISTRIBUTION OF
POPULATION MEANS,S THAT STANDARD
DEVIATION OF MEAN IS SMALLER
THAN THE STANDARD DEVIATION OF
POPULATION. IT IS DIFFERENT BY
REDUCING IT BY THE NUMBER --
SCROLL TO THE EN END. THIS IS TT
STANDARD ERROR OF MEAN YOU
ALWAYS SEE. WHAT IS FUNNY ABOUT
THIS THING IS IF YOU APPLY THIS
TO ANY DISTRIBUTION WHETHER
UNIFORM LIKE THE FIRST CASE,
TRUE GALSION DISTRIBUTION, OR A
SKEWED DISTRIBUTION WHETHER
NORMAL UNIFORM OR SKEWED,
WHENEVER YOU TAKE THE SAMPLE
MEAN, IT BECOMES MORE NORMA NORS
THE LARGER NUMBER OF SAMPLES YOU
PUT IN THAT MEAN, MORE NORMAL IT
GETS. THIS IS IMPORTANT FOR THE
REASON THAT THE MEAN ALWAYS
STAYS THE SAME AND SOMETIMES YOU
DON'T KNOW EVEN WITH THE
DISTRIBUTION YOU HAVE TO STILL
MAKE JUDGMENTS ON WHAT IS
PROBABLE. SO AGAIN THE MEAN
APPROACHES THE TRUE MEAN,
STANDARD DEVIATION IS BECOMES A
STANDARD ERROR OF THE MEANS WHEN
YOU GET LARGER POPULATIONS THEY
BECOME GALLION. WHY THIS SHOULD
MAKE SENSE IS WHILE ALL OF US
HAVE LIKE DIFFERENT WEIGHTS, IF
WE TAKE THE MEAN OF OUR WEIGHT,
IT IS KIND OF LIKE SAYING HOW
DIFFERENT ARE ARCH OF 30 PEOPLE
VERSUS JUST ONE OR TWO PEOPLE?
SO THAT YOU CAN SAY CHANCE OF
FINDING SOMEONE TALLEST IN THE
GROUP IS PROBABLY IF SOMEONE IS
TALL VERSUS THE REST, THAT IS A
BIG VARIANCE BUT IF I AVERAGE
THAT PERSON WHO IS TALL AMONG 30
OTHER PEOPLE, THE IMPACT OF
THEIR VARIANCE IS MINIMIZED. SO
THE REASON THIS IS IMPORTANT,
YOU CAN ACTUALLY ALWAYS COUNTER
THE SAMPLE MEAN AND ASSUME ITS
NORMAL AND THEN BACK CALCULATE
WHAT THE STANDARD DEVIATION OF
THE POPULATION WAS BECAUSE YOU
MOW NUMBER OF REPLICATES YOU DO,
YOU CAN GET STANDARD DEVIATION
OF YOUR SAMPLE MEANS. THEN YOU
CAN MULTIPLY IT BY THE END AND
GET ACTUAL DISTRIBUTION OF THE
POPULATION. IT IS CENTRAL TO LOT
OF STATISTICAL THEORY. SO WHAT
IS THE MEAN? WE USED ARITHMETIC
MEAN, THIS IS BASICALLY IF YOU
THINK ABOUT ANY DISTRIBUTION,
AND PLOT WEIGHTS IT IS THE
FULCRUM POINT TO BALANCE THIS
WHOLE DISTRIBUTION. THE
ARITHMETIC MEAN IS SUM OF ALL
INDIVIDUALS DIVIDE BY NUMBER OF
MEASUREMENTS. IT ACTUALLY IS
USEFUL ALL THE TIME, YOU CAN
ALWAYS CALCULATE IT. IT IS
AFFECTED BY EXTREMES. THE MOST
FAMILIAR WITH WHAT -- BUT THERE
ARE HOARE KINDS OF MEANS YOU CAN
CALCULATE IN TERMS OF CENTRAL
TENDENCY. ONE IS CALLED THE
MEDIAN. THAT IS IMPORTANT
BECAUSE BASICALLY WHAT IS THE
MIDDLE VALUE OF ANYTHING? THAT'S
INSENSITIVE TO REAL WIDE
DEVIATION BECAUSE MIDDLE VALUE.
THE MOST IS THE MOST FREQUENT
VALUE NOT SENSITIVE TO THE WIDE
RANGE BECAUSE IT IS CENTRAL OF
THE MOST COMMON VALU VALUE. ONET
IS AFFECTED BY EXTREMES IS MID
RANGE, AND THE WAY IT PRESENTS
IS IF YOU HAVE A NORMAL
DISTRIBUTION ALL THE MEANS ARE
THE SAME. BUT YOU CAN SEE IF
YOU HAVE A SKEWED DISTRIBUTION
TO THE LEFT, THE ACTUAL
ARITHMETIC MEAN IS HIGHER THAN
THE MODE. SO THESE ARE ONES THAT
PEOPLE DO CHECK ON. STANDARD
DEVIATION IS A MEASUREMENT OF
VARIATION. AND I THINK WE ALL
INTUITIVELY KNOW IF YOU HAVE
ONLY ONE SAMPLE, THEN IT HAS NO
STANDARD DEVIATION BECAUSE IT IS
A SINGLE ONE. IF YOU HAVE
DIFFERENT REPLICATES LIKE THIS,
THE DISTRIBUTED WEIRD, THIS
STANDARD DEVIATION SHOULD BE
FAIRLY LOW BUT HERE THERE IS
WIDE VARIANCE. SO STANDARD
DEVIATION IS SOMETIMES LARGER
THAN THE MEASUREMENT ITSELF. FOR
THE POPULATION, WHEN YOU GO
EPIFINTY THEY COLLAPSE TO SIGMA
THE MU AND TOTAL ENDS. IF YOU
HAVE A SAMPLE, ANYTHING SMALLER
THAN 30 YOU HAVE TO REDUCE
DEGREES OF FREEDOM BY ONE. SO
INSTEAD OF 30 SAMPLES YOU HAVE
DEGREE OF FREEDOM OF 29. SO
SCORES CLOSE TOGETHER WILL YIELD
SMALLER STANDARD DEVIATION. IT
IS INTUITIVE AND WE FORMALLY
HAVE DONE THAT. THE OTHER THING
THAT IS IMPORTANT FOR NORMAL
DISTRIBUTION IS TO UNDERSTAND #
9, 9568 RULE. WHAT IT MEANS IS
IN THREE UNITS THREE STANDARD
DEVIATION UNITS, 99% OF ALL THE
AREA UNDER THE CURVE IS
CONTAINED IN THAT. SO THAT IS
TRUE NORMAL BELL SHAPED
DISTRIBUTION IS. SO THAT IS HOW
WE ALWAYS SAY LIKE WE DO THREE
STANDARD DEVIATION THE MEAN.
THAT MEANS THE BULK OF THE
POPULATION ACTS LIKE NORMAL
DISTRIBUTION, IT IS ALL THE
SAME. WHAT IS DIFFERENT, ONE
PERCENT THAT IS DIFFERENT IS
THREE STANDARD DEVIATIONS AWAY
FROM THE MEAN. THAT IS HOW WE
CAME TO THAT NUMBER. THIS IS A
LITTLE BIT OF WHAT I INFERRED
FROM YEARS OF EXPERIENCE AND IT
IS NOT SOMETHING PEOPLE
EMPHASIZE BUT BRING TO YOUR
ATTENTION. ON THE TOP WHEN WE
SCREEN, THE REASON WHY IS HDS
DATA NORMALLY DISTRIBUTED AND
WHY TREAT IT LIKE THAT? I KIND
OF TALKED ABOUT IT SEVERAL
TIMES. ONE IS THAT RANDOM
COMPOUND COLLECTION 99% ARE NOT
ACTIVE. SO THEY SHOULD ACT LIKE
NOISE. THEY SHOULDN'T HAVE ANY
ACTIVITY. THE WAY TO MAKE IT
REAL, IF I DIDN'T -- WE DO THE
-- WHEN I WAS SUPERVISOR I COULD
HAVE TOLD ONE OF MY PEOPLE TO
SCREEN THIS LIBRARY BUT WHAT I
COULD HAVE DONE WAS TO FOOL THEM
COULD HAVE TAKEN A COMPOUND THAT
TEN MICROMOLAR IC 50 MADE
100,000 REPLY CASE AND SCREENED
THAT. WHAT THEY SHOULD FIND
USING THE STANDARD SCREEN IS
THAT THEY WILL FIND IF THEY
SCREEN TEN MICROMOLAR, ICD 10
MICROMOLAR THEY FIND
DISTRIBUTION THAT LOOKS NORMAL
BUT CERTAINTIED AT 50%
INHIBITION RATE. I BELABOR THIS
BECAUSE WHEN WE DO DOSE RESPONSE
CURVE THIS IS WHAT WE ARE DOING
TAKING REPLICATES TO SEE IF IT
IS EACH CONCENTRATION IN MY ALSO
GALSION AND IS THAT THE AVERAGE
THE OTHER REASON I BRING THIS UP
IS MANY TIMES YOU DO A SCREEN
AND YOU MANAGE, I SCREEN AND
CAN'T FIND ANYTHING, SCREEN
HIGHER CONCENTRATION YOU FIND
MORE THINGS. ACTUALLY IF YOU DO
THAT ALL YOU DO IS MOVING THE
POPULATION UP TO HIGHER AVERAGE
ACTIVITY AND COLLAPSING YOUR
WINDOW. THE REASON WHY REAL
SCREENS WORK, MOST OF THE
COMPOUNDS HUNDRED THOUSAND
COMPOUNDS, THAT YOU HAVE
POTENCIES LESS THAN ONE
MICROMOLAR. THE IDEAL SCREEN IS
WHERE THE CENTROID IS NEGATIVE
AND YOU HAVE SAMPLE LORING
SPREAD OF COMPOUNDS, SO WHAT I
LOOK FOR IN THE SCREEN IS
SOMETHING THAT LOOKS LIKE
CENTRAL TENDENCY IS GALLION BUT
THERE IS A SMEAR -- GALSION, I
LOOK AND THERE IS A SMEAR TO THE
RIGHT. THE OTHER WAY TO DO THIS,
IF I GIVE A RANDOM LIBRARY AND
TARGETED LIBRARY AND MIC THE TWO
AND IT WAS AGAINST A TARGET
WHERE AVERAGE ACTIVITY WAS ONE
NANOMOLAR I SHOULD HAVE BIMODAL
DISTRIBUTION IN MY SCREENING. SO
I BELABOR THIS BECAUSE THE
COMMON THING IS YOU DON'T FIND
ANYTHING SCREEN MUCH HIGHER
CONCENTRATION. THAT LEADS TO LOT
OF PROBLEMS IT SKEWS AND YOU
START TO EXPOSE ARTIFACTS OF
INSOLUBLE COMPOUNDS. SO AGAIN
BACK TO THIS ROBUSTNESS, THERE
IS DEBATE TRYING TO FIGURE HOW
TO DO THIS AND CALCULATE THIS.
SO THE FIRST THING YOU WANT TO
DO TO OPTIMIZE ASSAY IS REALLY
LOOK TO GET GAIN MIC RAKE HIGH.
SO THE BACKGROUND AND SIGNAL.
WHEN I STARTED MY CAREER
SCREENING GET BACKGROUND LOW
SIGNAL HIGH AND THAT IS A
PERFECT ASSAY. IT SEEMS
REASONABLE. BE AS I TALKED TO
STATISTICIANS THERE'S WAYS OF
DOING THIS. SO THE GROUP I HAD
AT DUPONT WE WERE ON ONE OF
THESE SOAS MEETINGS AND GOT ON
CRUISE BOAT AND WE WERE PRETTY
DRUNK. WE JUST STARTED TALKING
ABOUT THIS. AND MY SECOND IN
COMMAND SAID THERE SHOULD BE
REALLY SIMPLE PARAMETER, SHOULD
BE EASY TO REMEMBER. SO WE WERE
KICKING THE IDEA THIS IS A
COMMON THING. HERE IS AN ASSAY,
I WILL DEFINE WHAT IT IS IN A
SECOND BUT WE LOOK AT THIS
PARTICULAR ASSAY, IF THE SIGNAL
BACKGROUND IS HERE, THAT IS YOUR
BACKGROUND SIGNAL AND THEN YOUR
HIGH SIGNAL IS RIGHT HERE, THE
AVERAGE, THAT RATIO HERE FOR
THIS EXAMPLE IS TEN. SO THAT
SHOULD BE PRETTY GOOD ASSAY.
BUT LOOK AT THE SCATTER OF THE
DATA, ACTUAL COMPOUNDS AND
INTUITIVELY KNOW THAT IS NOT
GOOD. WHEREAS HERE YOU CAN SEE
THE HIGH SIGNAL CLUSTERS
TOGETHER, LOW SIGNAL CLUSTERING
TO AND WILL IS A FREE ZONE OF
BOUNDARY. SO THAT HAS SIGNAL TO
NOISE OF 12 AND SIGNAL TO
BACKGROUND OF ONLY FIVE BUT
CLEARLY LOOK AT THIS AND SAY
THAT IS A BETTER ASSAY. SO WE
WANTED TO FORMALIZE THIS. AND MY
GROUP TOLD ME THE SOLUTION AND I
THOUGHT THAT WAS OBVIOUS. I
THOUGHT IT WAS TRIVIAL AND I
SAID THAT'S STUPI STUPID. WHICHH
WE'LL USE IN THE GROUP BUT WHO
CARES. THE PERSON WHO WANTED TO
PUBLISH IT PUSHED AND PREDICTED
THERE WOULD BE MOST CITED PAPER
AND EVIDENTLY PROVIDE US
SOMETHING EASY FOR EVERYONE TO
USE SO THAT IS MY ADMISSION THAT
WASN'T THAT GOOD A MANAGER AT
THE TIME. SO I WANTED TO
FORMALLYZE THIS, THIS IT IS Z
PRIME YOU CAN LOOK IT UP. THE
WAY TO THINK ABOUT IT IS YOUR
BASICALLY THE HIGH SIGNAL AND
LOW SIGNAL CONTROLS, THE HIGH
CALL THIS LOW AND HIGH SIGNAL
CONTROLS, EACH HAVE A
DISTRIBUTION EACH HAS ITS
STANDARD DEVIATION AND SPREAD,
SO AS LONG AS EACH DISTRIBUTION
DOESN'T TOUCH EACH OTHER YOU
HAVE A GOOD ZONE OF SEPARATION
AND THE WAY TO THINK ABOUT IT IF
THE ZONE -- IF THIS VARIATION
WAS ZERO, STRAIGHT LINE, THIS
GOES TO ZERO AND Z PRIME WOULD
GO TO ONE. IT JUST MEANS IT IS
TWO LINES THEY DON'T EVER TOUCH.
THERE IS ONLY SEPARATION WILL BE
THE MAXIMUM IT CAN BE. DOESN'T
MATTER. THE OTHER PREMISE IS IF
YOU HAVE A -- THE ZONE OF
SEPARATION DISAPPEAR. WHAT THAT
MEANS IS THREE SIGMA LOW AND
THREE SIGMA HIGH BOTH TOUCH EACH
OTHER. AT TO POINT YOU STILL
HAVE ASSAY BUT IT IS BASICALLY
STILL ACCEPTED ASSAY BECAUSE YOU
ARE STILL -- WHERE THIS ZONE OF
SEPARATION APPEARS IT IS STILL
ONLY TOUCHING 1% VARIANCE. SO
YOU STILL HAVE THIS. BUT IF THE
TWO OVERLAP THEN THE -- IF THE
TWO ACTUALLY -- IF THIS ALL --
IF THE SEPARATION OF MEANS IS
EQUAL TO THIS NUMERATOR TERM,
THIS COLLAPSES, SO IF THIS IS
ZERO HERE, THE TWO MEANS TOUCH,
NO MATTER WHAT THE DISTRIBUTION,
DISTRIBUTION IS OVERLAPPED. SO
IF -- THIS IS SEE REON THE
BOTTOM, THIS GOES DOWN TO Z,
ZERO. SO IT MEANS NO MATTER WHAT
YOU DO, THE SEPARATION OVERLAP.
SO THAT IS NOT A USABLE ASSAY.
BY DOING THIS YOU CAN CALCULATE
THAT Z -- IF Z FACTOR IS 1,
PERFECT ASSAY. YOU CAN REALLY
NEVER ACHIEVE THIS BECAUSE THAT
MEANS THERE'S NO NOISE IN EITHER
BAND BUT .5 TO 1 IS ACCEPTABLE
SCORE. AND IF IT IS BETWEEN 0
AND .5, YOU NEED TO DO
REPLICATES. YOU CAN SALVAGE
ASSAY BUT CAN'T RUN SINGLE KIT.
IF 0 OR LESS THAN 0 IT MEANS YOU
CANNOT DO AN ASSAY. IF IT IS
ZERO THE ACTUAL BANDS OVERLAP SO
MUCH IT IS IN OR OUT OF THE
SCREEN. SO THAT IS A YES, NO
ASSAY. THAT IS THE ORIGIN OF Z
FACTOR. HOW YOU USE IN REAL
SCREENS YOU MOST A HISTOGRAM OF
THE DATA. FREQUENCY PLOT OR PLOT
TYPICAL AND LOOK BASICALLY TO
SEE IF THERE IS A SEPARATION
BAND YOU CAN SEE IT THIS WAY,
SCATTER GRAM EMPHASIZES EACH
INDIVIDUAL DEFLECTION WHEREAS A
HISTOGRAM GIVES YOU DISTRIBUTION
OF THE AVERAGE SO YOU CAN SEE
HERE, THIS IS IDEALIZED. SO THE
CONTROL SHOULD BE TRULY GALSION
AS WELL AS HIGH AND LOW
CONTROLS. YOU LOOK TO SEE WHAT
HAPPENS WHEN YOU HAVE REAL
COMPOUNDS. IF YOU LOOK AT REAL
DATA HERE IS AN EXAMPLE OF ASSAY
PROTEIN PROTEIN INTERACTION, WE
CALCULATED THE PARAMETERS FOR
THE PIE WILL THE SCREEN, THIS IS
WHAT YOU DO, YOU CALCULATE THE
METRICS FOR PILOT SCREEN AND GET
VALUES Z FACTOR PRETTY GOOD, HAD
PRETTY GOOD SIGNAL BACKGROUND
THEN DO IT FOR THE FULL SCREEN
SO 7,000 COMPOUNDS, IN THIS
PARTICULAR CASE YOU CAN SEE THE
Z MAINTAINS ITSELF, THE POSITIVE
CONTROL SIGNALS WITHIN LINE SO
THIS IS GOOD. WE GOT REASONABLE
HIT RATES. WHEN YOU PLOT THE
ACTUAL DATA, PARAMETERS, YOU CAN
SEE HERE VERY CLEARLY, THE
CONTROLS ARE GALSION. THIS IS
BIASED BUT LOOK AT THE ENVELOPE
OF HITS, THE MAIN ENVELOPE IS
GALSION BUT YOU SEE THIS TAIL.
THIS IS GOOD. THIS PARTICULAR --
THERE WAS ACTUALLY A WIDE RANGE
OF HITS. SO TYPICALLY WHAT YOU
SHOULD DO IS PLOT THIS AND THEN
PLOT BLOWUP OF INSET HERE. YOU
CAN SEE IT IN A SCATTER GRAM.
YOU CAN CALCULATE ON PLATE BY
PLATE BASIS SO THIS IS A GOOD
PORTION OF THE SCREEN, SO TEN
PLATE RUN AGO PLATE RUN ABOUT.
ABOUT 20,000 COMPOUNDS OR SO.
YOU CAN SEE FOR THIS ASSAY Z
FACTOR STAYED WITHIN SPEC,
ALWAYS GRADED .5 SIGNAL BACK
GROWN AND SIGNAL NOISE WORKS
CONSISTENT SO THIS GAVE US
CONFIDENCE THAT WE ARE PLATE BY
PLATE, RUN WAS GOOD SO WE CAN
AGGREGATE ALL THE DATA. THE
FINAL THING I WANT TO ASK YOU
WAS SO AGAIN THE REASON WHY WE
PICK THE ONE PERCENT, IT IS
THREE STANDARD DEVIATION AWAY
FROM THE POPULATION AVERAGE
WHICH IS RANDOM NOISE. SO IT IS
SIGNIFICANT THINGS. TYPICALLY IN
GROUPS YOU CAN SET A THRESHOLD
FOR CALLING A HIT STATISTICALLY
OR SOME DON'T CARE THEY JUST SAY
I WILL DO ARBITRARY 50% BECAUSE
IF I TESTED TEN MICROMOLAR AN
50% PERTURBATION IT IS A WEAK
MICROMOLAR HIT SO MOST COMPOUNDS
ARE PROBABLY MILLIMOLAR IC
50s. BUT IF YOU GO BY
STATISTICS IF YOU ARE HAVING A
THRESHOLD, IF YOU SET A
THRESHOLD AND THEN RETEST
COMPOUNDS, ANYTHING ABOVE THAT
THRESHOLD, IF YOU TEST THE
COMPOUND RIGHT AT THRESHOLD,
WHAT EVERY YOU CHOOSE FOR A
THRESHOLD, WHAT DO YOU THINK THE
CONFIRMATION RATE OF THAT
COMPOUND SHOULD BE? OR THOSE
COMPOUNDS? THEY ARE RIGHT AT
THRESHOLD. THE ANSWER IS HERE.
IT IS EXACTLY 50% BECAUSE YOU
RIGHT AT THRESHOLD SO IF YOU
RETEST IT NO MATTER HOW PRECISE
HALF THE TIME ABOVE, HALF THE
TIME BELOW. HOWEVER IF YOU HAVE
A COMPOUND THAT IS SAY FIVE
STANDARD DEVIATION FROM ORIGINAL
SIGMA, AND YOU RETEST THAT
COMPOUND, 500 REPLICATES AND
RETEST IT, IT SHOULD BE NORMAL
DISTRIBUTION. IT IS VARIATION
WILL BE DEPENDENT UPON THE
RANDOM ERROR OF YOUR SAY SAY.
THERE IS A WAY TO HAVE
CALCULATING IF YOU HAVE A TABLE
OF ALL YOUR HITS AND THERE ARE
PERCENT INHIBITION, YOU CAN
CALCULATE THE INHIBITION
CONFIRMATION RATE EXPECTED.
PRACTICALLY YOU WILL NEVER HIT
50% COPYMATION BECAUSE THAT
MEANS ALREADY HITS THRESHOLD.
WHAT IS REASONABLE FOR THRESHOLD
IS ABOUT 30% OVERALL
CONFIRMATION IS WHAT YOU CAN
GET. YOU CAN GET FEWER POTENT
HITS, MOST THRESHOLD. WHEN I DO
A SCREEN, IF I GET CONFIRMATION
RATES BELOW 30% I WORRY THAT IS
WHEN YOU WANT REPLY CASE AND GO
BACK TO CHECK CONDITIONS. LAST
IS FAST POSITIVE FALSE NEGATIVE.
IF YOU SET A THRESHOLD TOO HIGH,
TOO STRINGENTLY, THERE IS A
CHANCE YOU YOU WILL MISS
IMPORTANT COMPOUNDS. THE WAY YOU
DO THAT IS REDUCE ITS
STRINGENCY, LET THE THRESHOLD BE
LOWER SO MORE HITS ARE INCLUDED
IT DOES INCREASE YOUR FALSE
POSITIVE RATES BUT I THINK IF
YOU MISS A COMPOUND CLASS, THE
FALSE NEGATIVE IS MORE DAMAGING
THAN TOO MANY FALSE POSITIVES SO
YOU CAN FIND THAT YOU CAN DO A
SCREEN AND TEST HIT RATE COUPLE
OF DIFFERENT THRESHOLDS AN CHECK
YOUR CONFIRMATION AND SCAFFOLDS
YOU CAN COME UP WITH AND PLAY
WITH THAT. JUST TO SUMMARIZE ONE
THING I HEAR IS LOT OF PLACES
THEY HAVE A HIGH THROUGH PUT
GROUP FOCUSED ON HIGH PRODUCTION
AND THEY HAVE A DOSE RESPONSE
GROUP. BUT YOU SHOULD USE
PRIMARY ASSAY AS YOUR IC 50
GENERATING ASSAY. WHICH IS WHAT
WE DO. YOU CAN SEE IF YOU HAVE A
1536 WELL PLATE YOU CAN SUPPORT
QUITE A NUMBER OF DOSE RESPONSE
ASSAYS 10-POINT DOSE RESPONSE,
129 IN TYPICAL 36 WELL PLATE SO
NEVER LIMITING THE CYCLE TIME
WHENEVER WE DESIGN ASSAYS WE
DESIGN BOTH PRIMARY AND COUNTER
ASSAY IN THESE HIGH THROUGH PUT
FORMATS SO WE CAN SUPPORT THIS
THING. AND THEN WE FOCUS ON DO
THE DIFFERENT BIOLOGICAL ASSAYS
LOWER THROUGH PUT BUT TRIAGE
DOWN TO REAL SCAFFOLDS THAT ARE
SHOWING SAR. SO WHEN YOU CAN DO
THAT ON -- IN THE NCATS HAS DONE
THIS, BECAUSE THEY SCREEN BUY
LIBRARIES VERTICALLY THEY HAVE
FULL DOSE RESPONSE CURVE AND
TEACHING IS YOU CAN'T LOOK AT
THAT MANY CURVES YOU HAVE TO
CATEGORIZE THE CURVES TURNS OUT
THAT YOU ARE CATEGORIZE WITH
DESCRIPTORS THAT YOU LOOK UP IN
THE NCATS ASSAY GUIDANCE MANUAL
ON HGS IC 50 BASED HCS BUT THIS
IS IMPORTANT FOR COLLEAGUES
BECAUSE YOU CAN HAVE COMPOUNDS
GIVE YOU FULL NORMAL RESPONSE
WITH TWO LOW AND HIGH, ALSO ONES
THAT REACH LOWER ASIMTOTE OR
DON'T BOUND OR HAVE MORE THAN
ONE MAXIMUM. . BUT THEY HAVE
FORMALIZED ALL THIS. SO FINAL
COMMENTS, JUST THAT HGS RELIES
ON STATISTICALLY BASED ROLES AND
BIGGEST IS MOST COMPOUNDS ARE
INACTIVE SO THEY ACT LIKE
BACKGROUND NOISE. YOU CAN TREAT
THAT NORMALLY. THE OTHER THING,
YOU CAN SPEND TIME TRYING TO
MAKE THE ASSAY PERFECT BUT YOU
ONLY NEED TO MAKE IT GOOD ENOUGH
AND ROBUST ENOUGH TO BE
CONFIDENT THAT A SINGLE
CONCENTRATION ATTEMPT FINDING
THAT COMPOUND YOU HAVE A GOOD
CHANCE OF FINDING IT. THE OTHER
THING THAT PROTECTS ALL OF US IN
REDUCE SCREENING IS IT IS VERY
RARE YOU SCREEN CHEMICAL
COLLECTION IS IS THAT IS
COMPOSED OF SINGLETONS THAT
DON'T HAVE RELATED ANALOGS IN
THAT. GOOD LIBRARIES ARE BASED
UPON ISLANDS DIVERSITY AND
OTHERS SAR. AND YOU CAN MITIGATE
THE RISK OF FALSE POSITIVES,
FALSE POSITIVE BY SEEING HIGHER
THRESHOLD BUT YOU HAVE TO THINK
FALSE NEGATIVES YOU MIGHT HAVE.
FINALLY, WHEN WE ARE DOING
CONFIRMATION WE DO REPLICATES
BECAUSE THAT GIVES YOU A CHANCE
TO VERIFY WHAT YOU FOUND WAS AN
EFFECT WITHIN THAT POPULATION.
LAST THING, IF YOU FOLLOW THESE
RULES YOU SHOULD ORBED BE
CONFIDENT YOUR ASSAY FOUND
CHEMICAL COMPOUNDS YOU SHOULDN'T
HAVE BEEN ABLE TO FIND, IT IS
JUST AS IMPORTANT TO TELL
MANAGEMENT AND PRESERVE
RESOURCES THAT YOU SCREEN
CORRECTLY WHAT IS PROBLEM IS NOT
THE ASSAY, THE COLLECTION OF
COMPOUNDS YOU TEST. BECAUSE LIKE
FISHING IF YOU ARE IN A LAKE
THAT DOESN'T HAVE FISH, DOESN'T
MATTER HOW WELL YOU FISH.
ANYWAY, THAT IS MY LITTLE
TUTORIAL.
[APPLAUSE]
>>THANK YOU SO MUCH, TC FOR A
WONDERFUL TALK. QUESTIONS.
>>CAN YOU PLEASE COME CLOSE TO
THE MICROPHONE? I HAVE BEEN TOLD
TO SAY THAT.
>>THANK YOU VERY MUCH FOR THAT
WONDERFUL PRESENTATION. YOU
MENTIONED THAT FOR THE Z
STATISTICS WORK THERE RECOGNIZE
TO BE TEXT ON YOUR SLIDE THAT
NEEDS TO BE LARGE SAMPLE. I KNOW
THIS MIGHT BE NOT POSSIBLE TO
ANSWER BUT FROM YOUR EXPERIENCE
DO YOU HAVE A RULE OF THUMB FOR
HOW LARGE A SAMPLE YOU NEED TO
GET RELIABLE NUMBER?
>>DEFAULT TO THE SAMPLE MEAN,
SO IF YOU ARE GREATER THAN 30
YOU REALLY FIND. WHEN WE WERE IN
96 WELL PLACE, YOU ARE
CONSTRAINED. LOOKING AT ONE
PLATE YOU ARE CON VAINED TO
EIGHT BY 12 SO MAYBE FOUR AND
FOUR, THAT'S LOW BUT THEN WE
WOULD WHEN SET UP ASSAY TAKE 9 #
RUN HALF HIGH, HALF LOW. YOU
MAKE THE AIONAL WHEN YOU PUT
REAL COMPOUNDS ON YOU INCLUDE
SOME THOSE REPLICATES AS WELL.
WHAT YOU DO IS YOU DO PLATE BY
PLATE THEN AT THE END YOU CAN
LOOK AT ALL YOUR CONTROLS AND
PULL THEM TOGETHER AND THAT
GIVES YOU OVERALL STATISTIC AS
WELL. IF YOU RUNNING ANIST
ASSAYS A FOR FAN GP YOU HAVE TO
REFERENCE HEIGHTENED CONTROL, SO
TWO WAYS TO DO THAT, USE A Z
SCORE WHICH MEANS HERE IS MY
FLOOR OF THE ASSAY, AND POP UP
ABOVE THAT. BY EXPERIENCE YOU
CAN HAVE TIGHT THINGS AND THINGS
THAT RISE TEN PERCENT ABOVE
BASELINE BUT NEVER GIVE A HIT
BUT YOU CAN STATISTICALLY DO
THAT. THE OTHER WAY FAKE IS LOW
CELL COUNT FOR HIGH CELL COUNT
RECEPTOR AND FORCE THE SYSTEM
BUT YOU DONE KNOW UNLESS YOU
HAVE A REFERENCE ACTNIST.
>>I HAD A QUESTION GIVEN YOUR
COMMENT THAT MOST COMPOUNDS ARE
INACTIVE, 99% WHATEVER THE
NUMBER MIGHT BE, HOW MUCH OF THE
ASSAY OPTIMIZATION CAN BE
DEFERRED TO THE ACTUAL SCREEN?
LET ME ASK SPECIFIC QUESTION,
SPECIFIC EXAMPLE, SAY YOU TRY TO
ASSESS PLATE TO PLATE
VARIABILITY. IN THE ACTUAL
SCREEN YOU SHOULD SEE THAT
ANYWAYS AND BECAUSE YOU HAVE GOT
ALL THESE DIFFERENT PLACE
RUNNING SO DOES THAT NEED TO BE
DONE DURING OPTIMIZATION BECAUSE
YOU WILL SEE IT ANY WAYS IN THE
ACTUAL SCREEN?
>>THE OPTIMIZATION WITH SMALL
NUMBER OF REPRESENTED PLACE WILL
GIVE YOU THE LOWEST SOMETHING
CAN BE WITHOUT INTERFERENCE. YOU
HEARD WHILE COMPOUNDS --
ASSUMPTION HYPOTHESIS MOST
COMPOUNDS INACTIVE, THAT IS
BASED UPON ACTIVITY OF TARGET
INVOLVED IN BUT AS YOU HEARD
HERE THERE IS COMPOUND
INTERFERENCE THAT HAS NOTHING TO
DO WITH BIOLOG BIOLOGY. INTERFEG
WITH THE SYSTEM, NON-BIOLOGICAL
BUT REPRODUCIBLE. TO DEFER, BACK
-- YOU TAKE ALL YOUR COMPOUNDS
LOOK AT NUMBERS OF HEAT MAPS AND
LOOK AT THEM. IF THERE IS HIGH
COMPOUNDS ON THIS SIDE OR
STRIPING YOU WILL SEE THOSE
PATTERNS. IF U YOU SEE LIKE --
IF YOU HAVE IMAGE BASED ASSAY,
CENTER OF THE PLATE JUST BLOOMS,
YOU WILL BE WARNED. IF YOU ARE
AT A VALUABLE TARGET WHERE
EVERYTHING IS EXPENSIVE, YOU CAN
TAKE SHORT CUTS LIKE THAT.
>>ANY MORE QUESTIONS FOR TC? I
DON'T SEE MORE QUESTIONS THANK
YOU SO MUCH, TC. APPRECIATE YOUR
TIME.
[APPLAUSE]
OUR NEXT SPEAKER IS DR.
VISWANATH DEVANARAYAN, EXECUTIVE
DIRECTOR AND HEAD OF DATA
SCIENCE AT ASCI INC., ALSO
ADJUNCT PROFESSOR UNIVERSITY OF
ILLINOIS CHICAGO. HE HAS MORE
THAN 25 YEARS RESEARCH
EXPERIENCE OF GSK ELY LILLY,
MERC. DR. DEVANARAYAN IS A LONG
STANDING EDITORIAL BOARD MEMBER,
WE ARE EXCITED TO HAVE HIM HERE
TODAY. HE WILL BE SPEAKING
ABOUT REPRODUCIBILITY ASSESSMENT
OF IN VITRO SCREENING RESULTS.
FLOOR IS YOURS, DEVANARAYAN.
MANY TIMES SELENIAN ABIGALE PUT
IN HARD WORK ORGANIZING THE
WORKSHOP. BEEN DOING THIS A LONG
TIME AND EACH TIME A DIFFERENT
PLACE, THIS PLACE IS FANTASTIC,
VERY NICELY SPREAD OUT AND
COMFORTABLE AND THANK YOU ALL
FOR MAKING IT. TO THIS WORKSHOP
THIS IS MY FIRST TALK IN PERSON
SINCE THE PANDEMIC STARTED. IT'S
BEEN SO LONG, SO LONG BEFORE I
USED TO GIVE MULTIPLE TALKS
EVERY YEAR. IT'S BEEN TWO YEARS,
TWO OR THREE YEARS NOW AND BEEN
THE FIRST TIME IN PUBLIC. SO
REAL EXCITED, THAT WE ARE
GETTING CLOSER TO THE NEW
NORMAL. YOU HEARD ABOUT Z PRIME
FACTOR AND ALL THE IMPORTANT
STATISTICAL METRICS FROM TC.
FOR THE LAST 45 MINUTES. I'M
GOING TO EXTEND THAT FURTHER TO
THE SCREENING ASSAYS WHERE WE
WORRY ABOUT THE VARIABILITY OF
POTENCY RESULTS FROM VARIOUS
COMPOUNDS THAT ARE TESTED. EC 50
IC 50, K, AND SO FORTH. SO THERE
IS ANOTHER METRIC LIKE Z PRIME
NOT AS POPULAR. BUT RELEVANT FOR
COMPARING POTENCY RESULTS IN
COMPOUNDS. SO WHAT WE ARE DOING
IS DIFFERENTIATING COMPOUNDS IN
TERMS OF METRICS AND RANK
ORDERING THEM AND DECIDING WHAT
TO MOVE FORWARD. LOOK AT
STANDARD ERROR ASSOCIATED WITH
KI OR IC 50 OR DC 50 AND MAKE
APPROXIMATE JUDGMENT BUT IS
THERE A Z PRIME THAT WECAL QUEUE
RATE FOR SCREENING DAT DATA. AFR
CALCULATING THAT WE CAN FEEL
CONFIDENT THIS SCREENING DATA
ASSAY WILL BE RELIABLE FOR
GENERATING THOSE EC 50 IC 50
VALUES FOR THOSE COMPOUNDS. SO
THERE IS A METRIC LIKE THAT, IT
IS HARDER TO CALCULATE, THAN Z
PRIME, BUT WE GO TONA IN SOME
DETAIL NEXT 40 MINUTES. THIS IS
COVERED IN COUPLE OF CHAPTERS IN
THE AGM. THERE ARE SOME
MATERIAL IN THE SLIDES NOT
COVERED BUT IF YOU HAVE ANY
QUESTIONS FEEL FREE TO REACH OUT
LATER. HOW MANY HEARD Z PRIME?
MOST OF YOU HAVE. HOW MANY HEARD
ABOUT MSR? MEAN SIGNIFICANT
RATIO? ONE, TWO, THREE, FOUR.
AND I HAVE BEEN PRESENTING THIS
SINCE 2005 AT SBS AND SOAS AND
THESE WORKSHOPS AND AGAIN THIS
IS NOT AS EASY TO CALCULATE A Z
PRIME AND PROBABLY WHY NOT
CAUGHT ON YET BUT AS BACKGROUND
I USED TO WORK AT ELY LILLY,
MERCK SO FORTH ALL THESE PLACE
WHERE IS I WORKED WHEN THIS
CONCEPT WAS INTRODUCED MSR
CONCEPT WAS INTRODUCED, THERE
WAS ALWAYS SOME DELAY ADOPTING
THAT BUT ONCE PEOPLE GOT USED TO
IT, THEY JUST LOVED IT AND NOW
COUPLE OF YEARS BECAME REQUIRE
METRIC FOR EVERY SCREENING
ASSAY. IN THIS PIPELINE. SO ELY
LILLY MERCK AS WELL WHERE I USED
TO WORK IS REQUIRED METRIC
CALCULATED LIKE Z PRIME IS
CALCULATED MSR IS CALCULATED
BEFORE POSITION MADE SCREEN
COMPOUNDS WITH ASSAY. SO YOU
REALIZE FROM IN THE NEXT HALF
HOUR IT GETS DIRECTLY TO HEART
OF THE PROBLEM HOW WELL YOU CAN
DIFFERENTIATE POTENCY RESULTS
BETWEEN COMPOUNDS. SO Z PRIME
QUICK REVIEW, IT IS DIFFERENCE
BETWEEN SIGNAL AT TOP EXTREME
AND BOTTOM EXTREME HIGH
CONCENTRATION OF POSITIVE
CONTROL IN THE BACKGROUND. WE
CALCULATE VARIABILITY AROUND
THAT. THREE SDs CALCULATED AT
TOP AND BOTTOM. THIS GAP IS NUMB
RAY FOR OF Z PRIME FACTOR,
DENOMINATOR IS FULL WINDOW
SIGNALS TOP AND BOTTOM. SO THAT
IS YOUR Z PRIME. WHAT IS REALLY
IMPORTANT AND WHAT TC MIGHT NOT
HAVE TOUCHED UPON IS THAT THIS
VARIABILITY CALCULATE, STANDARD
DEVIATION CALCULATE IS HAS TO
TAKE INTO ACCOUNT ALL THE
SOURCES OF VARIABILITY IN YOUR
HDS. SO TYPICALLY WHEN Z PRIME
IS CALCULATED MOST PEOPLE
>>GENERATE DATA TOP AND BOTTOM
FROM SINGLE PLATE AND CALCULATE
THE MEAN AN STANDARD DEVIATION
FROM THE SINGLE PLATE.
CALCULATE Z PRIME FOR SINGLE
RATE. BUT THAT ONLY REFLECTS
VARIABILITY WITHIN THAT PLATE.
FOR HDS SCREENS YOU MAY NEED TO
WORRY ABOUT BETWEEN PLATE
VARIABILITY IF YOU ARE
UNDERSTANDING OVER TIME, IF SOME
PART OF THE HDS IS ONE LAB, WE
-- IF ROBOTICS ARE NOT INVOLVED
AS MUCH BUT MORE -- DEPENDING
UPON THE PARTICULAR TYPE OF HDS
AND DIFFERENT COMPONENTS OF
VARIABILITY INVOLVED IN THE HDS.
ALL THOSE VARIABILITY COMPONENTS
HAS TO BE INCORPORATED IN THE
CALCULATION OF THE VARIABILITY.
OTHERWISE WE OVERSTATE QUALITY
OF ASSAY, OVERESTIMATING Z PRIME
AND WE HAVE FALSE CONFIDENCE ON
THE ASSAY. T THAT Z PRIME VARIES
FROM .7 TO .4 DEPENDING HOW YOU
CALCULATE IT. IT IS IMPORTANT TO
MAKE SURE COMPONENTS OF
VARIABILITY IS RELEVANT FOR THAT
HDS INCO INCORPORATED IN Z PRIMO
THAT IS THE MESSAGE TO CONVEY
ABOUT Z PRIME BEFORE MOVING TO
THE OTHER TOPICS. ONE MORE
THING THAT ALSO SOMETHING TC
REVIEW, WE HAVE THREE ASSAYS AND
I MAKE THIS POINT THOUGH HE SAID
I WANT TO SAY AS WELL BECAUSE
THIS IS AN IMPORTANT THING AND I
SEE THE SAME MISTAKE BEING MADE
AFTER MANY YEARS NO MATTER HOW
MANY TIMES SAY IT PEOPLE USE
SIGNAL TO BACKGROUND ALSO SIGNAL
TO NOISE. AS WAY TO JUDGE
DEVELOP ASSAY OPTIMIZE ASSAY
CONDITIONS YOU SAW THIS POINT
MADE BY TC EARLIER. ASSAY ONE
EXCELLENT. SIGNAL TO BACKGROUND
OF TEN, SECOND IS EIGHT, THIRD
IS FIVE. SO GOING BY THAT PICK
THIS ASSAY CONDITION SAYING THIS
IS FANTASTIC, NICE WINDOW RATIO
OF TEN TO ONE BUT IF YOU TAKE
THE VARIABILITY INTO ACCOUNT, IT
IS ONE .4 FOR FIRST, .6 FOR
SECOND AND .75 FOR THE THIRD
ASSAY. IMPORTANT TO USE
APPROPRIATE METRIC WHEN WHAT
ASSAY CONDITIONS TO WORK WITH,
FORMAT TO WORK WITH, AND WHAT WE
ARE DEALING WITH, IN MAKING
POSITIONS ON IF WE START WITH
THE WRONG METRIC LIKE THIS
SIGNAL TO BACKGROUND WE END UP
WITH THE TOTALLY OPPOSITE
CONCLUSION. THIS PARTICULAR
CONTEXT YOU WANT TO TAKE
VARIABILITY INTO ACCOUNT, TO
CALCULATE Z PRIME USE IT TO
COMPARE THE DATA. ASP SINGLE
POINT HDS YOU ARE READY TO START
THE NEXT PACE OF SCREENING AND
CALCULATING WHAT THE POTENCY
METRIC S IC 50 KI SO FORTH, Z
PRIME IS NOT GOOD ENOUGH YOU CAN
HAVE Z PRIME .7 BUT WHAT DOES IT
MEAN IN TERMS OF HOW WELL WE CAN
DIFFERENTIATE THE POTENCY
VALUES? FOR THAT WE HAVE
SOMETHING CALLED THE MSR MINIMUM
SIGNIFICANT RATIO. SO BASICALLY
AS IT STATES BASICALLY SMALLEST
RATIO OR SMALLEST CHANGE ANY
POTENCY IN CELLS THAT YOU CAN
SEE IS STATISTICALLY
SIGNIFICANT. IF YOU HAVE TWO
COMPOUNDS WITH IC 5010 AND 20,
TWOFOLD DIFFERENT IS THAT
DIFFERENCE REAL? CAN YOU SAY
THAT THE COMPOUND TEN NANOMOLAR
IS SIGNIFICANTLY MORE POTENT
THAN 20 NANOMOLAR. THAT CEASE
WHAT MSL GETS AT. SO THE
FORMULA IS AS SHOWN HERE,
STRAIGHT FORWARD BUT CALCULATION
IS NOT SO STRAIGHT FORWARD SO WE
REVIEW THAT IN SOME DETAIL OVER
THE NEXT SLIDES. KEY PART OF
THE FORMULA IS STANDARD
DEVIATION YOU SEE THERE, SD.
THAT STANDARD DEVIATION IS
REPLICATE LOG IC 50 OR KI
WHATEVER THE METRIC IS. FROM
DIFFERENT SOURCES OF VARIABILITY
RELEVANT FOR SCREENING ASSAY. AT
THE COMMENT MADE ABOUT Z PRIME
HERE ALSO WE HAVE TAKE INTO
ACCOUNT INTRADAY INTERDAY
INTRALAB ANALYST ALL DIFFERENT
COMPONENTS RELEVANT FOR
PARTICULAR SCREENING ASSAY. ALL
THAT GOES INTO THIS CALCULATION
AND THERE ARE DIFFERENT WAYS TO
GO ABOUT IT SO WHAT DOES THAT
REALLY MEAN? MSR FOR ASSAY IS
FIVE. FOR ANY GIVEN COMPOUNDS
THAT MEANS THIS COMPOUND YOU
CANNOT BE SURE IT IS DIFFERENCE
FROM ALL THE OTHER COMPOUNDS
THAT HAVE ACTIVITY WITHIN FIVE
FOLD. OF THIS PARTICULAR
COMPOUND. FOR EXAMPLE, IC 50
PARTICULAR COMPOUND IS 100, THEN
WE DON'T HAVE 95% CONFIDENCE TO
SAY THIS COMPOUND IS DIFFERENCE
FROM ANY OTHER COMPOUND THAT HAS
IC 50 BETWEEN 20 AND 500. 100
DIVIDED BY FIVE, 100 TIMES FIVE
SO WILL IS AFIELD FOLD GRAY ZONE
FOR ANY GIVEN COMPOUNDS. THAT
IS HUGE, THREEFOLD CANNING BIG
DEPENDING ON OBJECTIVE OF THE
SCREENING ASSAY. THIS IS WHY I
SAID GETS DIRECTLY TO HEART OF
THE PROBLEM WHICH IS HOW WELL
HOW CONFIDENT AM ID IN SAYING
ONE IS BETTER THAN THE OTHER AND
HOW CONFIDENT AM I IN RANK
ORDERING THESE COMPOUNDS BASED
ON THIS DATA. SO THAT IS WHY MSR
IS AT THAT PARTICULAR CORE
QUESTION. OBJECTIVE OF SCREENING
DATA. Z PRIME DOESN'T. SO Z
PRIME TELLS YOU DIFFERENTIATE
ACTIVE FROM INACTIVE, WE HAVE
GONE PAST THAT WHEN YOU ARE
LOOKING AT POTENCY DATA AND
MAKING COMPARISONS IN TERMS OF
POTENCY VALUES. THAT IS WHY THIS
MSR READS SO THE QUESTION WE GET
IS CALCULATE MSR, WHAT ARE WE,
TWO, THREE, FIVE, WHATEVER? LIKE
Z PRIME OF .7 OR 1 POINT 3, YOU
SAY METRIC THAT REFLECT IT IS
QUALITY OF HDS FROM ALL THE
DIFFERENT COMPOUNDS THAT HAVE
TESTED IN HDS. SIMILARLY, MSR WE
ARE SAYING WHAT ARE WE
CALCULATING IT OUT TO BE, 2, 3,
5, THAT VALUE REPRESENTS
ROBUSTNESS OF DIFFERENTIATING
ANY TWO POTENCY RISK SALES FOR
ANY PAIR OF COMPOUNDS, HIGH LIPO
TENT COMPOUND OR EXTREME LIPO
TEN OR LESS POE TEN COMPOUNDS.
HOW CAN YOU SAY THAT WHEN
VARIABILITY OF IC 50 DEPENDS ON
IC 50. THIS IS A GRAPH HERE IS
IC 50 OF BATCH OF COMPOUNDS
AGAINST VARIABILITY OF IC 50.
YOU CAN SEE HOW VARIABILITY IS
NOT CONSISTENT ACROSS THE RANGE
OF COMPOUNDS. THIS IS TYPICAL,
THIS IS NOT THIS PARTICULAR
ASSAY ALONE. THIS IS NOT ANY
ASSAY WHEN YOU CORRELATE
COMPOUND VARIABILITY OF IC 50
THIS IS TREND YOU SEE,
VARIABILITY GOES UP AS IC 50
GOES UP SO CALCULATE A SINGLE
METRIC LIKE MV THAT REPRESENTS
ALL THESE COMPOUNDS. THE ANSWER
LIES HERE. IN THE LOG SCALE,
VARIABILITY IS UNIFORM. THIS IS
THE SAME DATA BUT IT IS MEAN LOG
IC 50 AGAINST VARIABILITY
STANDARD DEVIATION OF LOG IC 50.
YOU CAN SEE CONSISTENT ACROSS
ENTIRE RANGE. THIS IS TRUE FOR
ANY POTENCY DATA, ANY MEASURE,
CANI IC 50 EC 50. SO THAT IS WHY
ANALYSIS IS DONE IN TERMS OF THE
LOG POTENCY VALUES. THAT IS
WHAT MAKES THIS METRIC
REPRESENTATIVE OF ALL THE
COMPOUNDS FROM THAT PARTICULAR
ASSAY. AND WE HAVE Z PRIME
REPRESENTS THE BILLIONS OF
COMPOUNDS TESTED IN HDS, WHEN
YOU CALCULATE REPRESENTS ALL THE
COMPOUNDS, HUNDREDS OF THOUSANDS
OF COMPOUNDS TESTED IN THAT
SCREENING ASSAY. SO THEREFORE
MSR CALCULATIONS IS BASED ON LOG
POTENCY INSTEAD OF ORIGINAL TO
ENSURE VARIABILITY ACROSS ALL
COMPOUNDS, THE FORMULA IS
DERIVED FROM THE 95 PURCHASE
CONFIDENCE INTERVAL CONCEPT. SO
95% CONFIDENCE INTERVAL FOR
DIFFERENCE OF ANY TWO LOG
POTENCIES IS BASICALLY LOOKS
LIKE THIS. THIS IS Y 1 MINUS Y 2
DIFFERENCE OF TWO LOG IC 50s,
TO DEFINE 95% CONFIDENCE
INTERVAL FOR THE DIFFERENCE OF
LOG POTENCIES. THIS FACTOR IS
WHAT WE CALL MINIMUM SIGNIFICANT
DIFFERENCE OF THE LOG POTENCY OF
THE TWO COMPOUNDS. IF YOU TAKE
THE ANTI-OF THAT, THAT GIVES YOU
MSR. SO DIFFERENCE OF LOG IS LOG
RATIO. YOU TAKE ANTI-LOG OF
THAT, THAT'S THE RATIO. THAT IS
WHY YOU HAVE THE RATIO. WHY MRS
INSTEAD OF MSD. IS THAT CLEAR?
OKAY. SO WHAT CRITERIA DO WE
HAVE TO APPLY HERE? BASED ON MY
EXPERIENCE, TALKING WITH VARIOUS
CHEMISTS AND BIOLOGISTS OVER
YEARS AND DIFFERENCE DISCOVERY
PROGRAMS, I FOUND GENERALLY
PEOPLE ARE HAPPY, IF LESS THAN
THREE. NO QUESTIONS ASKED
USUALLY. WHAT EVEN IS GREATER
THAN THREE THEY START GETTING
CONCERNED AND THEN DEPENDS ON
CONTEXT. IF YOU ARE DEALING WITH
A BROAD RANGE OF ACTORS OR THIS
PARTICULAR ASSAY IS PROFILING
ASSAY, NOT A PRIMARY ASSAY,
SLIGHTLY HIGHER VALUE OF MSR
3.54.5 IS ACCEPTABLE IN THOSE
CASES BUT IF THAT IS TOO HIGH,
IF YOU DON'T HAVE TIME TO
REOPTIMIZE THE ASSAY, ONE
SOLUTION IS SCREEN COMPOUNDS
MORE THAN ONCE. THAT
AUTOMATICALLY REDUCES
VARIABILITY AND THEREFORE MSR OF
SAY FOUR MAY COME DOWN TO 2.5 IF
SCREENING ALL COMPOUND TWICE.
IF LOW THROUGH PUT ASSAY, CELL
BASED ASSAY, TAKES A LONG TIME
TO GENERATE THE DATA, WE CANNOT
SCREEN COMPOUNDS TWICE OR TWO OR
THREE TIMES THROUGH PUT SO THAT
CASE BEST SOLUTION IS REOPTIMIZE
TO IMPROVE VARIABILITY SO BOTH
SCENARIOS IN SOME THEY SAY QUICK
SCREEN AT LEAST SUBSET OF
COMPOUNDS MORE THAN ONCE AND GET
MSR LOW AND SATISFACTORY AND
MOVE ON. I HAVE ALSO SEEN THE
OTHER EXTREME, THE CELL BASED
ASSAY TAKES A LONG TIME GENERATE
SO BACK TO DRAWING BOARD SPEND
WEEKS TO REOPTIMIZE, BROUGHT
VARIABILITY DOWN AND MSR CAME
FROM POSITIVE 6 WHATEVER TO 2.5,
2.7 AFTER OPTIMIZE ASSAY OR
CHANGE FORMAT AND CELL LINES AND
DEPENDING ON THE CONTEXT YOU
HAVE TO WORK AROUND WITH IT TO
GET THE DATA YOU WANT. THIS IS
WHAT HAPPENS IF YOU SCREEN MORE
THAN ONCE, IF YOU GET FROM A
SINGLE SCREEN, IF YOU SCREEN
COMPOUNDS MORE THAN ONCE SAY
TWICE, AUTOMATICALLY COMES DOWN
TO 3.96 FROM 7 TO 4. SCREEN
THREE TIMES COMES DOWN TO THREE
AND PLATEAUS AFTER A WHILE. SO
NOT MUCH GAIN AFTER CERTAIN
WHILE. COMES DOWN TO 2.2, THEN 2
THEN PLATEAUS. YOU CAN HAVE MSR
OF TEN BUT COMPOUNDS FOUR TIMES
BRING IT DOWN TO ALMOST THREE.
DOESN'T MEAN SCREEN ALL
COMPOUNDS THAT MANY TIMES ONLY
ONES THAT ARE INTEREST TO YOU
THAT MEET ACTIVITY CRITERIA, IF
IT IS ONE MICROMOLAR MSR IS
FOUR, YOU CAN SCREEN ALL THOSE
COMPOUNDS THAT HAVE/LESS THAN
FOUR MICROMOLAR MORE THAN ONCE.
SO FOR THOSE COMPOUNDS, ACTIVITY
LESS THAN 4 MICROMOLAR YOU CAN
-- IF YOU SCREEN MORE THAN ONCE,
MSR 2.7 TO INTERPRET THE DATA
FROM THOSE COMPOUNDS. THE KEY
PART OF THE FORMULA HERE IS
DIVIDING BY SQUARE ROOT OF N.
THIS N HERE IS NUMBER OF TIMES
SCREEN COMPOUNDS. THAT IS WHY
ABLE CALCULATE ALL THE SCENAR
SCENARIOS. SO PLUCK THE VALUE N
OF TWO, THREE, FOUR, WHATEVER N
YOU LIKE AND THAT IS HOW YOU
GENERATE MSR VALUES. SO YOU CAN
SIGH BEFORE YOU SCREEN COMPOUNDS
YOU CAN GET IDEA WHAT MSR AND
ALSO MAKE A DECISION HOW MANY
TIMES YOU SHOULD SCREEN
COMPOUNDS TO GET ACCEPTABLE MSR
SO GREAT WAY TO FIGURE OUT HOW
MANY TIMES SHOULD I DO IT. I
HAVE SEEN THE SCENARIO ONCE BY
DEFAULT THE PROGRAM DISCOVERY
PROGRAM WAS ASKED TO SCREEN ALL
THE COMPOUNDS THREE TIME,
WITHOUT ANY RATIONALE, OBJECTIVE
RATIONALE, JUST EXPERIENCE,
BIOLOGIES DO IT THREE TIMES.
BIOLOGISTS SCREEN COMPOUNDS
DIDN'T FEEL GOOD, CAME TO ME AND
SAID THIS DATA IS ROBUST, BUT I
SUGGEST ALL COMPOUNDS THREE
TIMES DOES IT MAKE SENSE, COME
UP WITH RATIONALE FOR THAT? SO
WE CALCULATED THE MSR, TO THE N
OF ONE, SCREEN COMPOUNDS ONLY
ONE, MSR IS 2.1. IF YOU SCREEN
TWICE IT COMES DOWN TO 1.8, 1
POINT THREE TIMES, AROUND 1.6,
SO NOT MUCH TO BE GAINED FINAL
SCREENING ONCE VERSUS WISE
TWICE, CERTAINLY NOT MUCH GAINED
FROM THREE TIME TIMES. SO HE USS
DATA TO CONVINCE COLLEAGUES TO
SAY LOOK GUYS REDUCING THROUGH
PUT THREEFOLD GENERATING THIS
DATA OVER THREE REPLICATES.
LET'S DO IT ONCE, AND LIMIT
SECOND TESTING TO ONLY THOSE
COMPOUNDS INTERESTING SAY LESS
TAN 100 NANOMOLAR ACTIVITY. REST
OF THE TEAM AGREED. THIS IS HOW
YOU GO ABOUT JUSTIFYING, ANOTHER
QUESTION THAT COMES UP IS DO TEN
POINT DOSE RESPONSE CURVE, 8
POINT, 12 POINT DOSE RESPONSE
CURVE? ? YOU CAN GENERATE DATA
BEFORE YOU SCREEN THE COMPOUND
YOU CAN GENERALOR GENERATE --
RUN A BATCH OF COMPOUNDS USING
DIFFERENT CONCENTRATIONS, 8
POINT FORMAT, 15 POINT FORMAT
AND CALCULATE MSR FOR THE
DIFFERENT SCENARIOS. THE M SR
FOR THE 8 POINT FORMAT IS GOOD
ENOUGH YOU DON'T HAVE TO OVERDO
IT. THIS CONCEPT IS POWERFUL FOR
THESE POSITIONS EARLY ON AS
WELL. BUT OF COURSE LATER ON
THIS IS CONTROL CONTROL TRICK TO
KEEP CONTROL OF THE QUALITY OF
THE ASSAY. SO DIFFERENT WAYS OF
ESTIMATING, ONE IS TEST FREE
TEST MSR. BEFORE YOU SCREEN
COMPOUNDS YOU CAN RUN IT TWICE
AND THESE CAN BE SOME TEST
COMPOUNDS THAT YOU HAVE, YOU
HAVE FOUR OR FIVE COMPOUNDS YOU
CAN WORK WITH YOU CAN CREATE
REPLY CASE OF COMPOUNDS AND
PRE-SELECT DIFFERENT COMPOUNDS
SO YOU HAVE 20 OR 30 COMPOUNDS
FROM 405 COMPOUNDS AND TEST THEM
OVER TWO RUNS AND GENERATE --
USE THOSE DATA TO CALCULATE TEST
FREE M,R AND THAT GIVES YOU A
GOOD FEEL HOW IT WILL BE DOWN
THE ROAD, AND IF GOOD MUST HAVE
YOU CAN SCREEN COMPOUNDS IN THE
FULL MODE. THEN YOU HAVE A
MOVING MSR BASED ON POSITIVE
CONTROL AND WE HAVE
RETROSPECTIVE MSR WHERE WE USE
ALL THE DATA TESTED AND
COMPOUNDS TESTED FOR PERIOD OF
TIME INCLUDING THE POSITIVE
CONTROL AS WELL AS ALL TEST
COMPOUNDS YOU RUN OVER PERIOD OF
TIME AND THEN CALCULATE WHAT MSR
IS FOR ALL THOSE DATA. SCENARIOS
DEPENDING ON THE APPLICATION.
TEST FREE TEST AS MENTIONED
EARLIER, SMALL BATCH OF
COMPOUNDS TESTED TWICE, CAPTURES
WITHIN RUN VARIABILITY BECAUSE
TWO RUNS BUT EARLY FILTER TO
MAKE A DECISION WHETHER TO START
FULL SCREENING OF COMPOUNDS.
BECAUSE IF THIS MSR IS BAD THERE
IS NO POINT STARTING THE SCREEN.
IF YOU START THE SCREEN YOU HAVE
INTERPLATE VARIABILITY AND IT
WILL GET WORS WORSE. O SO THIS A
QUICK FOR DECIDING WHETHER START
SCREENING OR NOT. THERE IS A
TOOL AVAILABLE FOR CALCULATING
THAT. HERE IS OUTPUT FROM TEST
FREE TEST DATA SO WHAT WE HAVE
IS RATIO OF POTENCY RESULTS
AGAINST THE AVERAGE POTENCY. AND
THIS IS A MEAN RATIO OF ALL
THOSE COMPOUNDS THAT WERE
TESTED, 95% CONFIDENCE INTERVAL
WITH LIMITS OF AGREEMENT
PROVIDED AND MSR VALUE 8.54. H
IS 3.54 HERE MOSTLY WITHIN
VARIABILITY. IF YOU -- IF YOU
WON'T FOLLOW-UP WITH SCREEN AN
SCREEN ALL YOUR COMPOUNDS WITH
THIS ASSAY, IT IS GOING TO GET
WORSE. SO YOU MAY GO BACK TO
IMPROVE ASSAY BEFORE YOU SCREEN
COMPOUNDS THIS IS GREAT FILTER
FOR DECIDING WHETHER TO MOVE
FORWARD OR NOT WITH THE SCREEN
BEFORE YOU REALIZE TOO LATE. YOU
CAN USE THE DATA TO DECIDE HOW
MANY TIMES TEST COMPOUNDS, SO
THIS 3.54 BUZZ BASED ON SINGLE
RUN OF COMPOUNDS, IF YOU ARE --
DECIDE TO TEST COMPOUNDS TWICE
YOU CAN PLUG IN THE VALUE IN
THAT FORMULA I SHOWED YOU
EARLIER, PLUG IN THE VALUE N
EQUAL TO TWO AND 3.54 COME DOWN
TO 2.5. SO IF THAT IS WHAT YOU
DECIDE TO DO, THAT IS REASONABLE
AND YOU MIGHT SAY, AT LEAST FOR
THOSE COMPOUNDS THAT MEET
ACTIVITY CRITERIA, SCREEN THEM
TWICE, ACCEPTABLE MSR 2.5 AND
SEE HOW IT WORKS OUT DURING YOUR
SCREEN. QUICK ILLUSTRATION HOW
TO CALCULATE IT SO TEST FREE
TEST DATA BATCH OF COMPOUNDS
TEST TWICE, TAKE LOG VALUES OF
THOSE TWO. TAKE DIFFERENCE OF
THE LOGS. THEN TAKE STANDARD
DEVIATION OF DIFFERENCES OF THE
LOGS. AND PLUG INTO THE FORMULA
AND GET THE MSR. THIS CONCEPT
OF COMPARISON BATCH OF COMPOUNDS
IS USEFUL WHEN TRANSFERRING THE
ASSAY FROM ONE LAB TO ANOTHER, A
PRACTICAL SITUATION WHERE ASSAY
DEVELOPED ONE PLACE AND GETS
MOVED TO DIFFERENT PART OF THE
COMPANY OR MOVE TO CRO WHO TESTS
THE CON POUNDS. IF YOU RUN ASSAY
SUCCESSFULLY OR MAKE IMPORTANT
CHANGE TO REAGENT OR FOR MT. OF
ASSAY, THIS TEST FREE EXPERIMENT
I MENTIONED, ALL METRICS THAT I
REVIEWED, CAN BE VERY USEFUL FOR
THOSE PURPOSES. TO GET A BETTER
ESTIMATE OF MSR WE WANT TO
CAPTURE ENTERAND
INTRAVARYIBILITY. ONE WAY TO DO
THAT IS USE POSITIVE CONTROL
DATA OVER PERIOD OF TIME AND
CALCULATE WHAT WE CALL MOVING
MSR AND AFTER A WHILE USE
SCREENING DATA ITSELF TO
CALCULATE PERSPECTIVE MSR. SO
REVIEW WHAT MOVING MSR IS. MSR
AS IT WORKS YOU ARE CALCULATING
POSITIVE CONTROL MSR IN
INCREMENTS OF SIX RUNS USING THE
FIRST SIX RUNS CALCULATE THE
VARIABILITY OF POSITIVE CONTROL
IC 50 AND MSR TO SIX RUNS AND
RUNS TWO TO 7, THREE TO 8, SO
FORTH IN SLIDING INCREMENTS.
THAT IS WHY YOU HAVE THE MOVING
MSR AND YOU CAN BULK THE DATA
OVER TIME TO SEE HOW THE
VARIABILITY IS CHANGING OVER
TIME. HERE IS I WILL STRAIKS OF
THAT. THIS IS A POTENCY VALUE OF
DATA OF THE POSITIVE CONTROL,
PLOTTED OVER PERIOD OF TIME. WE
USE THE FIRST SIX RESULT TO
CALCULATE MSR VALUE. THAT IS
WHAT GIVES YOU THIS DOT HERE.
THEN RUNS TWO TO 7 GIVES YOU
THIS POINT HERE, RUNS THREE TO 8
GIVES YOU THE THIRD VALUE. SO
FORTH. SO EVERY DOT IS MSR
CALCULATED FOR SIX RUNS, SIX RUN
INCREMENTS OF ALL THESE POSITIVE
CONTROL IC 50 VALUES. SO REAL
TIME MONITOR HOW ASSAY BEHAVING
IN TERMS OF MSR THERE IS XL TOOL
IN THE AGM WEBSITE YOU PASTE THE
RUN DATE IN ONE COLUMN AND
CORRESPONDING IC 50 POSITIVE
CONTROL AND GIVES YOU THE MSR
VALUE, GRAPHS AND STATISTICS
NICELY. IT IS MUCH EASIER TO
FLAG ISSUES IN TERMS OF MSR THAN
POTENCY VALUE. HERE IS
ILLUSTRATION TO MAKE THE POINT.
HERE WE HAVE POTENCY DATA
PLOTTED OVER PERIOD OF TIME FROM
POSITIVE CONTROL FROM KINASE
ASSAY, LOOK AT CORRESPONDING
MOVING MSR GRAPH, FOR THE SAME
DATA, YOU CAN SEE SOME
VARIABILITY HERE BUT NOT SO
OBVIOUS THAT THERE IS REALLY BAD
OR ACCEPTABLE. LOOK AT
CORRESPONDING MSR FOR THE SAME
TIME WINDOW APRIL TO JULY, APRIL
TO JULY, IT IS MUCH WORSE IN
TERMS OF MSR, IT GOES UP FROM
THREE TO AROUND 7, 8, THEN COMES
BACK DOWN SO IN TERMS OF MOVING
MSR GIVES YOU MUCH BETTER FEEL
FOR HOW BAD HOW THE DATA IS THAN
SIMPLY LOOKING AT POTENCY VALUES
OVER TIME. THIS IS ANOTHER
EXAMPLE WITH THE SAME POINT, YOU
HAVE VARIABILITY TO START WITH
BUT IN TERMS OF MSR YOU GET A
APPRECIATION HOW BAD IT IS, SO
AROUND TWO, THREE, FOUR AND GOES
UP TO 12, 14, AFTER ISSUE IS
FIXED COMES DOWN TO AROUND 2.
ANOTHER ISSUE THAT COMES UP
LATER ON, WHERE YOU CAN SEE THE
DATA POTENCY VALUE GOING UP,
CORRESPONDING MSR IS ALSO VERY
HIGH. SO HELPS US TO MONITOR THE
QUALITY OF THE ASSAY OVER TIME
CONSISTENT POTENCY VALUES ARE
FOR POSITIVE CONTROL OVER TIME
AND HOW CONSISTENT MSR VALUES
ARE OVER TIME. AND IT IS
IMPORTANT TO DOCUMENT WHAT ARE
CHANGES HAPPENING IN THE ASSAY,
IMPORTANT TO DOCUMENT THAT AS
AND WHEN THEY HAPPEN. BUFFER
CHANGES OR REAGENT CHANGES OR
INCUBATION TIME OR OTHER
CONDITIONS ASSAY CHANGE. MAKE A
NOTE OF IT IN A TRACKING SHEET
AND LOOK AT CORRESPONDING MOVING
MSR VALUE, THE MOVING MSR GRAPH
YOU ARE TRACKING. IF IT LOOKS
LIKE THIS, AND MAKE A CHANGE IN
THESE NOT ONLY SEE MSR GO UP YOU
KNOW CHANGE IT WAS BEFORE CHANGE
REAGENT CHANGE THAT IS NOT A
GOOD THING. SO YOU HAVE TO GO
BACK AND SEE IF YOU CAN RESOLVE
THAT. THE THIRD MSR IS BASED ON
ALL DATA FROM SCREENING ASSAY.
AFTER FEW MONTHS GO BACK TO DATA
SET AND PULL OUT POSITIVE
CONTROL DATA AND ALL TEST
COMPOUNDS TESTED TWO OR MORE
TIMES. AGAIN ALL THE DATA IS
RUN THROUGH MIXED FX NOVA
NORMALIZING COMPOUND TO COMPOUND
DIFFERENCE, WHAT WE ARE LEFT
WITH IS VARIABILITY OF THE
SCREENING DATA AFTER NORMALIZING
COMPOUND DIFFERENCE. THAT IS
CAPTURED IN STUDENT VISUALS AND
PLOTTED OVER TIME. THIS IS THE
DATA ON SCREENING ASSAYS,
VARIABILITY OVER PERIOD OF TIME
FROM TESTING COMPOUND AND THIS
IS USED TO CALCULATE THE MSR.
THIS GIVES YOU GOOD FEEL OVERALL
VARIABILITY ASSAY AND TAKING ALL
THE DATA INTO ACCOUNT. SO
QUESTION THAT COMES UP WITH IS
IT REALLY NECESSARY TO USE ALL
THE DATA TO CALCULATE THE MSR.
OR ADEQUATE TO USE POSITIVE
CONTROL TO CALCULATE THE MSR? TO
ANSWER THIS QUESTION WE LOOK AT
TEN DIFFERENT ASSAYS, FIVE
KINASE ASSAYS AND CELL BASED
ASSAY AND BINDING ASSAY, MSR
FROM POSITIVE CONTROL THAT I
MENTION CALCULATEK SIX RUN
INCREMENTS YOU CALCULATE THE
POSITIVE CONTROL CELL OVER TIME
PERIOD TWO YEARS OR HOWEVER LOCK
SCREEN ASSAY IS. WE USE FOR
TESTING COMPOUNDS. CALCULATE THE
MSR FROM ALL THE POSITIVE
CONTROL RESULTS OVER PERIOD OF
TIME AND CALCULATE THE
CORRESPONDING MSRR. COMPARE THAT
TO RETROSPECTIVE MSR, AND TURNS
OUT IN ALL CASES WE HAVE SEEN
THEY TRACK WELL WITH THE MSR
CALCULATED USING ALL THE DATA
FROM THE TEST COMPOUNDS AS WELL
AS POSITIVE CONTROLS. SO THIS IS
FANTASTIC. THIS MEANS
VARIABILITY CALCULATE IN TERMS
OF POSITIVE CONTROL, IS PRETTY
GOOD REPRESENTATION OF THE
VARIABILITY YOU HAVE FROM ALL
THE DIFFERENT COMPOUNDS IN
SCREENING ASSAY. POSITIVE
CONTROL HAS TO BE THE ONE MOST
LOW IC 50 VALUE, LOW -- HIGH
LIPO TENT. YOU MAY WONDER DOES
IT PRESENT VARY -- HIGHLY
POTENT. YOU CAN SEE FROM THE
ASSAYS EVALUATED. THEY ARE IN
THE BALLPARK, RIGHT IN THE
MIDDLE MOST CASES. SO IN FACT
THIS MADE US FEEL SO GOOD ABOUT
IT THAT WE REDUCED THE FREQUENCY
RETROSPECTIVE MSR
A. CXFC DO IT EVERY FIVE MONTHS
AND MAKE SURE POSITIVE CONTROL
MSR IS CALCULATED AND KEEP TRACK
OF MOVING MSR OVER PERIOD OF
TIME WHEN WE GENERATE DATA. THIS
COME SEPTEMBER OF MSR IS
EXTENNED TO SO FAR WE TALKED
ABOUT COMPARING HOW RELIABLE YOU
CAN DIFFERENTIATE POTENTIAL
CELLS ANY PAIR OF COMPOUNDS IN
THE SCREENING ASSAY. THE OTHER
IMPORTANT METRIC IS SELECTIVITY
RATIO, SO YOUR NOT ONLY
INTERESTED IN ACTIVITY OF
AGAINST PRIMARY TARGET BUT MAKE
SURE COMPOUNDS DON'T BIND OR NOT
ACTIVE AGAINST OFF TARGET SO
SELECT RASH YES REFLECTS THE
RATIO OF POTENCY TO PRIMARY
TARGET VERSUS POTENCY AGAINST
THE OFF TARGET. IF YOU WANT
SELECTIVITY RATIO FOR ALL
COMPOUNDS SELECTED FROM OFF
TARGET, BUT THERE WE HAVE EVEN
MORE VARIABILITY. WE HAVE
VARIABILITY PRIMARY ASSAY,
PRIMARY TARGET ASSAY AND
VARIABILITY OF THE OFF TARGET
ASSAY. PROPAGATION OF ERROR AS
YOU ADD MORE DATA TO THE
DIVISION. SO THERE IS A SIMPLE
EXTENSION OF MSR IN SELECTIVITY
RATIO APPLICATION. BECAUSE MSSR
MSR SR STANDS FOR MINIMUM
SIGNIFICANT SELECTIVITY RATIO
AND MSR SR IS MINIMUM
SIGNIFICANT RATIO OF SELECTIVITY
RATIOS, VERY EASY TO CALCULATE.
USING JUST THE MSR YOU CALCULATE
FROM THE PRIMARY ASSAY, THE OFF
TARGET YOU CAN CALCULATE
METRICS. HERE IS MSSR THIS IS
FOR ASSESS WHETHER COMPOUND IS
DIFFERENT ON TWO TARGETS PRIMARY
TARGET OFF TARGET MSSR PRESENTS
SMALL CHANGE FROM POTENCY TO
PRIMARY TO PROTEN SHY TO OFF
TARGETOR TARGET. SO THIS CALLS
FOR PLUGGENING MSR VALUE FOR THE
ASSAY, ASSAY MSR EQUATION, THAT
GIVES YOU THE MSSR VALUE. MSSR
PRIMARY IS 2.5 AND FOR
SELECTIVITY ASSAY IS THREE. PLUG
THOSE INTO THE FORMULA, MSSR IS
2.75 SO HOW WE USE IN PRACTICE.
SUPPOSE CRITERIA IS 100 FOLD YOU
WANT COMPOUNDS 100 FOR SELECTIVE
TO THE PRIMARY TARGET AND
DURATION OFF TARGET. WE TALKED
ABOUT VARIABILITY. QUITE A LOT.
BOTH ASSAYS NOW AND WE TAKE
VARIABILITY IN BOTH ASSAYS INTO
ACCOUNT. THE MSSR IS 2.75, WHAT
DOES IT MEAN IN TERMS OF HUNDRED
FOLD CRITERIA? MEANS THAT I NEED
TO SEE AT LEAST 275 FOLD
SELECTIVITY TO BE 95% CONFIDENT
THAT I HAVE 100 FOLD
SELECTIVITY. IF THAT IS TOO MUCH
TEST COMPOUNDS TWICE AND BRING
MSSR DOWN TO 2.2 OR 2. THAT
MEANS IF CRITERIA IS 100 FOLD
SELECTIVITY YOU NEED AT LEAST
200 FOLD SELECTIVITY TO BE 95%
CONFIDENT YOU HAVE 100 FOLD
SELECTIVITY. OTHER THINGS ALSO
WHAT IF I HAVE FIVE FOLD
SELECTIVITY? DOES THAT MEAN I
SHOULD MOVE COMPOUND FORWARD?
THESE ARE BASED ON 95%
CONFIDENCE. SO WE CAN REVERSE
ENGINEER THIS AND CALCULATE WHAT
LITTLE CONFIDENCE CAN YOU HAVE
WITH COMPOUND THAT HAS 150 FOLD
SELECTIVITY. IT DOESN'T HAVE THE
275 FOLD SELECTIVITY BUT 150
FOLD SELECTIVITY, HOW MANY
CONFIDENCE DO I HAVE? I DON'T
HAVE 95% CONFIDENCE BUT DO I
HAVE 75%? THAT IS COVERED IN ONE
OF THE PAPERS I CITED IN THE
FIRST SLIDE, THE THIRD CITATION
OF THE PRESENTATION. THAT
PUBLICATION WE GO OVER THAT IN
DETAIL HOW TO CALCULATE
VARIABILITY OF COMPOUND BEING
MORE HOE SELECTIVITY OF RATIO
SIGNIFICANTLY HIGHER THAN THE
HUNDRED FOLD SELECTIVITY AS IN
THIS CASE. SO WE DONE HAVE TO
BLINDLY APPLY CONCEPTS IN TERMS
OF 95% CONFIDENCE YOU CAN
CALCULATE THE CONFIDENCE LEVEL
ITSELF FOR A GIVEN COMPOUND IF
IT DOESN'T MEET MSSR CRITERIA.
EXTENDING FURTHER NOW IF YOUR
HONOR PROGRAM CALLS FOR
COMPOUNDS HAVING BETTER
SELECTIVITY THAN POSITIVE
CONTROL, SUPPOSE POSITIVE
CONTROL HAS 50 FOLD SELECTIVITY
AGAINST OFF TARGE TARGET. WE WT
TEST COMPOUNDS MOVE FORWARD TO
BEAT POSITIVE CONTROL HAVING
MORE THAN 50 FOLD SELECTIVITY
RATIO THIS HAS. BETWEEN TEST
COMPOUNDS OR POSITIVE CONTROL
AND ALSO TEST COMPOUNDS. FOR
THAT WE NEED MSR SR AND WE PLUG
IN THOSE TWO VALUES AS IN THE
PREVIOUS SLIDE WITH A SLIGHTLY
DIFFERENT FORMULA. PURPOSE TO
COMPARE SELECTIVITY RATIOS. FOR
EXAMPLE, MSR PRIMARY ASSAY IS
TWO, OFF TARGET MSR IS THREE.
MSR IS 3.67. WE WANT COMPOUNDS
TO BE AT LEAST 50 FOLD SELECTED
THAT MEANS AT LEAST 180 FOLD
SELECTIVITY TO BE 95% CONFIDENCE
FOR 50 FOALED SELECTION. THIS
SEEMS LIKE A BIG NUMBER BUT IF
YOU TEST COMPOUND MORE THAN ONCE
WE CAN BRING THE VALUE DOWN AND
CALCULATE THE CORRESPONDING MSR
SR. WHEN YOU HAVE TWO
COMPOUNDS, ONE SELECTIVITY RATIO
OF 17, ANOTHER COMPOUND
SELECTIVITY RATIO OF 73. SO THIS
SECOND COMPOUND IS FOUR FOLD
MORE SELECTIVE THAN THE FIRST.
IF YOU TESTED ONLY ONCE, THE MSR
SR IS 5.6 SO NOT GOOD ENOUGH.
BUT IF YOU TESTED THESE
COMPOUNDS TWICE 5.6 COMES DOWN
TO 3.3. SO WE HAVE THE
CONFIDENCE THAT THESE COMPOUNDS
IS MUCH MORE SELECTIVE THAN THE
FIRST COMPOUND. SO THIS HELPS
DECIDE HOW MANY TIMES TO TEST
THE COMPOUNDS AND HOW WELL
DIFFERENTIATE THESE COMPOUNDS.
IN SUMMARY YOU HEARD Z PRIME IN
DETAIL OVER THE PREVIOUS
PRESENTATION AN HEARD HOW TO
EXTEND THE Z PRIME FACTOR TO
CONTEXT OF POTENCY DATA MAKING
IMPORTANT DECISIONS IN TERMS OF
ONE COMPOUND BETTER THAN THE
OTHER IN TERMS OF POTENCY VALUES
OR SELECTIVITY RATIOS WE HAVE
DIFFERENT TYPE OF M SR YOU CAN
CALCULATE. WE ALSO TALKED ABOUT
HOW WE CAN MONITOR THE QUALITY
OF ASSAY IN REAL TIME USING
POSITIVE CONTROL DATA AND NOT
JUST LOOKING AT POSITIVE POTENCY
OVER TIME BUT LOOKING AT THE
VARIABILITY MILLION TERMS OF MSR
POSITIVE CONTROL OVER TIME
MAKING OBJECTIVE POSITION ON IS
THERE CHANGE IN REAGENT OR ASSAY
MEANINGFUL, DOES IT REDUCE
QUALITY OF ASSAY SO IT GETS ALL
THAT AND WE TALKED ABOUT HOW TO
EXTEND THE CONTEMPT IN TERMS OF
RATIO AS WELL. SO WITH THAT I'LL
STOP. THANK YOU VERY MUCH.
>>THAT WAS A FANTASTIC TALK.
THE Z PRIME FACTOR EQUATION,
DESCRIBED THE SEPARATION BETWEEN
HIGH AND LOW CONTROLS. IT IS TC
SHOWED US WHEN WE LOOK AT THE
EQUATION, IT IS ANATOMY TELLS
EXACTLY HOW TO DEVELOP A HIGH
QUALITY ASSAY TO IDENTIFY HITS,
RIGHT? WE WANT TO MAXIMIZE THE
SIGNAL TO BACKGROUND, WE WANT TO
MINIMIZE THE VARIABILITY. I KNOW
YOU HAVE HAD EXPERIENCES OVER
THE YEARS WORKING WITH ASSAY
BIOLOGISTS, ON THE MSR WHICH
HELPS PEOPLE DEVELOP A GOOD
ASSAY COMPARE POTENCIES. THE
MSR UNLIKE Z FACTOR TELLS YOU
SEPARATION BETWEEN HIGH AN LOW
CONTROLS, THE MSR TELLS YOU
VARIABILITY OF POTENCY. BUT IT
IS LESS CLEAR FROM EQUATION WHAT
WE CAN DO IS AS BIOLOGIST TO
MAKE THE BEST POSSIBLE ASSAYS TO
OPTIMIZE POTENCY MEASUREMENTS OR
REDUCE VARIABILITY OF
MEASUREMENTS. WERE YOU ABLE TO
LEARN ANYTHING FROM ASSAY
BIOLOGIST COLLEAGUES ABOUT
THINGS THAT HAVEN'T THOSE ASSAYS
TO REDUCE MSR?
>>GREAT QUESTION. WE TALKED Z
PRIME OF .7 DOESN'T MEAN MSR OR
2 OR VERY GOOD MSR VALUE SO IF
YOU OPTIMIZE THE ASSAY IN TERMS
OF JUST Z PRIME DOES IT
GUARANTEE GOOD ASAYS AND HOW DO
YOU WORK AROUND THAT AND
OPTIMIZE ASSAY TO MAKE SURE YOU
HAVE GOOD MSR. THAT IS WHAT YOU
ARE ASKING. SO ACTUALLY HAD
SITUATION A NUMBER OF TIMES
WHERE THE MSR WAS BAD, EXAMPLE
WAS BRIEFLY MENTIONED ABOUT
EARLIER FOR CELL BASED ASSAY, I
CAN'T TELL THEM SCREEN TWO OR
THREE TIMES FOR LOW THROUGH PUT
ASSAY, YOU WANT TO REOPTIMIZE IT
YOU HAVE TO A MULTI-FACTOR DOE
APPROACH TO OPTIMIZE THE ASSAY.
THE CRITERIA THAT WE USE THERE,
THE REASON FIRST OPT MICE
BECAUSE MSR WAS AROUND 6 OR 7.
SO THEY WANT TO BRING DOWN TO
LESS THAN THREE. WHAT WE DID WAS
SET UP THE DESIGN IN SUCH A WAY
DOE IN A WAY TO GENERATE DATA
NOT JUST LOW AND HIGH BUT TWO
OTHER CONCENTRATIONS IN BETWEEN
BACKGROUND, ONE LEVEL OF LOW,
AND MID LEVEL, AND ANOTHER LEVEL
BETWEEN MID AND HIGH. THEN HIGH
THEN GENERATE REPLICATES AT EACH
OF THOSE THEN WE CALCULATE THE
VARIABILITY AROUND EACH PAIR
BACKGROUND TO LOW, BACKGROUND TO
MID, BACKGROUND TO MODERATE AND
BACKGROUND TO HIGH. AND WE USED
ALL THESE MEASURES IN THE DOE
ANALYSIS TO FIGURE OUT THE BEST
ASSAY CONDITIONS THAT WILL GIVE
YOU GOOD SEPARATION ACROSS ALL
THE WINDOWS THAT WE HAVE. FROM
DIFFERENT CONCENTRATIONS. THAT
WILL GET YOU CLOSER TO HAVING
REASONABLY GOOD DATA ACROSS THE
ENTIRE RANGE CONCENTRATION
RESPONSE CURVE. WE DON'T KNOW WE
HAVE ONE COMPOUND IC 50 COULD BE
DOWN HERE, ANOTHER COMPOUND IC
50 COULD BE UP HERE. WE WANT TO
MAKE SURE THE ENTIRETY ENTIRE
AGENT COURSE RESPONSE -- DOSE
RESPONSE CURVE WE HAVE
REASONABLY GOOD DATA WITH LOW
VARIABILITY AND ROBUST SIGNAL.
THIS IS A WAY THE
>>ABOUT IT. TO OPTIMIZE
QUALITY AT DIFFERENT SECTIONS OF
THE POSITIVE CONTROL
CONCENTRATION RESPONSE CURVE.
THAT WAY YOU CAN HOPE THAT
EVENTUALLY BEST ASSAY CONDITION
YOU COME UP WITH THAT OPTIMIZES
IT WILL GIVE YOU ROBUST IC 50
DATA. OBVIOUSLY IT WAS
SUCCESSFUL WE BROUGHT DOWN MSR
IN ONE CASE FROM CIRCUMSTANCES
OR SEVEN DOWN TO THREE OR FOUR.
OBVIOUSLY WE CAN ONLY DO BETTER.
CAN'T DO WORSE WORSE BUT THIS IY
TO GO AROUND CHALLENGE OF
RELIABLE IC 50 VALUES BUT WORK
DIFFERENT SEGMENTS OF THE
CONCENTRATION RESPONSE CURVE NOT
JUST HIGH AND LOW.
>>I THINK THIS MAYBE A GOOD
TIME TO ASK ABOUT BEST PRACTICE
FOR IC 50. I SEE SOME ISSUES,
SOMETIMES THE CURVE REACH 60%,
HOW DO YOU FIX THE BEST IC 50? I
HEARD SOME USE 100% MANDATORY TO
100 SOME USE AOC. SO THAT IS MY
FIRST QUESTION. MY SECOND
QUESTION, SOMETIMES ALSO THE
CURVE SEEMS TO DRIFT AND MINUS
20, IN THAT CASE WHAT WILL BE
THE BEST WAY?
>>PROBABLY ASKING ABOUT THOSE
ASSAYS WHERE YOU DON'T HAVE A
CLEAR IC 50 WHERE YOU DON'T HAVE
A CLEAR MAX. WE HAVE SOME
PERCENT EFFICACY.
>>SAY WE DO HAVE A GOOD
POSITIVE CONTROL TO GET 100%,
BUT FOR CERTAIN COMPOUNDS
DOESN'T GET --
>>SO SOME COMPONENTS YOU GET TO
70%, SOME 90, SOME 60. SO YOU
ASK HOW DO WE DEAL WITH THOSE
KINDS OF DATA AND HOW DO YOU
DEFINE THE EC 50 IN THOSE CASES?
>>COMPARE THEM.ONE IS TO HAVE
A THRESHOLD FOR ALL THE
COMPOUNDS, SO DEFINING THE EC 50
IN REALS TO THE MAXIMUM OBSERVED
-- IN RELATION TO THE MAXIMUM
OBSERVED ACTIVITY FOR THAT
COMPOUND BUT DEFINING IT AS
ABSOLUTE, ABSOLUTE 50% SCALE. SO
GIVEN COMPOUNDS MAXIMUM IS 70,
YOU CALCULATE CONCENTRATION OF
50% RAW 50% ACTIVITY, 50% OF 70
BUT 50% PERIOD. SO CALCULATE
50% A I LONG WITH AOCC. SOME MAY
NOT AGREE WITH IT BUT IF YOU
COMBINE WITH AOC, WE FIND IT TO
BE PRETTY GOOD. SO YOU CAN
CALCULATE END OF THE CURVE FOR
THE ENTIRE CONCENTRATION
RESPONSE CURVE USE AUC WITH
ABSOLUTE EC 50 TO COMPOUNDS.
(INAUDIBLE) ANOTHER WAY IS TO
CALCULATE RELATION TO MAXIMUM
OBSERVED ACTIVITY. AND THEN GO
ALONGTHAT IS THE POTENCY.
THE EFFICACY IS THE PLATEAU. IF
IT IS LIKE 50% PARTIAL AGONIST.
YOU CAN ALSO SUPER AGONIST WHICH
GOES ABOVE 100%. SO THAT IS A
PHARMACOLOGIC -- THE CASES WHERE
MATHEMATICAL CONSIDERATIONS ARE
IF YOU KNOW YOU HAVE AN
ANTAGONIST AND REFERENCE
ANTAGONIST YOU KNOW WHAT 100%
AND ASUM HUNDRED IS ANTAGONIST
AT BASELINE, WHEN IT DOESN'T GO
BASELINE IT IS NOT TYPICALLY
BECAUSE OF THE THE MAP, IT IS
BECAUSE COMPOUNDS SOLUTION,
THERE'S SOME REAL BIOLOGY TO
THAT. SO THING TO BE CAREFUL
ABOUT ARBITRARILY SAYING, 50% OF
TOTAL.
>>ARE YOU SAYING YOU PREFER
(INAUDIBLE)?
>>I'M DOING A GPCR LOOKING
FUNCTIONAL ASSAYS LOOKING EC
50s, I WOULD ALWAYS HAVE EC 50
AND A EFFICACY, TWO PARAMETER,
EFFICACY AND EC 50.
>>THAT IS WHAT I WAS SAYING SO
USE EC 50 ALONG WITH E MAX FOR
FUNCTIONAL ASSAYS. BUT I HAVE
SEEN SCENARIOS PEOPLE ARE
CONFLATE WITH THE ABSOLUTE EC
50. SO USING THAT NUMBER AND E
MAX TO GO WITH IT AND JUST
MAKING SURE COMPARING APPLES TO
AMOUNTS BY COMPARING TO THE
THRESHOLD OF 50%. AOC SEEMS TO
WORK WELL. SO WHEN DOING
MODELING, TO PREDICTION MODEL
AND REFINE SAR BASED ON THE QSAR
WHAT METRIC DO WE USE? EC 50
ABSOLUTE EC 50, E MAX? IN THAT
CASE I FOUND AOC WORKS WELL SO
WHEN YOU USE AOC AS RESPONSE TO
THEN BUILD PREDICTION MODEL QS
ARKANSAS MODEL, WE GET MORE
MEANINGFUL MODEL THAN JUST USING
A MODEL AGAINST ONE OF THE OTHER
PARAMETERS.
>>ALL RIGHT. THANK YOU VERY
MUCH, DEVAN FOR A WONDERFUL
TALK.
[APPLAUSE]
>>ALL RIGHT, EVERYONE, WE WILL
TAKE A BREAK NOW AND RECONVENE
AT 3:30. MAKE SURE YOU COME
BACK. THERE'S TWO AMAZING FINAL
TALKS TODAY. ONE IS ON DRUG
SCREENING WITH ENGINEERED TREATY
TISSUE MODELS. AND THE OTHER ONE
IS ON IN VITRO ASSESSMENT OF
PROPERTIES OF LINKED COMPOUNDS.
BE HERE AT 3:30 SO WE FINALIZE
WITH TWO AMAZING TALKS. ALL
RIGHT. HAVE A NICE BREAK.
OUR NEXT SPEAKER IS DR. MARC
FERRER, DR. FERRER IS CURRENTLY
THE DIRECTOR OF THE 3D TISSUE
BIOLABORATORY AT NCATS, A
MULTI-DISCIPLINARY GROUP
ESTABLISHING 2018 WITH GOAL OF
CREATING VALIDATING AND USING
TREATY BIOENGINEERED TISSUE FOR
DISEASE MODELING AND PREDICTIVE
DRUG DISCOVERY AND DEVELOPMENT.
HIS TALK IS ENTITLED DRUG
SCREENING WITH ENGINEERED 3D
TISSUE MODELS. THANK YOU FOR
BEING HERE. FLOOR IS YOURS.
THANK YOU FOR THE INTEREST AND
THANK YOU FOR STAYING. ALMOST TO
THE END. THE LAST TWO DAYS WE
LEARNED THAT DRUG DISCOVERY
PROBLEM YOU NEED TO HAVE A
BATTERY OF ASSAYS NOT ONLY A
PRIMARY ASSAY BUT SECONDARY
ASSAY FOR COUNTER SCREEN, FOR
READ OUTS.S. THESE NEED TO BE
ROBUST. THE FACTORS ARE MORE
THAN .5 AND MSR LESS THAN 3. SO
I WILL TRY TO CONVINCE YOU MORE
ASSAYS U YOU NEED ASSAYS THAT
ARE PHYSIOLOGICALLY RELEVANT.
THE REASON FOR THAT, WITH ALL
THESE ROBUST ASSAYS AND
BEAUTIFUL DOSE RESPONSES YOU
HAVE SEEN THESE TWO DAYS THE
RATE DRUGS GET APPROVED IS STILL
10% SO WHETHER TARGET
VALIDATION, WRONG TARGETS NOT
CLINICALLY VALIDATED OR THE
ASSAYS WE USE ARE TOO SIMPLISTIC
AND DON'T CAPTURE THE
ENVIRONMENT OF THE TARGET IN THE
ACTUAL BODY SO BEAUTIFUL TALKS
ALONG THE DAY WE USE ENZYMATIC
ASSAYS AND MANY TIMES YOU SCREEN
WITH CATALYTIC SIDE BUZZ FULL
LENGTH IS NOT MOST GPCR PROBABLY
SCREENING ARE CHOICE HEALTH SO
THAT IS NOT THE CELLS IN YOUR
BODY SO WE THINK PART OF THE
PROBLEM IS THAT BY CREATING
THESE ASAYS THAT ARE SO ROBUST,
WE MAKE COMPROMISES THE
COMPROMISE IS THEY LOSE THAT
PHYSIOLOGICAL RELEVANCE THAT WE
NEED IN THESE ASSAYS AND TOUCH
ON ASPECT OF BIOLOGYCAL
RELEVANCE BUT THE TRUTH IS THAT
BY GROWING CELLS ON GLASS NOT
PHYSIOLOGICALLY RELEVANT THE
CELLS IN BODY GROW TALKING TO
OTHER CELL TYPES, THEY GROW WITH
TYPES DIFFERENT (INAUDIBLE)
THERE'S MECHANICAL FORCES, THE
VASES QUEUE LATURE. SO SIGNALING
GENE EXPRESSION CELLS NEW TARGET
IN BODY IS PROBABLY NOT WHAT YOU
FIND IN OVEREXPRESSING THE
FAVORITE GPCR IN CELLS WHICH YOU
OPTIMIZE TO HAVE A WHOPPING
SIGNAL AND GOOD Z FACTOR AND
REPRODUCIBILITY AS YOU HEARD
BEFORE. SO WE ARE NOT THE ONLY
ONES, YOU PROBABLY HEARD ABOUT
3D MODELS TISSUE IN A CHIP,
ORGANOIDS STEROID, REALLY A
BROAD RANGE OF DIFFERENT 3D
MODELS AND THE IDEA IS THAT BY
CREATING THESE ASSAYS AND
PUTTING CELLS MORE TISSUE LIKE
DRUG RESPONSE WILL BE MORE LIKE
IN YOUR BODY. AS ALWAYS THIS
COMPROMISES WE CAN MAKE IT AS
COMPLEX AS YOU CAN OR SIMPLE AS
YOU CAN. SIMPLICITY BRINGS
THROUGH PUT WHICH IS IMPORTANT
FOR THE WORK THAT WE ARE DOING.
AS YOU INCREASE FIZZ CROW
LOGICAL COMPLEXITY, YOUR THROUGH
PUT GOES DOWN BECAUSE THESE ARE
TECHNICALLY COMPLEX TISSUE
MODELS WHERE YOU HAVE FLUIDICS,
YOU HAVE MUCH OF THE MODELS ARE
PHYSIOLOGICALLY MORE RELEVANT,
THEY ARE DEVELOPED WITH PRIMARY
CELLS, OR IPS DERIVED CELLS CELE
TRY TO AVOID THE CELL LINES THAT
WE USE IN NORMAL 2D BECAUSE
THESE CELLS HAVE PASSAGE IN
PLASTIC AND NOT WHAT YOU FIND IN
YOUR BODY. SO I WON'T GO INTO
DEAIL TO THE DIFFERENCES
SIMILARITIES PROS AND CONS, WE
HAVE A REVIEW MANY REVIEWERS BUT
WE HAD TABLE THAT EXPLAINS THE
DIFFERENCES IN SIMILARITIES WITH
DIFFERENT MODELS. WHAT IS
REALLY CRITICAL TO ALL OF THEM
IS THE QUESTION YOU ALWAYS GET,
HOW PREDICTABLE -- CLINICALLY
PREDICTIVE ARE THESE MODELS
COMPARED TO THE 2D MODEL. THIS
IS SOMETHING YOU HAVE TO KEEP IN
MIND AS YOU START DEVELOPING ANY
OF THESE 3D MODELS ORGANOIDS THE
OTHER ASPECT IS HOW MUCH
PHYSIOLOGICAL COMPLEXITY DO YOU
NEED TO INCLUDE IN THESE
MODELINGS TO HAVE THE
PREDICTABILITYIBILITY YOU ARE
LOOKING FOR FOR A SPECIFIC
DISEASE, SPECIFIC TARGET YOU ARE
INTERESTED IN. WHAT CELL TYPES
DO YOU NEED TO INCLUDE IN YOUR
MODEL? HOW YOU HAVE ALL THESE
DIFFERENT CELL TYPES AND WHAT
ARE YOU GOING TO USE, EVERY CELL
YOU BUY FROM A DIFFERENT VENDOR,
COMES WITH DIFFERENT MAGIC
MEDIA, SOMETIMES YOU DON'T KNOW
WHAT IS IN THE MEDIA. HOW YOU
BRING THESE TOGETHER MAKE SYSTEM
3DISH TISSUE SYSTEM THAT IS
FUNCTIONING BY WHATEVER CRITERIA
YOU DECIDE TO HAVE. AND AS YOU
KNOW, MANY DIFFERENT PLATFORMS,
DIFFERENT VENDORS HOW DO YOU
CHOOSE? AT THE END OF THE DAY
DECIDING BASED ON APPLICATION
AND THE QUESTIONS YOU ARE TRYING
TO ANSWER. YOU HEARD ABOUT
VALIDATION. BIOLOGICAL
VALIDATION, IN OUR CASE, YOU
WILL SEE IN EXAMPLES, I WILL
SHOW WE ARE TALKING ABOUT CELL
MARKERS WHAT CELLS DO I HAVE IN
MY TISSUE, DO THEY EXPRESS SAME
MARKERS THAT I WOULD EXPECT IN
THIS SAME CELLS IN A HUMAN
TISSUE. CELL COMPOSITION
MORPHOLOGY, DOES THE TISSUE HAVE
MORPHOLOGY YOU WOULD EXPECT IN A
HUMAN. WE DO A LOT OF
PROTEOMICS. HOW YOU VALIDATE A
LOT OF IMMUNOHISTOCHEMISTRY,
IMAGING YOU WILL SEE. WHETHER IN
SINGLE CELL SEQUENCING BECAUSE
AT THE END OF THE DAY YOU WANT
TO BENCHMARK THESE TISSUES YOU
ARE MAKING TO THE CLINICAL
SAMPLES. WE WANT TO BE CLOSE TO
CLINIC, SHOW US THAT YOU CAN DO
IT HOY CLOSE BY MORPHOLOGY
FUNCTION OMICS. HOW CLOSE WILL
YOU TO THAT HUMAN SAMPLE. ONCE
YOU GET AND CONSEQUENCE YOURSELF
YOU HAVE A TISSUE WITH THE RIGHT
MARKERS, THE RIGHT FUNCTION,
RIGHT MORPHOLOGY, YOU WANT TO
USE THEM TO BE ASSAY TO TEST
COMPOUNDS. SO USER TO WORRY
ABOUT EVERYTHING SAID ABOUT Z
FACTOR DOS THEY HAVE ROBUSTNESS.
REPRESENTATIVE FROM DAY TO DAY
WELL TO WELL PLATE TO PLATE.
MANY TIMES YOU USE EXPERIMENT
ITSELF SO A LOT OF PRIMARY CELLS
NO LONGER AVAILABLE I'M CHANGING
LOGS DO I GET THE SAME? THE
ANSWER IS NO. YOU DON'T KNOW
WHY. BUT YOU HAVE TO ALMOST
RE-EVALUATE AND REDEVELOP ASSAY
FROM THE CRUTCH BECAUSE CELLS
WERE DIFFERENT. AGAIN WE WANT TO
USE THESE ASSAYS FOR SCREENING,
WE WANT TO DEVELOP FUNCTIONAL
END POINTS. YOU CANNOT DO A
SCREEN WITH SECTIONING AND
IMMUNOHISTOCHEMISTRY. YOU CAN DO
-- YOU NEED ASSAYS THAT ARE
AMENABLE TO THE ALTERATIONS AND
READERS THAT YOU USE TO REGULAR
2D ASSAYS SO WHAT TYPE OF ASSAY
READ OUTS WE USE, WE USE A LOT
OF HIGH CONTENT SCREEN, USE A
LOT OF ASSAYS THAT PERHAPS YOU
CAN SEW IN A 1536 WELL PLATE BUT
FOR THIS TYPE OF THROUGH PUT,
IT'S OKAY TO WANT ELISA,
ACTUALLY POWERFUL SO WE DO
MULTIPLEX ELISA, LUMINEX TYPE OF
ASSAYS. THE HOPE IS THAT WE WILL
BE ABLE TO APPLY THE SAME
ROBUSTNESS CRITERIA THAT TC
DESCRIBED IN HIS TALK CAN WE
MAKE ASSAYS IN TISSUE MODELS AND
ENGINEER TISSUE MODELS WITH Z
FACTOR .5. I WILL SHOW IN SOME
ASSAYS WE CAN GIVE THEM. AND
FINALLY, AS DEVAN WHEN IN THE
LAST TALK TAKING CONTROL
COMPOUNDS AND DOING IC 50 AND
LOOKING AT POTENCY AND EFFICACY
OF COMPOUNDS IN THESE TISSUE
MODELS AND COMPARING IT TO 2D,
SO WE ALWAYS WE DON'T WANT MORE
WORK ON OURSELVES IF WE DON'T
HAVE TO. WE TRY TO BENCHMARK
TISSUES AGAINST CELLS IN 2D.
ADVANTAGE TO TAKE IPS DERIVED
NEWSPAPER AND PUT IN CONTEXT OF
TISSUE VERSUS SCREEN NEURON IN
2D. YOU HAVE TO GO BACK AND
BENCHMARK TO SEE ADVANTAGE OR
NOT. NOT ONLY IN THE BIOLOGY,
BUT ALSO IN THE PHARMACOLOGY OF
THE COMPOUNDS. WE DECIDED TO
INVEST IN 3D TISSUE
BIOFABRICATION. IT IS REALLY AN
EMERGING TECHNOLOGY IS THAT IS
POWERFUL AND ALLOWS YOU TO
ASSEMBLE COMPLEX ISSUES WITH A
SPECIAL CONTROL WHATEVER CELL
COMPOSITION YOU WANT TO INCLUDE
IN YOUR TISSUE. SO IT IS
ADDITIVE MANUFACTURING. FOR US
IT WAS INTERESTING BECAUSE WE
COULD HAVE SPATIAL CONTROL
MULTI-CELL TYPE STRUCTURES IF
YOU WANT A DISEASE TISSUE WE CAN
CHOOSE WHAT CELLS WE INCLUDE
NORMAL VERSUS DISEASE AND
UNDERSTAND A LITTLE BIT WHAT
TYPE OF CELLS IN THAT PARTICULAR
TISSUE WAS SEPARATING THE
DISEASE MODELS OR PHENOTYPE.
BIOPRINTING ALLOWS PHYSIOLOGICAL
FEATURES LIKE VASCULATURE,
INCLUDE IMMUNE CELLS, SO VERY
VERSATILE TECHNOLOGY TERMS OF
BEING ABLE TO INCLUDE WHATEVER
PHYSIOLOGY YOU WANT TO INCLUDE
IN YOUR PRESENTATION. MOST OF
THE TIME WE USE PRIMARY CELLS OR
IPS DERIVED CELLS, THE TIME TO
DEVELOPMENT AND HAVING MATURE
TISSUE IS SHORTER THAN PERHAPS
MAYBE ORGANOIDS FOR THOSE OF YOU
DEVELOP ORGANOIDS MIGHT TAKE
WEEKS AND NON-HAVE MATURE
ORGANOID, THAT IS FUNCTIONAL.
HERE WE CAN GO THREE TO FOUR
WEEKS, STILL LONG TIME, COMPARED
TO 2D MODEL BUT IT IS RELATIVELY
SHORT WHEN YOU TALK 3D MODEL.
WE CAN DO THAT THIS TISSUE IN A
REPRODUCIBLE WAY UP TO 96 WELL
PLACE, WHICH IS FOR US HIGH
THROUGH PUT. SO BIOPRINTING 101,
IT IS USED, ADDITIVE
MANUFACTURING TECHNOLOGY,
PROBABLY HAVE BEEN USED FOR
PRODUCTION OF ORGANS OR
REGENERATIVE MEDICINE. WE USE IT
TO CREATE THE COMPLEX 3D TISSUE
MODELS. THIS NOW SEVERAL BRANDS
OF BIOPRINTERS IN THE MARKERS SO
COMMERCIALLY AVAILABLE. THEY
RANGE FROM 50,000 TO HALF
MILLION DOLLARS DEPENDING ON
FEATURES YOU WANT IN THE
BIOPRINT BUT MOST HAVE LIKE
DROPLETS OF CELLS. HAVE
EXTRUSION YOU MIX YOUR CELLS
WITH HYDRA GEL AND PRESSURIZE
SQUEEZE YOUR KETCHUP OR MISON
YOUR HOT DOG. AT THE END OF THE
DAY, THE TECHNOLOGY WE USE IS
REALLY HYDRO GEL BASED EXTRUSION
FOR MOST OF OUR TISSUES. WHAT
YOU DO HERE IS MIX YOUR CELLS OF
INTEREST WITH HYDRO GEL. WE USE
GELATIN BASE WE ADD COLLAGEN, A
LOT OF DIFFERENT GELS BEING
DEVELOPED AT THE MOMENT BUT THE
IDEA IS THAT THIS HYDRO GEL HAS
TO HAVE THE RIGHT IMMUNOLOGICAL
PROPERTIES SO YOU CAN SQUEEZE IT
THROUGH MIDDLE OF YOUR SYRINGE.
SO YOU DON'T WANT TO KILL CELLS
BY EXTRUDING THEM THROUGH THE
NEEDLE. IT HAS TO PROMOTE CELL
ADHESION, YOU DON'T WANT CELLS
TO BE FLOATING IN THE GEL. YOU
WANT CELLS ATTACHING TO THE GEL.
NON-TOXIC AND PRINTABLE
PROPERTIES, IN MANY CASES YOU
WANT YOUR HYDRO GEL TO BE
BIODEGRADABLE, YOU WANT IT TO
MAKE A STRUCTURE WITH THE
PRINTER BUT YOU WANT THE CELLS
TO -- IF YOU MAKE A TISSUE AND
WANT THE CELLS IN YOUR TISSUE TO
BE SECRETING NATIVE EXTRA
CELLULAR MATRIX. THAT'S THE
ULTIMATE GOAL SO YOU HAVE A
BALANCE BETWEEN YOUR GELATIN OR
FIBRIN TO CREATE AT THE SAME
TIME THE TISSUE MAKES ONLY ECM
SO LOOKING AGENT THE RIGHT
BALANCE ESSENTIALLY IF THE GELS
DEGRADE TOO FAST YOU GET PANCAKE
BECAUSE EVERYTHING FLATTENS. SO
IT IS AN ART TO THE ASSAY IN THE
TISSUE, TO GET GOOD SPATIAL
CONTROL REPRODUCIBILITY AND DO
MEASURES. THIS IS JUST SOME
MOVIES, EXTRUSION BASE, THIS IS
-- THIS WAS SOME SORT OF INITIAL
EXPERIMENTS WE KID TO
DEMONSTRATE YOU CAN -- WE DID TO
DEMONSTRATE YOU CAN CREATE
PATTERNS WITH A COMPUTER DESIGN
SOFTWARE TO 2D SO WE MADE THIS
PIZZA WITH RED AND GREEN
FIBROBLASTS. YOU CAN MAKE
SPATIAL ARRANGEMENTS. CELLS
THAT ARE LIVE AND DO IT ALSO IN
3D SO THIS IS IS THE SHAPES WE
DID AT THE BEGINNING WHEN WE
STARTED WORKING USING THE
BIOPRINTER SO I WILL TELL YOU
ABOUT TWO MODELS THAT WE HAVE
BEEN WORK, ONE IS SKIN, THE
OTHER ONE IS A NEUROVASCULAR
UNIT THAT WE HAVE BEEN WORKING.
WE HAVE BEEN USING TO GENERATE A
MODEL OF GLIOM GLIOMA. SKIN ON
SIMPLISTIC WAY YOUR DERMIS WHICH
IS LOWER LAYER WHICH IS MOSTLY
FIBROBLASTS AND ECM BUT MAKE IT
AS COMPLEX AS YOU WANT WITH
VASCULATURE, WITH HAIR, WITH
NERVES AND SWEAT GLANDS AND
EPIDERMIS ON TOP IS ESSENTIALLY
KERATINOCYTES WHICH HAVE BEEN
STRATIFIED IN DIFFERENTIATED
WHEN EXPOSED TO THE INTERFACE SO
MANY YEARS PEOPLE HAVE BEEN
DEVELOPING PROTOCOLS TO MAKE
SKIN EQUIVALENTS BY USING THUD
TRANSWELL PLACE WITH INSERTS AND
YOU CAN MAKE -- SO YOU PUT YOUR
SKIN TISSUE OVER DERMIS WITH
FIBROBLASTS AND YOU ADD A LAYER
OF KERATINOCYTES AND YOU DO
SUBMERGE AFTER A FEW DAYS ONCE
FORMS THE TISSUE YOU BRING THE
WHOLE TISSUE TO LIQUID
INTERFACE, ESSENTIALLY YOU
REMOVE THE MEDIA FROM THE TOP
AND KERATINOCYTES DIFFERENTIATE
MAKING YOUR EPIDERMIS. SO WITH
THIS BASIC PROTOCOLS WE CREATED
A PLATFORM OF DIFFERENT SKIN
TISSUE. HERE IS WHERE
INVESTIGATING WHAT PHYSIOLOGICAL
COMPLEXITY DO I NEED IN MY MODEL
FOR WHATEVER DISEASE, WHATEVER
BIOLOGY I WANT TO STUDY SO YOU
CAN MAKE SKIN EPIDERMIS BY
KERATINOCYTES PUTTING IN RIGHT
MEDIA AND EARLY INTERFACE. YOU
CAN MAKE FULL SKIN, USING A
BIOPRINTER, BY MIXING
FIBROBLASTS WITH KERATINOCYTES,
WE HAVE'S SKYLARIZED DERMIS BY
ADDING ENDOTHELIAL CELLS
RECENTLY WE HAVE BEEN WORKING ON
INCLUDING IMMUNE CELLS IN
MACROPHAGES THROUGH ELEVATED
PHYSIOLOGICAL COMPLEXITY WE NEED
FOR MODELS WE ARE INTERESTED IN
AND AGAIN FOR ALL THESE MODELS
WE ARE DEVELOPING EXTENSIVE
CHARACTERIZATION USING HISTOLOGY
IMMUNOHISTOCHEMISTRY, USING A
LOT OF HIGH CONTENT IMAGING WITH
CONFOCAL MICROSCOPY. AT THE SAME
TIME AS MENTIONED BEFORE WE WANT
FUNCTIONAL -- CELL VIABILITY,
ELECTRICAL RESISTANCE
MEASUREMENTS. WE TAKE
SUPERNATANTS AND MEASURE
CYTOKINES, AND USE HIGH CONTENT
IMAGING AS I MENTIONED BEFORE.
WHAT IS NICE ABOUT THIS PLATFORM
IS THAT IT IS MODULAR, VERSATILE
SO WE DEVELOP NORMAL SKIN TISSUE
OF WHATEVER COMPLEXITY WE WANT
AND WE ADD DIFFERENT INSULTS TO
CREATE DISEASE MODEL. THE INSULT
CAN BE A CYTOKINE, A CANCER
CELL, IT CAN BE A VIRUS, OR IT
CAN BE IRRITANT IF YOU ARE
INTERESTED IN SPECIFIC -- BUT
THE BOTTOM LINE IS WHAT TAKES A
LOT OF TIME IS DEVELOPING THE
INITIAL TISSUE AND EVALUATING
THE INITIAL TISSUE. THEN IT IS
ADDING THAT DISEASE INSULT
NORMALLY DOESN'T TAKE AS LONG
THOUGH IT IS STILL AN ART IN
TERMS OF DEVELOPING THE RIGHT
CONDITIONS. SO TODAY I WILL
SHOW YOU AN EXAMPLE OF ECTOPIC
DERMATITIS MODEL, ECZEMA AND ONE
OF THE CANCER MODELS. SO WE HAVE
BEEN INTERESTED IN DEVELOPING
CANCER MODELS BEYOND TAKING
PROLIFERATION CELLS AND GROWING
THEM IN ASSAY. GROWING TUMORS IN
CONTEXT OF TISSUE WHERE THE
TUMOR GROWS SO WE ARE INTERESTED
IN SKIN MODEL SO WE ARE
INTERESTED IN LOOKING AT HOW DO
MELANOMA CANCER CELLS GROW IN
THE CONTEXT OF THE SKIN. OR
ANOTHER WE HEARD IS HOW DO SMALL
CELL CARCINOMA CANCER CELLS GROW
IN CONTEXT. THEN THE QUESTION
LIKE OKAY, SO IF YOU HAVE
COMPOUNDS THAT DO WE HAVE
COMPOUNDS THAT WORK MORE
PREDICTIVE IN TERMS OF THE
ANIMAL MODELS AND HOPEFULLY
HUMANS. AGAIN, IT IS THE IDEA OF
GROWING THE CANCER CELLS, TUMOR
CELLS IN CONTEXT OF TISSUE. YOU
CAN MAKE IT AS COMPLICATED AS
YOU WANT, T-CELLS, MACROPHAGES
SO STEP BY STEP WE ARE BUILDING
IN THIS PHYSIOLOGICAL COMPLEXITY
MANY THE MODELS. FOR THAT, THE
ENGINEERING APPROACH IS VERY
GOOD. WE PUBLISH THIS PAPER
COUPLE OF YEARS AGO, THIS WAS
CANCER CELL CARCINOMA, WE
STARTED WITH THE FIRE BREATHING
PROTOCOLS, WE ADDED SEC CELLS,
IN THIS CASE LABELED. WITH
FLUORESCENCE AND YOU CAN SEE
THAT WE CAN SEE THE CELLS
GROWING IN THE TISSUE. WITH
HISTOLOGY, VALIDATED BY
ALTERNATIVE ASSAY METHOD. THEN
WE DEVELOP ASSAY, CANCER CELLS
WERE LABEL WITH GFP OR RFP, RED
FLUORESCENT PROTEIN. YOU CAN SEE
AFTER TWO DAYS, YOUR SILL YOU
CAN SEE REDUCTION OF THE TUMORS
AND YOUR SILL IS USE #-D IN
CLINIC FOR SEC. WE WENT AHEAD
AND DEVELOPED THE ASSAY IN A 96
WELL PLATE AND THIS IS MY SLIDE
TO SHOW YOU CAN MAKE THIS ASSAY
IN A 96 WELL PLATE, NOT -- Z
FACTORS ARE .66 AND FOR THE
GREEN WHICH IS THE FIBROBLAST
LABEL GREEN SO THAT WAS OUR
INTERNAL CANCER SCREEN SO WE HAD
THE CANCER CELLS IN RED AND
FIBROBLAST IN GREEN SO KILL
CANCER CELLS BUT NOT FIBROBLAST
SO WESTERN ABLE TO HAVE A GOOD
ASSAY WINDOW FOR THE FIBROBLAST
AND .36 DOABLE ASSAY SO NOT BAD
FOR A BIOPRINT TISSUE MODEL WITH
CANCER GROWING IN THEM. THEN WE
DID PROOF OF CONCEPT WITH COUPLE
OF CONTROLS, EURO SILL AND CARBO
PLATIN. WE TAKE THE SAME CANCER
CELLS IN 3D AND TRADITIONAL 2D
MODEL. WHAT WAS INTERESTING IS
THE FIVE NEUROUROSILL IN 2D
DIDN'T HAVE ANY EFFECT IN CELL
PROLIFERATION BUT PUT IN CONTEXT
OF 3D IT HAD AN EFFECT. SO THAT
WAS VERY INTERESTING FOR US
BECAUSE IT SAYS THAT IN 3D YOU
CAPTURE BIOLOGY THAT YOU MIGHT
NOT CAPTURE IN A 2D ASSAY WHERE
CELLS ARE PROLIFERATING. BUT
COMPOUNDS WERE THE SAME, DIDN'T
MATTER. SO THESE DIFFERENCES IN
ASSESSING WHAT WORKS AND WHAT
DOESN'T WORK AND WHAT TYPE OF
ASSAY, WHERE WE ARE. THIS IS A
STATUS WHERE WE ARE NOW WITH THE
3D MODELS SO DEVELOP PROTOCOLS
TO MAKE VASCULATURE. BY MIXING
ENDOTHELIAL CELLS, FIBROBLASTS
AND PERICYTESS. WE CAN USE
BIOPRINTING TO CREATE PATTERNS
THIS ZIG ZAG PATTERN, WHY DOING
THAT? NOT BIOLOGICALLY RELEVANT
BUT IT IS ACTUALLY A VERY GOOD
PATTERN BECAUSE IT ALLOWS US TO
USE HIGH CONTENT IMAGING TO
READILY ASSESS AND QUANTITATE
VASCULAR FORMATION OF BIOGENESIS
SO BY CREATING THE EMPTY SPACES
WITH A HYDRO GEL YOU CAN SEE
VESSELS FORMING IN THIS SPACES
THEN WE CAN GO EASILY WITH THE
MASKS OR THE HIGH CONTENT READER
AND ASSESS HOW MUCH VASCULATURE
WE HAVE IN THESE SPACES. WE CALL
THAT ANGIOGENESIS. SO ABLE TO
DO VASSALRIESED SKIN, HAVE A LOT
OF CONTENT IMAGING LOOK AT THE
DIFFERENT LAYERS OF THE SKIN
IMMUNOHISTOCHEMISTRY. SO THIS
MODEL WE DECIDED TO REPRODUCE A
MODEL OF ECTOPIC DERMATITIS.
IMMUNE DISEASE, IT IS TRIGGERED
BY SOME INSULT, THROUGH THE SKIN
AND THAT GETS ACTIVATES TH 2
CELLS THAT TWO BACK TO THE SKIN,
AND THEN THIS IL 4 AND IL 13 THE
OTHER THING WE ARE LEARNING IS
DIFFICULT TO REPRODUCE DISEASES
END TO END IN THESE MODELS
BECAUSE WE HAD SKIN BUT NO LYMPH
NODE SO WE WOULDN'T BE ABLE TO
REPRODUCE THE WHOLE DISEASE END
TO END. WHAT WE DECIDED TO DO IS
LOOK LET'S START SIMPLE, WE WILL
USE IL 4 TO ACTIVATE THE DISEASE
PHENOTYPE. THIS WAS PUBLISHED
LIKE AGAIN COUPLE OF YEARS AGO,
SO WE CREATED THE VASCULARIZE
SKIN AND HAVE NORMAL AND THEN
INDUCE DISEASE PHENOTYPE WE
ADDED IL 4. PARTICULAR PROTOCOL
ADDED THE SAME DAY TISSUE WAS
ADDED TO THE INTERFACE. YOU CAN
GO BACK AND SEE BY HISTOLOGY,
THIS IS A NORMAL AND THIS IS THE
ECTOPIC DERMATITIS BY ADDING IL
4 YOU USE HYPO DISRUPTED
DIFFERENTIATION OF THE
KERATINOCYTES IN THE EPIDERMIS
THEN LOOK AT MARKERS OF
DIFFERENT DIFFERENTIATIONS SO
YOU CAN SEE LOWER IS WIN OF
THEM, NORMAL YOU SEE HIGH, IN
ECTOPIC DERMATITIS DISAPPEARS,
SAME FOR E CAD HEREIN. THE
QUESTION IS LOOKS BY HISTOLOGY
WE HAVE A GOOD MODEL FOR ECZEMA
PROOF OF CONCEPT, IL 2 SIGNAL
JAK STAT PATHWAY SO LET'S ADD
JACK INHIBITORS TO SEE WE CAN
PREVENT THESE DISEASE PHENOTYPE
HERE SHOWING DIFFERENT JAK
INHIBITORS WE ALSO TRIED A
IMMUNOSUPPRESSER. SO THE JACK
INHIBITORS CORRECTED THE DISEASE
PHENOTYPE, NORMAL,K ECZEMA AND
JAK 1, 2 AND 3 INHIBITORS AND
THEY CORRECT THE DISEASE
PHENOTYPE BY HISTOLOGY. WE
DIDN'T HAVE ANY P CELLS, THAT IS
THE TARGET OF CELLS SO MAKES
SENSE MAKES IT EVEN WORK IN THIS
ASSAY. THIS GOES ABOUT YOU ALSO
UNDERSTANDING WHAT IS THE
BIOLOGY OF ETIOLOGY YOUR ASSAY
CAPTURES? YOU CAPTURE WOULD MAKE
SENSE AND CAPTURE WHAT DOESN'T
MAKE SENSE AND THIS IS
DEMONSTRATED HERE. WE LIKE
ELECTRICAL RESISTANCE, IN 96
WELL PLATE, THE NORMAL IS HIGH,
THE ECZEMA LOW AND THEN J,K
INHIBITORS WERE CORRECT EVEN AND
CONFIRM WITH ADDITIONAL LIKE
MARKERS OF DIFFERENTIATION. SO
AGAIN VALIDATION PROOF OF
CONCEPT WITH SOME COMPOUND WE
THINK SHOULD WORK AND SOME
COMPOUNDS WE THINK SHOULDN'T
WORK. WHAT WAS INTERESTING IS
THAT WE WENT BACK TO WE HAVE ALL
THESE DIFFERENT MODELS WE HAVE
VASCULARIZED SKIN, DOESN'T MAKE
A DIFFERENCE.
DO WE HAVE TO DO A FULL
THICKNESS VASCULARIZED SKIN FOR
ECZEMA? THE TRUTH IS THAT WHAT
WAS REALLY INTERESTING IS THAT
IF YOU LOOK AT ELECTRICAL
RESISTANCE, AND LOOK JUST AT THE
DERMIS, BY ADDING THE IL 4 IT
DOESN'T HAVE EFFECT ON TIER
VALUE FOR JUST EPIDERMIS. THE
JAK INHIBITOR ESSENTIALLY KILL
THE EPIDERMIS, THAT WAS
INTERESTING AND BETWEEN THE
NON-VASCULARIZED AND
VASCULARIZED DIDN'T MAKE A
DIFFERENCE FOR THE READ OUT. SO
DON'T NEED VASCULATURE IF I WANT
TO LOOK AT THIS TIER EFFECT ON
THE ECZEMA MODEL. WHEN YOU LOOK
AT ONE OF THE CYTOKINES, SO WE
ALSO TOOK SUPERNATANTS AND
DIFFERENT CYTOKINES SO ATTACK
SIN IS ONE OF THE CYTOKINES AS A
BIOMARKER FOR ECZEMA. WHAT IS
INTERESTING IF YOU TAKE
KERATINOCYTES AND FIBROBLASTS
AND EVEN EPIDERMIS,
DIFFERENTIATED AND STIMULATE
WITH IL 4, YOU SEE NOTHING.
THERE IS NO SECRETION OF TOXIN.
ONLY WHEN YOU START WITH THE
FULL SIGNALS THAT INCLUDE
FIBROBLASTS LAYER, THAT'S WHEN
YOU START SEEING SECRETION OF AO
TAXIN AND REVERSE WITH JAK
INHIBITORS SO FOR US THIS WAS AN
EXAMPLE OF LIKE HITTING THE
SWEET SPOT FOR THAT PARTICULAR
READ OUT AND THAT PARTICULAR
EFFECT AND DISEASE PHENOTYPE.
YOU COULDN'T DO THAT IN 2D. NOW
WE HAVE OTHER EXAMPLES WE SEE
SIMILAR EFFECTS THAT ONLY WHEN
YOU HIT THE RIGHT PHYSIOLOGICAL
COMPLEXITY YOU SEE A PHENOTYPE
THAT IS RELEVANT. CLINICALLY
RELEVANT. GOING TO GIVE AN
EXAMPLE OF ANOTHER MODEL WE
DEVELOPED. THIS IS NEUROVASCULAR
UNIT. ANOTHER VERY NICE THING
METHOD WE USE IS THAT YOU CAN
REPURPOSE THE PROTOCOLS FOR
DIFFERENT TISSUE SO THE PROTOCOL
FOR VASCULATURE IS REDUNDANCY IN
NATURE. ENDOTHELIAL CELLS,
FIBROBLASTS OR IN THE BRAIN
ASTROCYTES TO FORM VASCULATURE
NETWORKS SO YOU CAN USE THE SAME
BASIC PROTOCOL AND USING BRAIN
ENENDOTHELIAL CELLS AND BRAIN
ASTROCYTES AND MIX THAT WITH THE
SAME HYDRA GELS, GELATIN BASED
FIBRIN BASED HYDRA JELL AND
CREATE PATTERNS AND FORM
VASCULATURE SO IN THIS
PARTICULAR ASSAY, AGAIN WE
CREATED THIS RING DONUT RATHER
AND THE PURPOSE IS SO YOU CAN
MEASURE READILY VASCULAR GENESIS
AN ANGIOGENESIS IN THE MODEL.
NOT FOR OTHER PURPOSE, VERY
PRACTICAL BUT ESSENTIALLY WHAT
YOU DO IS YOU BITE DONUTS IN
EACH WELL AN COVER WITH EMPTY
HYDRA GEL. WE WAIT ESSENTIALLY
FOR 21 DAYS AND YOU CAN SEE THIS
BEAUTIFUL VASCULATURE, THIS IS A
CARTOON, JUST BEFORE WE SHOW YOU
THAT FORMATION OF VASCULATURE,
TO GIVE YOU A SENSE THE
PREDICTABILITY IS REPRODUCIBLE
FROM WELL TO WELL TO WELL, WE
HAVE GOOD CV ON THE AREA, OF THE
DONUT AND THE ROUNDEDNESS,
PRETTY REPRODUCIBLE PROCESS. IF
YOU WAIT LONG ENOUGH RIGHT
CONDITIONS YOU CAN USE HIGH
CONTENT IMAGING CONFOCAL ENOUGH
EVEN THESE TISSUES ARE 200
MICROTWO PHOTON CLEARING YOU CAN
ACTUALLY STICK IN THIS CASE WE
HAVE A PHOENIX SO YOU CAN
MEASURE REAL TIME LIKE FORMATION
OF VASCULATURE NETWORK AND YOU
CAN QUANTITATE THAT CLEARLY WITH
ALGORITHMS FROM THE INSTRUMENT
AND YOU CAN SEE FORMATION OF THE
VESSELS AND YOU CAN QUANTITATE
FORMATION OF THE VESSELS. WE
HAVE NORMAL, WHAT CAN WE DO WITH
THIS. TO INTRODUCE A GLIOMA CELL
TO CREATE A MODEL OF GLIOMA IN
CONTEXT OF NEUROVAS QUEUE
ALREADY A UNIT. IT IS NOT
PERFECT, DOESN'T HAVE NEURONS.
THAT IS THE NEXT STEP BUT TRYING
TO SHOW THE PROCESS IS VERSATILE
AND MODULAR YOU CAN CHOOSE WHAT
CELLS YOU WANT TO INCLUDE AND
SEE, SO IN THIS CASE WE WANT TO
CANCER CELLS GROWING IN THE
RELEVANT TISSUE VASCULATURE. WE
ADDED A GLIOMA CELL IN THE
MIDDLE OF DONUT THEN WE LOOK AT
FORMATION OF VASCULATURE.
FORMATION IN THIS CASE
ENDOTHELIAL CELLS LABEL WITH
GFP. THE PATIENT DERIVED GLIOMA
CELLS LABELED WITH CHERRY SO WE
CAN USE IMAGING TO LOOK AT THE
EFFECTS, GROWTH OF THE VESSELS
AND GROWTH OF THE TUMOR. THEN
YOU CAN PLAY GAMES AND SAY WHAT
DIFFERENCE -- DOES THE
VASCULATURE MAKE A DIFFERENCE IN
THE GROWTH OF TUMOR? DOES TUMOR
MAKE A DIFFERENCE ON THE GROWTH
OF THE VESSEL, SO THESE ARE
INTERESTING QUESTIONS BECAUSE WE
ARE TRYING TO ASSESS WHETHER
THIS CROSS TALK BETWEEN VAS VIEW
LATURE AND TUMOR WHETHER THAT
AFFECTS EACH ONE OF THEM. THEN
ASSAY WORKSHOP. WE DID THAT IN A
96 WELL PLATE AND FOCUS SCREEN.
HOW MANY COMPOUNDS, THERE
WEREN'T TOO MANY LIKE 20 OR
SOMETHING. THE IDEA HERE WAS TO
THINK OF IT LIKE A XENOGRAPH. A
DAY 13 WE START SEEING TUMORS
GROWING AND THEN THIS IS ALL YOU
CAN DO REAL TIME MEASUREMENTS
AND YOU CAN MEASURE THE TUMOR
DAY 13 AND MEASURE TUMOR DAY 21,
LOOK AT DIFFERENCES IN GROWTH.
WITH OR WITHOUT COMPOUNDS. SO
HERE I SHOW YOU PLATE ON DAY 13
ON TOP AND PLATE MAP HERE WE
HAVE THE DMSO CONTROLS ON THE
ONE SIDE AND THEN WE HAVE FOUR
DOSES ON TOP AND FOUR ON THE
BOTTOM. OF EACH PLATE. WHAT YOU
CAN SEE EASILY IS DAY 21 AFTER
SEVEN DAYS OF BIDING THE
COMPOUND MOST VASCULATURE, THESE
COMPOUNDS WERE SELECTED
CHEMOTHERAPEUTICS MORE TARGETED
COMPOUNDS, MEC INHIBITORS, WE
WERE TRYING TO LOOK AT EFFECT OF
COMPOUNDS WE KNEW SORT OF ALSO
AFFECTED THE SORT OF
MICROENVIRONMENT TUMOR CELLS IN
THE MICROENVIRONMENT. AT THE END
OF THE DAY, MOST OF THE EFFECTS
WERE REALLY IF WE HAD AN EFFECT
ON THE TUMOR HAD EFFECT ON
VASCULATURE. WE WERE VERY EASILY
SEE WHAT COMPOUND IS FLUORESCE
SENT BECAUSE IT FROM SLIDES, SO
YOU CAN REMOVE THAT EASILY. THE
BOTTOM LINE IS THAT THE ASSAY
WAS QUITE ROBUST. WE DIDN'T GET
THE RESULTS WE WERE EXPECTING
BUT AGAIN ALL THOSE COMPOUNDS
WERE DEVELOPED IN 2D SO MAYBE
THAT IS WHAT WE EXPECT. MEMBER
WHAT WE OUGHT TO DO IS START
SCREENS WITH ASSAYS AND FIND NEW
TARGETS AND NEW BIOLOGY THAT
WORK IN 3D AND NOT NECESSARILY
IN 2D BUT HOPEFULLY THEN WILL BE
MORE RELEVANT WHEN WE GO INTO
ANIMAL CLINIC. ALL I WANT TO
SHOW YOU HERE IS GENERATING DOSE
RESPONSES, PROBABLY HAVE THAT,
WE WOULD LIKE TO YET BUT THIS IS
WHERE WE ARE GOING TO, CAN STILL
-- CAN WE ALREADY ASSESS
ACTIVITY POTENCY AND EFFICACY OF
COMPOUNDS USING BIOPRINTED
TISSUES. SO THIS IS MY LAST
SLIDE. WANT THE GIVE A TASTE OF
WHAT WE ARE DOING WHERE ARE WE
GOING THE QUESTIONS WE ARE
TRYING TO ASK. IT IS NOT JUST
PRINTING THE TISSUES. I THINK
OUR BIGGEST CHALLENGE IS GETTING
THE RIGHT CELLS. AS I MENTION TO
MAKE THESE TISSUES MORE
PHYSIOLOGICALLY RELEVANT WE USE
PRIMARY CELLS AND IPS DERIVED
CELLS. THERE IS NO CLEAR WINNER.
IF YOU CAN YOU WANT TO USE
PRIMARY CELLS, FROM LOT TO LOT
SOMETIMES WORK, SOMETIMES IT
DOESN'T WORK. SOMETIMES THE
CELLSES LOOK OKAY BUT NO
VASCULATURE. WE DON'T KNOW WHY.
IPS DERIVED CELLS THEY ARE
EXPENSIVE. EITHER MAKE THEM IN
HOUSE OR BUY THEM ALREADY
DIFFERENTIATED. SO THE PROCESS
OF SCALING UP BATCH TO BATCH IS
BIG ISSUES FOR US AND A BIG
ISSUE FOR THE FIELD.
BIOPRINTING THE HYDRA GELS, I
THINK THAT IS SOMETHING WE ARE
WORKING ON. IT IS NOT BIG
HURDLE. TUSH SHOE VALIDATION IS
REALLY CRITICAL AS WELL, WHETHER
YOU USE IMMUNOHISTOCHEMISTRY,
SINGLE CELL SEQUENCING. WE ARE
LEARNING A LOT OF PLASTICITY IN
THE CELLS. AND ENDOTHELIAL CELLS
IS VERY DIFFERENT FROM
ENDOTHELIAL CELL FORMING TUBES
AND FORMING VASCULATURE.
UNDERSTANDING THESE DIFFERENCES
I THINK IS VERY IMPORTANT AND
BENCHMARKING THESE CHANGES TO
REAL CLINICAL SAMPLES I THINK
THAT IS VERY IMPORTANT AS WELL.
DOING SINGLE CELL SEQUENCING TO
UNDERSTAND PLASTICITY,
BENCHMARKING TISSUES. A LOT OF
IMMUNOHISTOCHEMISTRY WHEN WE CAN
PHARMACOLOGY. USE HIGH THROUGH
PUT SCREENING A LOT AND GENERATE
A LOT OF DATA. SO IT PERSON
BECOMES YOUR BEST FRIEND, THE
INFRASTRUCTURE YOU NEED TO STORE
AND ANALYZE THAT DATA, JUST
INCREDIBLE. IT IS A WHOLE POCKET
WE NEED TO SET UP TO REALLY BE
ABLE TO BE FUNCTIONAL DURING
THESE KINDS OF STUDIES. I THINK
THAT IS IT. THANK YOU VERY MUCH
[APPLAUSE]
>>THANK YOU SO MUCH, MARC. SO
COOL TO SEE YOU PRESENT.
QUESTIONS FOR MARC?
>>THAT WAS COOL. WHEN YOU ARE
SHOWING THE EXAMPLE, YOU CAN GO
NINE DAYS FOR THE BIOPRINTED
TISSUES AND THE ASSAY WAS 48
HOURS TWO DAYS FOR THE OTHER. SO
WONDERING ONE, COULD THAT
ACCOUNT FOR THE DIFFERENCE IN
ACTIVITIES FOR FIVE FU AND MAYBE
LEARN SOMETHING ABOUT THAT TOO.
ANY COMMENTS ON THAT?
>>ABSOLUTELY RIGHT. I THINK
MUCH CLOSER TO THE CLINIC AND
WHAT IS CLOSER TO ANIMAL MODELS
AND I THINK PROLIFERATION
ASSAYS, CELLS ARE GOING FAST,
THEY ARE A LOT OF MEDIA. THE
FACT THAT A COMPOUND DIDN'T WORK
FOR WHATEVER REASON, MEANS
(INAUDIBLE) MIGHT BE LONGER TIME
BUT IT IS JUST IN ONE WE DON'T
HAVE ANY EFFECT SIZE SO WE DON'T
KNOW THE RIGHT SIDE, THESE ARE
THE DIFFERENCES THEY ARE TRYING
TO ASSESS NOW.
>>THAT BRINGS UP A POINT I
DIDN'T REALLY CATCH. DO YOU
TREAT FOR NINE DAYS WITH 5 FU
FOR THAT NINE DAY EXPERIMENT?
>>SO WE TEND (INAUDIBLE) AND WE
-- (OVERLAPPING SPEAKERS)
>>OKAY. THANK YOU. SO HIGHER
DOSE DENSITY JUST TO --
>>LONGER TIME. (INAUDIBLE)
>>DO YOU MIND TALKING INTO THE
MICROPHONE?
>>HI, MARC. I HAVE A QUESTION
AND IT MIGHT BE STUPID. THE 2D
SYSTEM YOU HAVE THE MATRIX IN
LIQUID FORM. AND 3D IN A GEL
FORM. MY UNDERSTANDING. MY
QUESTION IS WITH REGARDS TO WHEN
YOU ADD THE COMPOUND, DOES IS
SOLUBILITY OF COMPOUND CHANGE OR
HAVE YOU NOTICED ANY
VARIABILITY?
>>I GUESS WHEN WE SEE ACTIVITY
WE KNOW COMPOUND GETS IN. WHEN
WE DON'T SEE ACTIVITY WE HAVEN'T
DONE POSITIVE ANALYSIS OF
COMPOUND PENETRATION IN THESE
TISSUES. DYES GO IN THESE
BIOPRINTED TISSUES. IT IS
DIFFERENT FROM ORGANOIDS. AND
STEROIDS ORGANOIDS AN STEROIDS
ARE MORE DENSE AND THEN YOU HAVE
TIME, YOU HAVE A HARDER TIME
GETTING DYES INTO THE SYSTEM AND
PROBABLY COMPOUNDS TOO. THESE
TISSUES ARE NOT AS DENSE. SO I
THINK COMPOUNDS GO IN BUT WE
DON'T HAVE THE DATA TO SAY
POSITIVELY THAT IS WHAT HAPPENS.
THANK YOU.
>>
(OFF MIC)
VASCULATURE IN MY MIND IS BLOOD
VESSEL WITH ALLELE CELLS WITH A
CERTAIN ARCHITECTURE, WHEN YOU
ARE SHOWING THAT -- WHAT IS THE
NETWORK, WHAT IS THE NETWORK
LOOK LIKE? IS SOME CELLS LINED?
>>THE ONES YOU CAN FUND VAS
QUEUE LATURE, IT IS HOE LOW AND
WE CAN SEE THAT LITTLE
SECTIONING AND THAT DOES NOT PRO
FUSE. SO IN ORDER TO HAVE
PROFUSION YOU NEED TO GO TO A
DIFFERENT PLATFORM. MORE
MICROFLUIDIC TYPE OF PLATFORM.
BUT TO ANSWER YOUR QUESTION,
THESE ARE REAL VESSELS IN THE
SENSE -- YOU CAN SEE WHEN YOU
SECTION YOU CAN SEE ACTUALLY
THAT.
>>PRETTY COOL.
>>ANY MORE QUESTIONS?
>>THANK YOU SO MUCH, MARC.
[APPLAUSE]
WE HAVE TWO WORKSHOPS TALKING
ABOUT 3D MODELS. ONE ORGANIZED
WITH HELP OF DR. FERRER IN
COLLABORATION WITH BILL AND
MELINDA GATES FOUNDATION AND 3D
ANTI-DRUG DEVELOPMENT TWO FULL
DAYS OF 3D MODELS TALKS RECORDED
AVAILABLE FOR YOU TO VIEW. SO IF
YOU ARE INTERESTED IN THIS TYPE
OF RESEARCH I HIGHLY ENCOURAGE
YOU TO LOOK AT THOSE VIDEOS. WE
HAVE ANOTHER WORKSHOP ON CELL
BASED ASSAYS THAT HAD ONE DAY ON
COMPLEX VISUAL MODELS AS WELL.
SO ALL THESE RESOURCES ARE
AVAILABLE FOR YOU TO VIEW. ALL
RIGHT. OUR FINAL SPEAKER IS DR.
XIN XU, SENIOR SCIENTIST AND
DIRECTOR OF PHARMA KINETICS IN
THERAPEUTICS DEVELOPMENT BRANCH
DIVISION OF PRE-CLINICAL
INNOVATION AT NCATS. SHE IS ALSO
A LONG STANDING EDITORIAL BOARD
MEMBER OF THE AGM, WE ARE
EXCITED TO HAVE HER HERE TODAY.
HER TALK IS ENTITLED IN VIVO
ASSESSMENTS OF ADME PROPERTIES
OF LEAD COMPOUNDS. XIN, THE
FLOOR IS YOURS.
>>CAN YOU HEAR ME? I THANK YOU
FOR MY TALK ESPECIALLY AFTER
EXCITING TALKS MARC. WHAT I WILL
TALK ABOUT TODAY IS IN VITRO
ASSESSMENT OF THE ADME IS THE
FOUR IMPORTANT PROCESS OF DRUG
INVOLVEMENT. I WOULD LAKE TO
START ACKNOWLEDGMENT SLIDES,
COMBINED AWARD FROM MANY
SCIENTISTS IN THIS FIELD AND
ALSO LIKE TO THANK DR.
(INAUDIBLE) PHARMACOKINETIC
CHAPTER IN THIS ASSAY GUIDANCE.
HERE IS THE OUTLINE OF MY TALK.
FIRST GO OVER BASIC ADME
CONCEPTS IN DRUG DISCOVERY AND
DEVELOPMENT. AND I WILL GO OVER
SOME IN VITRO ADME ASSAYS IN THE
DRUG SELECTION LEAD COMPOUND AND
HELP DESIGN NEW MOLECULES IN THE
DRUG LIKE MOLECULES. I WILL TALK
ABOUT IN SILICO ADME BECAUSE
AFTER YOU GET THE DATA IN VITRO
ASSAYS, THE MODEL, ONCE YOU HAVE
A MODEL YOU CAN USE THE MODEL TO
START TO PREDICT OR HELP WITH
DESIGN THAT MAKES THE MOLECULE
AND I WILL END MY TALK WITH A
TAKE HOME MESSAGE. SO AS WITH E
KNOW, THERE ARE HURDLES FROM
DRUG DISCOVERY TO A FINAL
PRODUCT. SO WHAT WE ARE DOING
HERE ADME IS ONE HURDLE HERE.
WHAT HAPPEN IS BEFORE I GO TO
ADME I WILL TALK PHARMACO -- I
DON'T KNOW HOW MANY PEOPLE KNOW
WHAT IS THE PHARMACOKINETICS. IT
IS SCIENCE DESCRIBES THE DRUG
MOVEMENT IN THE BODY. WE USE A
MATHEMATICAL EQUATION TO
DESCRIBE THIS PROCESS. FOR
EXAMPLE, FOR ORAL YOU SEE AFTER
ORAL DOSE THE DIVIDE BY UPTAKE
OF IV DOSE DOSE. YOU CAN GET
ESTIMATE WHAT PERCENTAGE DRUG
INTO THE BIOLOGY SO MATHEMATIC
DESCRIBE CONCENTRATION AND TIME
RELATIONSHIP. WHY PHARMACO
KINETICS IS IMPORTANT? BACK IN
THE '90s YOU CAN SEE MAJORITY
OF DRUG FAILED IN THE CLINICAL
TRIALS DUE TO PHARMACO KINETICS.
SO HERE LIES THE IMPORTANT
PHARMACOKINETICS ALL PHARMACY
DISCOVERED RESEARCH PUT OWL
EFFORT IN PHARMACOKINETIC TRYING
TO SELECT COMPOUND FROM VERY
EARLY ON SO YOU CAN SAVE TIME
AND MONEY. SO SEEMS EFFECTIVELY
SEE THIS HIGH INCREASE
DRAMATICALLY, AS WE KNOW 20
YEARS AGO DATA TODAY IS ANY DUE
(INAUDIBLE) VERY, VERY LOW. HOW
WITH DO IT? THIS IS A CURVE THAT
SHOW PROFILE AFTER
ADMINISTRATION IN THE BODY. YOU
WANT THE CURVE HAS THE BLUE LINE
HERE, THAT MEANS THEY GAVE ABOVE
THE (INAUDIBLE) LEVEL BUT NOT
GOING TO BE HIGH TALKS LAB WHAT
IS REALLY THE FACTOR DETERMINING
THIS OCCUR AFTER GIVE ORAL
ADMINISTRATION, WE DIVIDE TO THE
FULL PROCESS, WE TALK ABOUT
ADME, ABSORPTION, DISTRIBUTION,
METABOLISM AND SECRETION. WE
WILL TALK ABOUT FACTORS IMPACT
WHOLE PROCESS. WE DEVELOP IN
VITRO ASSAYS. WE CAN COMBINE
THESE REGENERATING WHICH WE WILL
CALL RESULT BASED MODELING AND
PUT BACK MT. BODY BECAUSE WE
HAVE ALL THESE ORGANS THERE, AND
BROOD FLOW THERE, REGENERATED IN
VITRO YOU CAN PLUG IN AND YOU
CAN START TO PREDICT. THIS TIME
WE WILL TALK SMALL MOLECULE,
SMALL MOLECULE IDEALLY YOU GIVE
ORAL ADMINISTRATION DUE TO
SCREENS. FOUR OUR
ADMINISTRATION, THE DRUG WENT TO
SMALL BIOMARKERS GO THROUGH
SEVERAL BARRIERS IN ORDER TO
REALLY GET TARGET CONCENTRATION
WHEN WE WANT IT. THE FIRST IS
THE STOMACH. FOR COMPOUNDS NOT
CHEMICALLY STABLE THEY WILL
DEGREE DATE IN THE STOMACH. SO
ALSO SOLUBILITY IS IMPORTANTMENT
TO BE ABSORBED TO BLOODSTREAM
HAS TO BE SOLID ROLE SO
RATIONALE IN THE GI TRACK DOES
NOT HAVE A CHANCE TO BE ABSORBED
SO WE HAD TO LOOK AT SOLUBILITY
AT DIFFERENT PH. OTHER FACTOR
INVOLVE SUCH LIKE FULL --
CONDITIONS IN (INAUDIBLE) EFFECT
SEVERITY FROM DIFFERENT SEGMENT
OF GI TRACK. THE SIMILAR PILOT
JUNCTION BETWEEN CELLS GIVE INTO
YOUR BLOOD CIRCULATION. SO
THERE'S SEVERAL PROCESS INVOLVED
IN THE ABSORPTION PROCESS HERE.
THE MOST COMMON ONE IS PASSIVE
DIFFUSION. THIS CONCENTRATION
GRADIENT PROCESS. THERE ARE MANY
DRUGS THAT WILL BE CALLED ACTIVE
TRANSPORTER INVOLVED. THEN FOR
THE LARGE MOLECULES THEY HAVE
MECHANISM CALLED ENDOCYTOSIS,
PIE NOCITOSIS, THE CELLS BROUGHT
INTO THE COMPOUND INTO THE NEXT
STEP OF ABSORPTION. ANOTHER
BARRIER IS ME TAB LIMB EFFLUX
TRANSPORTER STILL IN THE GI
TRACK HER HERE. WE KNOW THERE AE
MANY DRUG ENZYMES IN THE
ERYTHROSITES. HEPATOCYTES AND
THERE IS A TRANSPORTER THERE,
THEY CAN HAVE EVE FLUX. EVEN THE
DRUG INTO THE SYSTEM, THE
LUMINAL SIDE, BUT THEN THROUGH
THE EFFLUX TRANSPORTER CAN PUMP
UP SO STILL NOT INTO THE BLOOD
SIDE. NEXT ONE WILL BE THE
LIVER. LIVER IS LARGEST ORGAN,
HAVE ALL -- AFTER OF ALL THE
DRUG, METABOLISM ENZYME, THE
BIGGEST DEFENSE MECHANISM.
COMPOUND (INAUDIBLE) SO THE
LIVER HAS TWO ROWS THERE, ONE IS
THE ENZYME TRYING TO DO BILE
TRANSFORMATION, ANOTHER ONE IS
THROUGH THE SECRETION. THAT IS
REALLY THE IMPORTANT ORGAN WE
ALWAYS FOCUS ON WHEN WE LOOK AT
DRUG ABSORPTION AND (INAUDIBLE)
NOW YOU CAN SEE FOR DRUG TO BE
SUCCESSFULLY ABSORBED INTO THE
BLOOD CIRCULATION THEY HAVE THE
PASS SO MANY BARRIERS HER HER HE
BOWEL WILL BE PROCESS --
BIOAVAILABILITY FOR THE ORAL
ABSORPTION, BAR IS PRETTY HIGH.
THE NEXT PARAMETER WE ARE
TALKING ABOUT IS TISSUE
DISTRIBUTION. MANY OF THESE
CHEMICAL PROPERTIES IMPACT THE
DISTRIBUTION, SUCH AS SIZE,
WEIGHT, CHARGE AND LIPO
PHYLLISTYTY. PROTEIN BINDING IS
ALSO A FACTOR. BLOOD IS DRIVER
DISTRIBUTE YOUR COMPOUND TO THE
TISSUES SO IF YOUR DRUG HAS HIGH
PROTEIN BINDING OR RESIDE INSIDE
RBC, AVAILABLE THE FREE DRUG
GAVE INTO THE TISSUE BENEFITS SO
WE'LL HAVE TO DETERMINE THAT.
ONCE DRUG IS INTO TISSUE SERVE
PURPOSE O TO DESIGN TARGETED
BINDING AS WE LIKE TO HAVE BUT
UNFORTUNATELY HAVE TISSUE
BINDING THERE. FOR SOME ONCE
BANDED TISSUE AND HARD FOR --
THE NEW MODALITY OF (INAUDIBLE)
SO DRUG METABOLISM WE TALK ABOUT
IDEALLY SO SIMPLE BUT IF YOU
COME TO THE DETAILS SO MANY
REACTIONS GOING ON SO WE DIVIDE
TO TWO MAJOR CLASSES ONE CALLED
THE PHASE 1 METABOLISM, SECOND
IS PHASE 2 ME TAB LISP, PHASE 1
IS IF MOLECULE, THESE DAYS
MOLECULE TENDS TO BE POTENCY
DRIVEN. THAT MEANS VERY
PROPHETIC. HIGH AFFINITY FOR
TARGET. SO FOR THOSE MOLECULES
HARD TO PARTICIPANT SPLIT OUT.
POOR SOLUBILITY. THEY ARE TRYING
TO ADD SOME POLAR GROUP TO THE
MOLECULE THERE. SOFA SILL AT A
TIME EITHER GOING TOWARD PHASE 2
REACTION, THAT IS REALLY POLAR
GROWTH CELL BASED REALLY BECOME
SOLUBLE MOLECULE, THE MOLECULE
CAN BE EXCRETED TO THE BILE INTO
THE URINE. SO IT IS REALLY TWO
MAJOR FAMILIES OF DRUG
METABOLISM REACTIONS. FOR PHASE
1 METABOLISM, THERE AGAIN SO
MANY ENZYMES THERE IN
TRANSFORMATION REACTIONS THERE,
WHY WOULD ALWAYS -- MEDIATED
INTERACTION, BECAUSE LOOK AT THE
APPROPRIATE DRUG MORE THAN 75%
DRUG INVOLVE SIF METABOLISM SO
THEY BECOME VERY IMPORTANT. IN
ADDITION TO P 450 SOME
(INAUDIBLE) MEDIATED METABOLISM
ESPECIALLY WHEN WE CALL AD HIGH
OXIDASE. THEY SHARE SOME
SIMILARITY WHEN THEY DO IT
CHEMICAL REACTION AS P 450.
PEOPLE KNOW IT IS IMPORTANT
TRYING TO DESIGN THE MOLECULES
AVOID MEDIA METABOLISM SOMEHOW
PUSH SCAFFOLDS AND GOING TO BE
THE SUBSTRATE OF AO. SO WHEN WE
DO ALL THESE ASSAY DEVELOP WE
HAD TO KNOW WHICH FRACTION OF
THE CELL, ENZYMES. RESIDE IN THE
MECHANISM FRACTION, HOWEVER FOR
(INAUDIBLE) IN THE SIDE OF THE
FRACTION CELLS. FOR THE PEOPLE
NOW GOING TO P 450 IT IS FLAVIN
CONTAINING PROTEINS, THERE'S
SEVERAL HERE IF YOU HAVE NEW
DRUG FOR APPROVAL THEY PROBABLY
ASK YOU ALL THESE CYPs IN
TERMS OF ACTIVITY. BECAUSE THEY
WANT SO MANY DRUG SHARE ENZYME
HERE THEY WERE ABOUT TO HAVE ANY
DRUG INTERACTION SO THE COMMON
ONE IS 3, 4, THEY ACCOUNT FOR
ALMOST 50% OF THE DRUG THAT HAVE
THE MARKER INVOLVED. CYP 3A 4.
WE ALSO HAVE 3A 43A 5 ARE
SIMILAR, SHARE A LOT OF SAME
PROPERTY FOR DRUG METABOLISM. IN
ADDITION TO 4 AND 5, AND ALSO WE
ARE WORKING ON THE 3A 7, IT IS
THE ENZYME FOR INFANT AND AGE OF
ONE. SO FOR NEWBORN BABY, YOU
RARELY SEE THIS. AND ACTUAL ONE
YEAR OF AGE IS ACTIVITY DECREASE
IN 3A 4 START PICKING UP. WHEN
WE DO ALL THIS METABOLISM STUDY
REALIZE THE SPACIOUS DIFFERENCE
THERE BECAUSE WE START DOING PK
IN ANIMAL FIRST. THEN KNOW
ENZYMES THERE'S SPECIES
DIFFERENCE SO WE HAVEN'T GO
AHEAD AND MOVE FORWARD FOR THE
HUMAN THAT IS THE MECHANISM WE
ARE INTERESTED REALLY START
HUMAN ENZYME ASSAY EARLY ON.
FOR EXCRETION WE TALK ABOUT IT
BRIEFLY ALREADY, THE ADDITION
RANDOM EXCRETION IS ALSO
IMPORTANT. THE KEY FACTOR IMPACT
EXCRETION SIZE, AND
TRANSPORTERS. WE TALK ABOUT
MEDICINE IN NEED OF METABOLISM,
DURING COVID ONE CLASS COMPOUND
SHOW UP AS NEW ANALOG. OR
ANTIVIRAL DRUG. WE ALSO HAVE
METABOLISM FOR MANY DRUG, THAT
IS PRO DRUG. EXCRETION IS COPING
FOR. SO NOW TALK ABOUT FACTORS
THERE, THAT IS HOW GOING TO
ADDRESS FACTORS THAT DESIGN
BATTERY OF IN VITRO ASSAY. FOR
ABSORPTION, LOOK AT SOLUBILITY,
PERMEABILITY AND EFFLUX
TRANSPORTER IN INTESTINE. FOR
METABOLISM WE TALK ABOUT ALL
THESE P 450 METABOLISM OR OTHER
TRANSLOCATIONS THERE, AND ALSO
TALK ABOUT BILIARY SECRETION AND
RHYTHM SECRETION. SO ALL THESE
ASSAY, IN VITRO, GO IN THE
COMPOUND. FOR THE DISTRIBUTION
THOSE ARE VERY IMPORTANT. THAT
IS HOW FOCUS ON THESE IN VITRO
ASSAYS. SCREEN OUT MOLECULE.
ONLY THE WINNER ONE WILL BE
MOVED FORWARD TO THE NEXT STAGE
SO MANY ASSAYS IF YOU RUN FOR
EVERY COMPOUND, CALLS WILL BE
VERY HIGH SO THEY START
INVENTING WITH THE PARADIGM. I
BELIEVE MOST IN THIS WORKSHOP
PROBABLY DOING THIS CELL BASED
ASSAY OR SOME SAY ASSAYS FOR THE
TARGET. SO WHEN ADVOCACY
STARTED REALLY DOING SCREENING,
FOR DMPK FUNCTION WE ALSO START
SCREENING BUT THAT WE CALL -- WE
ARE LIMITED ONLY JUST A FEW
ASSAYS THERE. THAT IS THE BASIC
ONE. FOR EXAMPLE SOLUBILITY AND
PERMEABILITY AND MICROSOMAL
STABILITY. IN SOME CASES LOOK AT
SIP INHIBITION. SO ONLY WHEN
PAST THIS HIGH SUPER SCREENING
THERE, WE SEND TO THE TEAM AND
TEAM TAKE A LOT, THEY ARE GOING
TO DESIGN THE NEXT SIZE MOLECULE
WE CALL THE STRUCTURE
OPTIMIZATION. THOSE ACTIVITY
THERE. THEN YOU DIVE IN TO EACH
TEAM AND UNDERSTAND WHAT IS THE
TARGET, WHAT DRUG WE NEED TO GET
TO THAT TARGET, DESIGN TIER 2
ASSAY. THAT IS THE PROJECT ASSAY
THERE IS. BUT AFTER THAT WE LEAD
AND GO INTO THE IN VIVO. IN VIVO
USUALLY START WITH RODENT.
ESPECIALLYRODENT.ESPECIALLY YOUO
CONCEPT STARTING. WHATEVER IN
VITRO POTENCY LOOKING AT IN VIVO
ANIMAL DISEASE MODEL ELLE MODELK
THERE. ONLY AFTER YOU VALIDATE
TARGET THEN THE COMPANY,
MANAGEMENT BE MORE INTERESTED
REALLY PUT MORE MONEY THERE TO
SUSTAIN THAT PROGRAM. SO THE
PROOF CONCEPTS BECOME IMPORTANT.
THEN WE HAVE TO REALLY DETERMINE
PHARMACOKINETICS AT THAT STAGE
MAKE SURE THE DRUG WILL HAVE THE
CONCENTRATION ABOVE IN VITRO
ASSAY SPECIFIC ESPECIALLY IN THE
TARGET ORGAN. THEN COME THROUGH
REALLY THE NEXT STEP, THEY PASS
THAT AND DOING ENABLING STUDIES
AND REALLY (INAUDIBLE). WE WERE
FOCUSED ON THE ASSAY PART,
TALKING TIER 1 ASSAY, REALLY THE
HIGH THROUGH PUT SCREENING
ASSAYS. WHICH IS QUICKLY GO OVER
ALL COMPOUNDS SYNTHESIZED, OUT
IN TERMS OF KEY PARAMETERS
LISTED HERE, SOLUBILITY,
PERMEABILITY, MICROSOMAL
STABILITY AND CYPs SO IT IS
FASTER PACE, RAPID TURN AROUND
TIME SO COMBINATION IS SIMPLE.
NCATS WE USE A (INAUDIBLE)
PLATFORM AND SCREEN COMPOUNDS
SYNTHESIZED BY CHEMISTS, AVERAGE
100 COMPOUNDS PER WEEK. SO WE
SCREEN EVERY COMPOUND WITH THESE
THREE ASSAYS. THEN SOME COMPOUND
EVEN LOOK AT 3 OR 4. SO OVER THE
TEN YEARS NCATS JUST UNDER TEN
YEARS ANNIVERSARY SO FOR TIER 1
WE WERE TO HAVE DATA FOR OVER
30,000 COMPOUNDS. WE
(INAUDIBLE) BUT HOW WE ARE GOING
TO MANAGE HOW FOOD GOOD ASSAY
WILL BE, WE MONITOR EACH PLATE
AND THE VARIATION THERE, WE USE
THE MSR EARLY PRESENTATION IN
THIS WORKSHOP. SO THIS IS JUST
TABLE SUMMARIZE THE DATA WE HAVE
FOR OVER 500 PLACE. AND WHAT IS
THE MSR THERE. IN GENERAL THE
ASSAYS ARE PRETTY GOOD FOR TIER
1 ASSAY. ONCE YOU HAVE THE DATA
GO TO THE TEAM MEETING AND SEE
THESE TABLES VERY OFTEN, THAT
MEANS THE COMPOUNDS CHEMISTS
WITH THOSE COMPOUND STRUCTURE ON
YOUR (INAUDIBLE) THERE AND WE
HAVE ALL THESE AVME PROPERTIES
THERE AND THEY HAVE ALL THESE
ADVOCACY DATA THER THERE. SO THN
GAVE COLOR CODED, THE GREEN IS
THE ONE, THIS IS GOOD AND YELLOW
MEANS WARNING AND RED IS
DEFINITELY NOT GOING TO MOVE
FORWARD. THAT'S HOW BASED ON
THIS DATA THEY GO BACK TO SELECT
THE NEXT MOLECULE FOR NEXT
COMPOUND MOVING FORWARD. FOR
THE TIER 2 ASSAY AS MENTIONED
MORE SPECIFIC FOR THE PROJECT
NEEDS. IF YOU ARE DOING THE
PROJECT, THEN YOU PROBABLY GOING
TO LOOK AT STABILITY. LIKE
REMDESIVIR, SO MANY AS RACE
THERE. ANOTHER ASSAY WANT TO
MENTION HERE IS (INAUDIBLE) THAT
TELL YOU IF COMPOUND IS STABLE
WHICH MOLECULE IS PRONE FOR MOVE
METABOLISM, WE CALL THAT
METABOLIC (INAUDIBLE) ONCE YOU
IDENTIFY SOFT SPOT. TO GUIDE TO
BLOCK VULNERABLE SITE. THIS IS
ONE EXAMPLE WE USE TO SHOW HOW
THE MED IP GUIDE STRUCTURE OF
THE ORGANIZATION. IT IS ONE
PROJECT WE CALL FOP PROJECT
DISEASE, MET ID. OUR
COLLABORATOR ABOUT MASS GENERAL,
COME TO OUR, THEY THINK THEY HAD
LEAD COMPOUND ALREADY, THIS IS
STRUCTURE SHOWN HERE, LDN
193189. HOWEVER WHEN WE LOOK AT
IT, THAT HAS -- WE LOOK AT
STRUCTURE AND JUST DID SOME
SIMILAR, IT IS NOT COMPLETE
SCAFFOLD BUT ONE PORTION OF THE
MOLECULE WE LOOK AT THIS PART,
WE KNOW THERE IS SOME ADD
FORMATION THERE, WHAT WE CALL
DRAFT METABOLITE, THEY ARE NOT
STABLE, THEY ARE PROTEIN OR DNA,
WE CALL THAT REACTIVE
METABOLITES. SO WE SAY WELL WE
ARE NOT SURE YOUR MOLECULE WILL
BE -- BECAUSE WE ARE (INAUDIBLE)
AND WE SPEND SO MUCH TIME
CONVINCE PEOPLE THAT'S GOING TO
HAVE ISSUE, WE PUSH THE
(INAUDIBLE) STILL CANNOT PASS.
SO TRYING TO AVOID THIS KIND OF
THING HAPPENING SO WE SAY WHAT
DO WE DO WITH MET ID FIRST? THE
DATA COME BACK, WE SEE SEVERAL
LIABILITY FOR THIS MOLECULE. WE
SEE REACTIVE METABOLITE FORM.
ALSO SEE TRACKING AGENT. CAN
TELL YOU THERE'S REACTIVE
METABOLISM FORM. WE SEE FROM
ANOLI. THAT IS SPECIES RESEARCH,
ALWAYS SOME TOXICITY THERE. BUT
WHAT SURPRISED US MOST MAJOR
METABOLIC PATHWAY IS ON THIS
SIDE, OXIDATION SIDE. THIS
ENZYME IS REALLY NOT BY PEOPLE
50, RATHER BY AOO. WHY AO IS A
CONCERN THERE. THIS ENZYME, IT
IS SPECIES DEPENDENT. DOG
DOESN'T HAVE ACTIVITY FOR THIS
ENZYME BUT IF YOU LOOK AT
SPECIES SELECTION THESE DAYS YOU
HAVE MORE THAN (INAUDIBLE) BUT
THIS ONE IS NOT GOING TO WORK
BECAUSE THEY DON'T HAVE THIS
ENZYME ACTIVITY METABOLITE YOU
SEE IN HUMAN. EVEN IN THE
SPECIES LIKE RODENTS, AND HUMAN
HAVE ALL THESE ENZYME THERE,
THEY HAVE LARGE INTERSUBJECT
VARIATION, IN HUMANS AND SIX
MORE DIFFERENCE THERE. IMAGINE
SUCH A VARIATION THERE WILL BE
HARD FOR YOU GOING TO DECIDE
THOSE (INAUDIBLE) SO THERE IS A
LIABILITY THERE. AFTER THAT, THE
TEAM DECIDE WED GO BACK, WE ARE
GOING TO FIX ALL THESE
LIABILITIES. AFTER FEW YEARS
EFFORT WE DIDN'T COMPLETELY
ELIMINATE AO BUT ATTENUATED SO
THE MAJOR METABOLIC PATHWAY
CANONICAL IS (INAUDIBLE) THIS
COMPOUND IS IN CLINICAL PHASE 2
TRIAL IN EUROPE. SO THIS IS JUST
A STORY TELLING HOW MET ID CAN
BE NEWER STRUCTURE OPTIMIZATION.
THE OTHER STUDY IN THE TIER 2
ASSAY IS A CYP. THE CYP STUDY IS
COMPLICATED BECAUSE SO MANY
ISOFORMS THERE. FOR THE TRUE
LEAD COMES TO THE IND ENABLING
START YOU ARE ADDING SO YOU HAVE
TO FOLLOW ADME GUIDANCE FOR THE
DBI ASSESSMENT SO IN THAT THEY
LISTED THE ENZYME THEIR METADATA
SO YOU HAVE TO DO THE CYP
INHIBITION FOR THESE ENZYMES
THERE. I DON'T GO OVER DETAIL ON
THE ASSAY PROTOCOL NOW BECAUSE
YOU CAN ALL GET DETAILS ONLINE
BUT NEVERTHELESS CYP, IN THE
DISCOVERY STAGE HIGH THROUGH PUT
SCREENING WE PROBABLY JUST DO
ONE CONCENTRATION SAY 10
MICROMOLAR, PRETTY HIGH FOR IN
VIVO CONCENTRATION SO WE SAY AT
THAT CONCENTRATION IF YOU HAVE
IF YOU DON'T HAVE INHIBITION
THERE YOUR COMPOUND SAFE BUT IF
YOU HAVE THEN YOU MAY HAVE THE
TITRATE WITH CONCENTRATION TO
REALLY SEE HOW POTENT IT IS. SO
THAT IS THE DETAILED ASSAY
VERSUS HIGH THROUGH PUT
SCREENING ONE CONCENTRATION
MAYBE TEN MICROMOLAR, BEFORE THE
DETAILED ASSAY YOU HAVE TO HAVE
SEVERAL CONCENTRATIONS THERE,
BUT YOU CAN SIT CONCENTRATION
PUSH HIGH AS LIKE 50 OR 100
MICROMOLAR DEPENDS ON SOLUBILITY
BECAUSE SOME ANTIBIOTIC DRUGS,
THOSE ARE HIGH ESPECIALLY HIGH
POTENTIAL ON TDI TO EXTENT OF
OTHER DRUG SO WE HAVE TO HAVE
SAFETY WINDOW SO WE PUSH HIGH
CONCENTRATION AND BROAD RANGE
TIER 2 ASSAYS. ALSO IN THE
DEMONSTRATION YOU HAVE TO LOOK
AT SUICIDE INHIBITION. MOST
INHIBITION IS COMPETITIVE
INHIBITION, DRUG A DRUG B WORK
FOR THE SAME ENZYME, SOMETIMES
IF ONE DRUG A ENZYME
IRREVERSIBLE WE SAUL SUICIDE
ENZYME, SO WE NEED TO KNOW THAT
INFORMATION EARLY ON. THE NEXT
ASSAY IS THE INDUCTION. THAT
MEANS IF YOUR DRUG WERE INDUCED
CYP ACTIVITY IN VIVO, IS THERE
TWO CONCERNS THERE, ONE IS IF
YOU INDUCE YOUR ENZYME ACTIVITY,
YOUR DRUG (INAUDIBLE) STANDARD
DOSAGE THERE, NOW GAVE
EFFICACIOUS ANY MORE. THE SECOND
IS LONG TERM THIS INDUCTION MAY
HAVE A SAFETY CONCERN ESPECIALLY
ON THE LIVER AND THOSE TOXICITY
ISSUE THERE SO DETAILED GUIDANCE
THERE. FOR THE ASSAY, THERE'S
LOTS WE USE mRNA ASSAY OR DO
WE USE FUNCTIONAL ASSAY? THE
ARGUMENT IS THAT EVEN IF YOU
HAVE INDUCTION BUT IDEALLY HAVE
ACTIVITY THERE, SO IT IS NOT
GOING TO ALTER DOSE REGIM REGIMT
NOW OVER THE YEARS YOU CAN SEE
YOUR 2020 ADME GUIDANCE PRETTY
MUCH SETTLED IN mRNA ASSAY
THERE. THIS IS ASSAY GAVE YOU
HOW DO YOU CALCULATE, LOTS OF
DETAIL CALCULATIONS. SO FOR
PEOPLE REALLY COME TO IS THAT
STAGE ENCOURAGE GUIDANCE YOU
PROBABLY HAVE LEAD COMPOUND BONE
TO CANONICAL BUT LOTS OF DETAIL
IN THE DATA ON INDUCTION PART.
THESE ARE TWO GUIDANCE ISSUE IN
THE 2022. FIRST ONLY ONE
GUIDANCE ON -- BUT 2020 GUIDANCE
TWO, ONE IN VITRO FOR THE ASSAY
WORK YOU PROBABLY LOOK AT IN
VITRO PART BUT SECOND IS
CANONICACANONICAL. DRUG DRUG
INTERACTION ASSESSMENT.
TRANSPORTER ASSAY, OUR BODY HAS
SO MANY TRANSPORTERS,
(INAUDIBLE) INITIALLY WE ARE
INTERESTED IN PGP, PG FOR US IS
IMPACT WITH ABSORPTION, THAT CAN
PUMP IT OUT. SO THE SECOND PART
IS THE BRAIN UPTAKE T NOT -- IF
YOUR TARGET INSIDE, YOU HAVE
SOME TRAVEL THERE BUT NOW WE
KNOW ACTUALLY REALLY WORKING ON
THIS ANIMAL WE REALIZE ALL THESE
TISSUE DISTRIBUTIONS ARE IMPACT
BY TRANSPORTERS. IF YOU WORK ON
ASSAY DEVELOPMENT THAT IS -- YOU
CAN SPEND MORE TIME THAN COLLECT
DATA INFORMATION SO THESE ARE
STANDARD SLIDE SHOW THREE MAJOR
ORGANS THERE, THAT IS INTESTINE,
LIVER AND KIDNEY, ALL THESE
TRANSPORTERS CAN BE INVOLVED IN
EACH ORGAN. THIS IS PRETTY
COMPLICATED ALREADY, SO ALLOWS
TRANSPORTERS REGULATE AND THE
GUIDANCE SO THEY ARE DEMANDING
YOU HAVE TO SUBMIT ALL THIS
DATA. SO THAT IS REALLY THE
TRANSPORTER ASSESSMENT. BUT FOR
OURS AS RESEARCH AND WE WANT TO
UNDERSTAND HOW THESE TRANSPORTER
REALLY WORK. FOR EXAMPLE, ONE
NCATS PROJECT IS CYCLE
(INAUDIBLE) IN VITRO NOT MUCH
METABOLISM THERE, (INAUDIBLE)
ANIMAL PK INTO THE CANONICAL WE
SEE THE PEAK. SO WE KNOW IT IS
NOT DUE TO VASCULARIZATION,
BECAUSE THERE IS MOME TAB LIMB,
SO LIKELY DUE TO TRANSPORTER AND
ALSO DISTRIBUTION TRANSPORTER
ALONG THE INTESTINES. AGAIN WE
DON'T HAVE DATA TO DEMONSTRATE
THAT BUT JUST BRING PEOPLE'S
ATTENTION WORK ON ASSAY WILL
REALLY HELP YOU CAN REALLY WORK
ON THE TRANSPORTERS THAT WE ARE
GOING TO START COLLECTING THE
DATA LIKE IN THE EARLY 'T 90s
WE STARTED ON THE CYP REGULATION
GUIDANCE AND ALL THE CYP,
TRANSPORTERS. THIS ONE SHOW YOU
THE TRANSPORTERS HAVE SO MANY
DIFFERENT (INAUDIBLE) SOME
UPTAKE, SOME EFFLUX, SOME HAVE
BOTH ACTIONS IN AND OUT FOR THE
EFFLUX TRANSPORTER WE USE THIS
TRANSWELL SET UP BY ALSO HAD
TRANSWELLS. TRANSWELLS YOU HAVE
FROM (INAUDIBLE) AND MEMORY
HERE, CELL MEMBRANE AND THEN YOU
HAVE B SIDE HERE SO LOAD DRUG
FROM A SIDE DETERMINE DRUG ON
BOTH SIDE AFTER CERTAIN
INNERVATION TIME OR LOAD DRUG ON
B SIDE THIS CHANNEL HERE AND
LOOK AT DRUG CONCENTRATION ON
BOTH SIDES, THEN YOU CALCULATE
WHAT IS CALLED THE EFFLUX SO BY
BOTH REACTIONS AND THEN LOOK AT
EFFLUX RASH YES THERE. YOU HAVE
BACKGROUND NOISE BUT IF YOU
RATIO GREATER THAN 203 FOR SURE
YOU HAVE EFFLUX TRANSPORTERS.
WHEN WE DO THIS TRANSWELL ASSAY
WE NEED THE ASSESS MEMBRANE --
ONE PARAMETER TO MEASURE IS
CALLED TIER MEASUREMENT. BECAUSE
IF MEMBRANE NOW FORM TIGHT
JUNCTION THERE, YOUR THE
TRANSPORT THROUGH MEMBRANE THERE
ACTUALLY IS A PATIVE DIFFUSION
BECAUSE YOU HAVE HOLE THERE. SO
YOU HAVE THE MAKE SURE YOU HAVE
THE BETWEEN CELL THAT FORM TIGHT
JUNCTION AND REALLY GOES LIEU
CELL MEMBRANE THROUGH DIFFUSION,
OR THROUGH TRANSPORTERS THERE.
TO MEASURE THE TIERS, MEASU
MEASUREMENT, BEFORE ONLY CELL 24
THE CELL TRANSPORT CELLS NOW YOU
SEE DRIVE FOR PACE AND RATE.
PROVIDE 96 WELL TRANSWELL
ALREADY. HOWEVER FOR THE PEER
MEASUREMENT, PRETTY CLASSICAL
THEY USE THE MANUAL ONE. REALIZE
THE GAP HERE NCATS SBR FUNDING
WE CONTACT APPLIED BASICS THAT
IS A COMPANY THAT HAVE 24 CELL
MEASUREMENT DEVICE THERE. WE ASK
IF THEY CAN MODIFY WHAT THEY
HAVE THERE AND MAKE FOR 96
WELLS. SO I SEE (INAUDIBLE)
THERE, MY GROUP, HE IS FORCE
BEHIND DEVICES. REALLY WORK WITH
COMPANY, CASCADE VALIDATE THE
DEVICE IS PRETTY MUCH IN
DATABASE. IN THIS DEVICE MEASURE
EACH WELL ONE SECOND,
AUGUSTN'TED TO FINISH
MEASUREMENT WE THINK TWO MINUTES
AND ALSO THIS DEVICE IS SO
COMPLEX THEY CAN PUT INSIDE
INCUBATOR SO YOU HAVE LOTS OF
FLUCTUATION TEMPERATURE WHEN YOU
MEASURE HERE. NOW I WILL TALK
ABOUT COMPLICATIONAL (INAUDIBLE)
BASED TOOL TO DESIGN AND PREDICT
DRUG CONTENT STRIAT CONCENTRATIS
NOT CHEAP. NEVERTHELESS WHEN WE
HAVE COLLABORATION WITH IP
CONSORTIUM IT IS A
PHARMACEUTICAL SCIENTIFIC WHEN
THEY GET TOGETHER AND TALK ABOUT
SCIENCE PART. THEY ALSO MENTION
VERY HELPFUL TO PUBLIC IF WE
HAVE SOME AVAILABLE SOFTWARE. SO
WITH THE DATA WE HAVE OVER TEN
YEARS AS I MENTION TIER 1 WE
HAVE DATA OVER 30,000 COMPOUNDS,
AT THIS MOMENT WE HAVE MODEL FOR
PARAMETERS SO THE NEXT STEP IS
TO START A WEBSITE WHERE WE
START DISSEMINATE MODELS, THESE
RETROSPECTIVELY VALIDATE AND
SUCCESS MARKET DRUGS, AND ALSO
THIS MODEL YOU CAN JOIN YOUR
STRUCTURE WITH ENZYME MOLECULE
OR (INAUDIBLE) THERE. FOR THE
PREDICTION OUTPUT, GIVE YOU SOME
CONFIDENCE OF FORCE AND
IMPORTANTLY YOU WANT PEOPLE USE
-- WANT SOMETHING BIG GUY BEHIND
THE SCREEN WE ARE DOING THERE SO
YOU CAN DOWNLOAD IT AND WE WILL
SEE DOWNLOAD YOU CAN REALLY
DESIGN AND PREDICT FOR YOURSELF
PROTOCOL REMEMBER TIER 1 ASSAY
ALL THESE PROTOCOL WE POSTED AT
(INAUDIBLE) TOGETHER WITH
PARTIAL DATA SETS, WE CANNOT
REROLLS ALL YET BECAUSE ALL
THESE ARE ACTIVE ONGOING
PROJECT. WE HAVE SOMETIME HAVE
THIS CREDA AGREEMENT AND WE
CANNOT RELEASE ALL STRUCTURE
FROM COMPOUND YET. THIS IS A
SCREEN SHOT OF THE WEBSITE. I
GAVE YOU THE WINDOW, YOU CAN
JOIN YOUR STRUCTURE HERE OR
REALLY DO THE BATCH, SOME --
LIKE COMPANIES THEY HAVE DESIGN
COMPOUND PRECONDUCT SO WE CAN
HAVE STABILITY MANAGE UP TO
(INAUDIBLE). SO YOU CAN DOWNLOAD
IT, YOU DON'T NEED TO WORRY
ABOUT THE PRIVACY ISSUES THERE.
THIS IS JUST WE ARE -- CURRENTLY
WE HAVE NANOMOLARS THERE BUT AS
WE GROW WE WILL ADD MORE MODEL
THERE ITHERE IS. YOU CAN SEE MM
STABILITY, (INAUDIBLE) DIFFERENT
PH AND SOLUBILITY AND CELL
MICROSTABILITY THESE ARE
PROTOCOLS FOR ASSAYS, WE UPLOAD.
SO THAT ONE MODEL WHAT WE TALK
ABOUT HELP CHEMISTS DESIGN THE
STRUCTURE AND ALSO THESE DAYS
CAN DESIGN SO MANY, HOW MANY
REALLY MAKE.
SO REALLY HELP WITH THE GRAND
COMPOUND, PROBABLY GAVE THE ONE
LOOKS LIKE MORE LIKE A
(INAUDIBLE) HIGH PRIORITY FOR
THOSE STRUCTURE, WE HOPE WE CAN
ACCELERATE DISCOVERY OF THE TIME
NEEDED. ALSO THEY CAN HELP TO
EXPAND DISCREPANCY WHEN YOU DO
THE ENZYME VERSUS CELL BASED
ASSAY, SOMETIMES PROBABLY DUE TO
SOLUBILITY. P YOU HAVE STRAFER
MOLECULE WE CAN PREDICT
SOLUBILITY THEN YOU ARE SEEING
HOPEFULLY IN VITRO DATA THERE.
ALSO I KNOW THESE DAYS
ESPECIALLY DURING THE COVID
REPURPOSES LIBRARY SCREEN BECOME
POPULAR. SO COMPANIES START
PURCHASE BIG LIBRARY COMPOUNDS
THERE. .
SO BEFORE YOU DO PURCHASING YOU
SEE THESE TOOLS HOW TO HELP YOU
TO SELECT COMPOUNDS. SO THEY ARE
NOT EFFICACIOUS THERE. WHAT WE
TALK ABOUT NOW IS REALLY JUST IN
VITRO, DYNAMIC OPEN IN VITRO IS
CLOSED. SO AGAIN WE NEED TO HAVE
IN VITRO IN VIVO CORRELATION
THERE. IN VIVO PHARMACO -- THESE
ARE JUST COMMON PARAMETER WE
LOOK AT IN EXPOSURES, LIKE
VIABILITY ABSORPTION,
DISTRIBUTION, AND CLEARANCE AND
HALF LIVES AND SO ON. SO ONCE WE
GET ALL THESE IN VITRO ASSAY
ANIMAL DATA THERE, WE CAN START
THESE CELLS BASED MODEL. IF YOU
LOOK AT BUILDING LOCKS FOR THIS
MODEL, THE FOREST PART IS
EASIER, YOU CAN SEARCH FROM THE
THE FIRST PART IS EASIER, YOU
CAN SEARCH -- RELATED PARAMETER
FOR YOUR ANIMAL, THE ORGAN
VOLUME, BLOOD FLOW , TISSUES AND
ENZYMES AND ORGANS. YOU CAN DO
LIST RESEARCH FOR YOUR OWN DATA
AND WHEN WE TALK ABOUT SOME DRUG
BIOLOGICAL PHYSICAL CHEMICAL
PROPERTIES TALK ABOUT ALREADY,
FOR EXAMPLE IF IT IS COMPOUND
MADE YOU ALREADY HAVE PHYSICAL
CHEMICAL PROPERTY, SOLUBILITY
WEIGHT, PERMEABILITY.
(INAUDIBLE) ANIMAL STARTED
PROTOCOLS THERE, ANIMAL DATA
ALREADY SO FROM ALL THIS YOU
PLUG IN ALL THIS INFORMATION
INTO HERE AND CHANGE NOW TO THE
HUMAN MODEL RATHER THAN ANIMAL.
THE FIRST THING IS TO ACTUALLY
ESTABLISH MODEL WITH ANIMAL
BECAUSE THAT ANIMAL YOU CAN HAVE
DATA. WE USE (INAUDIBLE) TO
CONFIRM THE MODEL SO AFTER THAT
YOU CAN HAVE THE CONFIDENCE THAT
YOU CAN PREDICT. SO FOR THIS
KIND OF MODEL HAVE LOTS OF
APPLICATION. WE ARE JUST
STARTING DOING OUR LAB NOW BUT
YOU CAN THINK ABOUT PEOPLE
PUSHING PKPD MODEL, IF YOU DON'T
HAVE PK MODEL FORGET PD -- SO DO
PK FIRST THEN ADD PB COMPONENTS
WHATEVER TARGET YOU HAVE THERE.
THAT IS PKPD MODEL SO THE
PATIENT PK MODELING WE CAN USE
THE FORECAST OF HUMAN PK BEFORE
PHASE 1 CLINICAL TRIALS. WE CAN
ASSESS THE POPULATION SPECIAL
PHARMACOKINETIC AND DIFFERENT
POPULATIONS LIKE CHILDREN OR
PEOPLE HAS RENAL DEFICIENCY
HEPATIC FAILURE. HOWEVER TO TALK
ABOUT DBI YOU HAVE DRUG DRUG
INTERACTION THERE BUT THAT IS IN
VITRO BUT THESE ARE REALLY IF
THEY HAVE SOME POTENTIAL DRUG
DRUG INTERACTION HOW WE CAN
JUDGE THOSE REACTIONS TO USE
THIS MODEL TO DO SOME EXCISE
THERE AND FINALLY WE TALK ABOUT
PK PD OR EVEN TALKS RELATIONSHIP
WITH ZERO CONCENTRATION. THIS
IS THE MODELS. THAT IS PRETTY
MUCH ALL I HAVE HERE. I HOPE
THROUGH THIS TALK IF YOU LEARN
THINGS HOW WE DO USE IN VITRO
ADME ASSAY TO SUPPORT DRUG
DISCOVERY AND DEVELOPMENT ALL
THE WAY TO THE FINAL MARKET.
REALLY SEPARATED ASSAY STAGE
WISE IN THE DRUG DISCOVERY STAGE
WE USE HIGH THROUGHPUT PUTT
SCREEN ASSAYS BECAUSE YOU HAVE
SO MANY COMPOUNDS THERE YOU ARE
TRYING TO FILTER OUT WHAT ARE
THE SCAFFOLD, WHAT ARE THE
LIMITED COME POUNDS THERE, THAN
YOU DIVE TO THE TIER 2 ASSAY,
THAT IS REALLY GOING TO FOR YOU
TO CHOOSE THE IND SELECTIVE
NEEDS THERE AND WE CAN STORE ALL
IN VITRO DATA THOSE INFORMATION
CAN HELP BUILD THE MODEL, THAT
CAN PREDICT THE GUIDES TO THE
CLINICAL TRIALS. SO I WILL ENDS
THIS TALK AGAIN PROBABLY TO MAKE
DATA VALID YOU HAVE TO HAVE
(INAUDIBLE). SO NEED TO HAVE
CONTROLS AND REPLICATE RUNS FOR
EACH ASSAY. THANK YOU.
[APPLAUSE]
>>THANK YOU SO MUCH FOR A
WONDERFUL TALK. QUESTIONS.
>>THANKS FOR AMAZING
PRESENTATION. THAT WAS
INSIGHTFUL. FOR THE IN SILICO
ADME DETERMINATIONS, IS THAT THE
OUTPUT OF THAT, IS THAT
SUFFICIENT FOR ANY DOWNSTREAM
REGULATORY CONSIDERATIONS? OR DO
YOU HAVE TO DO THEM AGAIN?
>>(INAUDIBLE)
>>SORRY?
>>THIS IS EARLY STAGE. FILE IND
THROUGH THE DATA.
>>YOU STILL NEED THE WET LAB.
>>AS I SAID, IT IS NOT -- I
MEAN (INAUDIBLE) AND FOR EXAMPLE
YOU PROBABLY HAVE ANIMAL PK
DATA. YOU HAVE PK DATA ANY WAY,
LOOK AT EXPOSURE THERE. RIGHT?
SO YOU HAVE INFORMATION THERE
ALREADY. SO REALLY TO HELP YOU
TO SAY FOR EXAMPLE, HOW MUCH IS
DUE TO THE METABOLISM. AND IF
YOU HAVE THE LIVER FAILURE AND
HOW MUCH IMPACT IT WILL HAVE SO
YOUR IN VITRO DATA HELPS. THEY
HAVE ALL THESE SOFTWARES THEY DO
SOME MODELING THERE TOO. THEY
WILL SEE THERE'S ANYTHING THE
TRIGGER CHANGE THE LABEL, OR
EVEN ON THE MARKET. (INAUDIBLE)
BLACK BOX.
>>ESSENTIALLY A GREAT TOOL TO
HELP YOU SELECT WHICH COMPOUNDS
UP TO SUBSEQUENTLY --
>>YES.
>>BUT YOU STILL HAVE TO DO WET
LAB.
>>ACTUALLY I FELT MOST PEOPLE
RESPOND AID SAY DEVELOPMENT PART
PROBABLY EARLY STAGE, EARLY
STAGE ACTUALLY IS REALLY FOR YOU
TO DESIGN THE MOLECULE. I CAN
GIVE EXAMPLE, THERE IS NO
(INAUDIBLE) HERE. WE HAVE A
(INAUDIBLE) THEY ARE A SMALL
COMPANY. CRO THAT COMPOUND YOU
CAN JOIN. THOSE PEOPLE FORM
SMALL COMPANY THEY HAVE LOTS OF
EXPERIENCE IN THE PHARMA
ALREADY. (INAUDIBLE) THEY MADE
200 COMPOUND IN ONE OF THE
(INAUDIBLE) IN CHINA. THEY HAD
DATA FOR 75 COMPOUNDS. SO NOW
THEY START (INAUDIBLE) THEN
AGAIN THERE IS A MODEL. SO HOW
ABOUT YOU GIVE (INAUDIBLE) YOU
KNOW HOW IT IS RIGHT? SO FOR
STABILITY WE HAVE 95%
PREDICTION. AGAIN WE DON'T SEE
THE STRUCTURE.
>>
(OFF MIC)
>>YEAH IT IS ONLINE. SINCE WE
ALL -- I DID HAVE THE NEWER
SLIDES, WE LAUNCHED NCATS CALLED
ADME NCATS, THERE YOU CAN GO YOU
CAN TRY THINGS WE JUST LAUNCH A
YEAR AGO LAST YEAR, NOW WE HAVE
A -- OVER 2500 (INAUDIBLE)
AROUND 77 COUNTRIES IN ONE YEAR
AND AMONG THAT 500 RETURNING
(INAUDIBLE) SO PEOPLE ARE
TRYING. AGAIN OR CONTINUE ADDING
MORE. THE BEAUTY IS THAT IS AN
EFFORT THEY HAVE A LIBRARY, BUT
HERE WE ARE CONTINUING TO ADDING
NEW DATA. ACTUALLY FOR PHARMA
BECAUSE THEY HAVE BIGGER
(INAUDIBLE) THEIR MODEL 24 HOURS
UPDATE BECAUSE EVERY DAY NEW
DATA COME IN. ONCE YOU HAVE THIS
DESCRIPTOR SET ALL YOU NEED IS
(INAUDIBLE) TRAINING VERIFYING
ALL THESE. ONCE YOU HAVE THE
MODALITY, MODULE SET IT UP, VERY
-- I DON'T THINK WE HAVE SO MANY
COMPOUNDS SO WE ARE DOING MAYBE
MONTHLY. SO BUT STILL OUR -- THE
BEAUTY OUR DATA IS IT IS COVER
OVER 300 PROJECTS THE SCAFFOLDS
IS A VERY DIVERSE CHEMICAL SPACE
HERE. THOUGH THEY HAVE MILLIONS
COMPOUNDS THERE. YOU CAN LOOK AT
THIS REALLY WHAT (INAUDIBLE)
YOUR STAFF FOCUS ON THAT TARGET
RIGHT? SO YOU MAY HAVE MORE BUT
WE ARE NOT WORKING ON THAT
DISEASE ANY MORE, WE WILL SWITCH
OURS SO THERE IS ACTUALLY WE DO
THIS DATA MODELING ASSESSMENT WE
CALL (INAUDIBLE) THE RECENT DATA
IS MOST VALUABLE.
>>I HAVE A QUESTION ABOUT THE
ADME NCATS ALSO. THAT SOUNDS
LIKE A REALLY INTERESTING TOOL
AND WONDERING WHICH DATA GOES
INTO IT, IS IT JUST YOUR FIRST
PASS SCREENING WHICH I THINK IT
INCLUDES PAMPA BUT NOT CACO 2
RIGHT? (OVERLAPPING SPEAKERS)
>>PAMPA IS 2 PH THAT, IS
ORIGINAL IN THE LAB. BUT LATER
ON REALIZED FOR G ABSORPTION YOU
NEEDS TO HAVE LOWER PH SO WE DID
(INAUDIBLE) WE FIGURED, CAN DO
MORE PH LEVEL BUT BUILD
DATABASE. SO WE FIGURED THAT
WOULD BE (INAUDIBLE) FIVE IS
REASONABLE RIGHT? SO THEN WE
CORRELATE WITH IN VIVO
BIOAVAILABILITY AND TOGETHER YOU
HAVE A CORRECT ORDER METABOLISM.
IF YOU JUST USE THAT ONE
(INAUDIBLE) SOMETIMES NOT
ACCURATE. BECAUSE A (INAUDIBLE)
ADDING METABOLISM DATA THERE YOU
HAVE BETTER PREDICTION.
>>THAT IS INTERESTING BECAUSE
YOU DONE HAVE ANY TRANSPORTERS
IN THERE.
>>NOT YET. TRANSPORTERS, OKAY.
>>GIVEN THE CACO 2.
>>CACO 2 WE HAVE A FEW HUNDRED
COMPOUND NOW BUT USUALLY FOR NEW
MODEL MINIMALLY YOU NEED A FEW
THOUSAND COMPOUNDS. THE FIRST
MODEL IS I THINK IT (INAUDIBLE)
THAT WE HAVE ABOUT OF 07,000
COMPOUNDCOMPOUNDS. EVEN WITH TE
DON'T FEEL COMFORTABLE BUT NOW
WE HAVE OVER 31, 3200 SOLID
COMPOUNDS,
>>THANK YOU.
>>I DON'T SEE ANY MORE
QUESTIONS. THANK YOU SO MUCH,
XIN FOR YOUR WONDERFUL
PRESENTATION.
[APPLAUSE]
>>I HAVE ONE MORE SLIDE TO
SHARE. IF YOU BEAR WITH ME.
THANK YOU FOR BEING HERE, THANKS
FOR PRESENTERS FOR AMAZING
TALKS. Y'ALL FOR ATTENDING.
WOULD LIKE YOU TO FILL OUT THE
FEEDBACK SURVEY WE ARE GOING TO
SEND THROUGH EMAIL, CORRECT?
WOULD LOVE TO HEAR YOUR
FEEDBACK. PLEASE TELL US WHAT WE
DID GOOD, WHAT WE DIDN'T DO
WELL, WHAT DO WE WANT TO HEAR,
MORE FROM ASSAY MANUAL WHERE WE
CAN GO FUTURE TO LOVE TO HEAR
YOUR FEEDBACK. THE VIDEO OF
THIS WORKSHOP WILL BE AID MADE
AVAILABLE ON THE NIH VIDEOCAST
WEBSITE, WE WILL SHARE THAT WITH
YOU AS WELL, WE WILL ALSO SHARE
THE LINKS I TACKED ABOUT OR
RESOURCES, THE ASSAY GUIDANCE
MANUAL HAS, THE PRESENTATIONS OF
THE SPEAKERS, AT THEIR
DISCRETION OF COURSE IF WILLING
TO SHARE WITH Y'ALL. AND THAT IS
IT. THANK YOU SO MUCH FOR
ATTENDING. HOPEFULLY THIS WAS
HELPFUL. AND HAVE A WONDERFUL
NIGHT. I WILL ADJOURN THE
MEETING. THANK YOU.
[APPLAUSE]