>> OKAY.

THANK YOU FOR COMING TO ANOTHER

PROTEOMICS INTEREST GROWTH GROUP

LECTURE FROM THE SERIES.

TODAY'S SPEAKER IS DR. STAWRT

MAUDSLEY FROM THE NATIONAL OF

AGING.

HE'LL TALK ABOUT THE

PHARMACOLOGY OF THE PROTEIN

KAPPA RECEPTOR.

>> THANKS FOR YOUR INVITATION.

SO THANKS EVERYONE FOR COMING

TODADAY.

I HOPE THAT YOU'LL ENJOY THE

TALK.

IT'S PRETTY PROTEOMICS LIGHT BUT

PHARMACOLOGY HEAVY, MY PRIMARY

BACKGROUND.

THE REASON I GOT INTO PROTEOMICS

WAS TO TRY AND UNDERSTAND THE

EVER INCREASING COMPLEXITY OF

TE GPCR SYSTEM.

10, 11 YEARS AGO WE OPENED PAN

DOOR'S BOX AND REALIZED

RECEPTORS WHICH UNDERSCORE

ALMOST ALL KNOWN FORMS OF

PHARMACOLOGY, PHYSIOLOGY, WAY

MORE COMPLICATED THAN WE

THOUGHT.

IS THIS ISN'T A HINDRANCE.

IT'S A HUGE BONUS FOR US BECAUSE

IT GIVE IT IS CAPACITY TO

DRAMATICALLY EXPAND THE PHARMACO

SPHERE.

WE'LL TALK ABOUT GPCR FROM

DISCOVERY TO EXPLOITATION OF

PHARMACOLOGICAL ENTITIESCH ONE

THING I TELL MY STUDENTS IS ONE

INTERESTING THING ABOUT GPCR IS

THEY'RE THE NANOMACHINES

PROTOTYPE.

WE KNOW SO MUCH ABOUT TEEN-BASED

FUNCTION OF RECEPTORS, VIRTUALLY

MORE THAN ANY OTHER

TRANSMEMBRANE PROTEIN, THERE ARE

ALMOST PAPERS IN WHICH YOU CAN

CATEGORIZE A MUTATION OF EVERY

AMINO ACID IN A SINGLE GPCR SO A

VAST AMOUNT OF DATA IS PRODUCED.

WHAT I'M GOING TO TALKED TODAY

ABOUT IS THE CONTEXT WHICH

GPCR'S EXIST AND HOW IMPORTANT

THIS IS FOR THAT PHARMACOLOGY

AND ALSO FOR THE RESEARCH INTO

DRUGS WHICH EXPLOIT THESE

IMPORTANT RECEPTORS.

SO I WAS PRIVILEGED TO WORK WITH

BOB WHO DISCOVERED THE FIRST

ONE.

JUST WHILE I WAS BORN ACTUALLY.

SO THIS WAS A BETA 2 RECEPTOR, A

40 KDA PROTEIN SO IT LOOKS LIKE

AMINO ACID SEQUENCE, LOOKS

BENIGN BUT THIS OPENED UP ALMOST

HALF THE CURRENT KNOWN DRUGS.

TARGETED TARDZ THESE PROTEINS.

SO AN ASTOUNDING DISCOVERY.

SO YOU DO A HYDROXY PLOT ON

THESE GUYS THEY HAVE THE

CLASSICAL HEXA HELICAL

HYDROPHOBIC REGIONS.

BUT HELIX 7 IS NOT PARTICULARLY

FATTY.

THIS 7TH HELIX IS FUNKY IN THE

MAJORITY OF RECEPTORS.

SO THIS IS THE PROTOTEEP

RECEPTOR.

IF YOU RUN THROUGH PREDICTION

PROGRAM YOU CAN SEE STRONG ALPHA

HELICAL PREDICTIONS BUT APART

FROM NUMBER 7 WHICH IS FAR LESS

HYDROPHOBIC.

THIS IS A KERNAL PROPERTY IN THE

MAJORITY OF GPCRs THAT WE

KNOW.

WHEN I WAS DOING MY Ph.D. AND

MY UNDERGRADUATE WE USED TO MAKE

HELICAL WHEEL DIAGRAMS BEFORE WE

HAD A CRYSTAL OF GPCR AND YOU

CAN ALIGN THEM BASED ON

CONSERVATIVENESS OF AMINO ACIDS

AND POLARITY.

AND WE GENERATED THIS CONCEPT OF

A POLAR POCKET INSIDE THE

HELICAL REGIONS OF GPCR WHICH

HYDROPHYLIC REGIONS INSERT

THEMSELVES AND INTERACT WITH THE

RECEPTOR ITSELF.

SO THE PROBLEM WITH GETTING A

DETAILED STRUCTURE WAS BASED ON

EXPRESSION PROFILES.

MAJORITY OF GPCRS ARE EXPRESSED

TO LOW LEVELS AND JUST IN THE

PLASMA MEMBRANE WHERE THEY'RE

FUNCTIONALLY RELEVANT.

BUT RHODOPSIN IS A GPCR LIKE

PROTEIN CLASSICALLY NOT A G

PROTEIN CUPPED RECEPTOR BUT A

PROTOTYPE OF GPCR.

THIS EXPRESSED AT TREMENDOUSLY

HIGH LEVELS.

SO IT'S BASICALLY A GOOD SOURCE

TO GET PROTEIN TO TRY TO GET A

CRYSTAL FOR THIS IMPORTANT CLASS

OF PROTEINS.

SO HERE IS AN ALIGNMENT OF

RHODOPSIN WITH HUMAN BETA 2

RECEPTOR SO IT ALIGNS NICELY.

NOT A BAD ANALOG FOR BETA 2

RECEPTOR.

SO FINISHING Ph.D. GOT A FIRST

CRYSTAL WITH RHODOPSIN AT

5-ANGSTROM RESOLUTION.

YOU BEGIN TO SEE DENSE AREAS

INDZ KAYTIVE OF TRANSMEMBRANE

REGIONSCH THIS IS REFINED AND

YOU START TO SEE 1, 2, 3 4, 5, 6

, 6, 7, ALPHA HELICES.

CRUNCHED INTO A BARREL.

YOU CAN MAKE THE 3-D MODELS SO

YOU CAN START TO SEE THE ALPHA

HEELEY SEIZE FORMING.

AND VEBTLY IN 2000 CAME UP WITH

A DESEN CRYSTAL 1.5-ANGSTROMS

WHERE THEY GET GOOD SPATIAL

RESOLUTION OF ALPHA HELICAL

REGIONS.

SO BRIAN (INDISCERNIBLE) CRACKED

IT IN '07 AND 2011 AND GOT A

DECENT HUMAN BETA 2 RECEPTOR.

HIGHLY MODIFIED PROTEIN, NOT THE

WILD TYPE BUT THIS WAS AS GOOD

AS IT'S GOING TO GETS.

SO YOU CAN FINALLY UNDERSTAND

WHAT THIS PROTEIN LOOKS LIKE AND

WE HAVE DECENT MOLECULAR

COORDINATES FOR THE INTERACTION

OF THE O THE HELIXES.

THE ESTIMATES ARE 1 TO 2,000

GPCRs IN THE GENOME.

IN US IT'S ABOUT 1% OF THE WHOLE

GENOME IS GPCR WHICH IS

ASTONISHING CONSIDERING THIS IS

ONE CLASS OF TRANSMEMBRANE

PROTEINS.

LOWER SPECIES YOU GET 5%.

THINGS LIKE ELEGANCE WORMS ARE A

MAJOR DOMINANT FACTOR OF THE

ENTIRE ORGANISM.

THESE ARE FOUND IN EVERY FORM OF

LIFE THAT WE KNOW OF AT THE

MOMENT.

ASTONISHLYPLAST NICK THEIR

ABILITY TO INTERACT WITH LIGANDS

FROM PHOTONS TO COMPLEX

PEPTIDES.

ESSENTIALLY THIS IS ONE OF THE

CORNERSTONES OF EVOLUTION.

WE THINK THE FACT THAT GPCRs,

THE FIRST ONE WE THINK THAT WAS

PROTOTYPE IS BACTERIA RHODOPSIN.

THIS IS A PROTON COMPOUND, NOT

REALLY A G COUPLED RECEPTOR BUT

A MECHANISM FOR THE EARLY

PROKARYOTIC AND EUKARYOTIC

ORGANISMS TO SENSE PROTONS,

PERHAPS THE MOST FUNDAMENTAL IN

A MOLECULE AND THE ENTIRE

UNIVERSE.

SO IT'S VERSATILE TRANSMEMBRANE

PROTEIN AND IT COUPLES THE

VIRTUALLY ALL FORMS OF SIGNAL

TRANSDUCTION.

AS MENTIONED AT THAT TIME TOP

50% OF THE COMMON THERAPEUTICS

ARE TARGETED TOWARDS

MANIPULATING GPCR FUNCTION SO

IT'S A PLATFORM WE CAN'T IGNORE.

THE MAJORITY FORM IT IS

PHYSIOLOGY OF CONTROLLED IN SOME

WAY DIRECTLY OR INDIRECTLY

THROUGH REGULATION OF GPCRs.

THE CLASSIFIED, THIS IS A RECENT

CLASSIFICATION SYSTEM

FREDERICKSON, CALLED THE GRASS

SYSTEM.

I WANTED TO SHOW YOU HOW

ENORMOUS AND VARIED THIS

RECEPTOR FAMILY IS.

THIS IS THE GRAPH SYSTEM HERE.

THIS LITTLE PINK ARROW IS WHERE

WE LIVE AND BREATHE.

THIS IS RHODOPSIN LIKE FAMILY

WHICH IS THE MAJORITY OF THE

PROTEINS WE STUDY AND KNOW A LOT

ABOUT.

ALL THESE OTHER GUYS WE KNOW

THAT FUNCTION BY ANALOGY, THEY

ARE VERY LOW HOMOLOGY AT THE

IDENTITY LEVEL OF THE AMINO

ACIDS TO THE RHODOPSIN.

SO THIS IS WHERE IT STARTED.

EVEN THIS IS IMMENSELY COMPLEX

STILL.

THIS IS THE SIMPLE STUFF, WE

HAVEN'T GOT INTO THE ACTUAL

FUNCTIONAL BASIS OF PHARMACOLOGY

YET.

THIS IS JUST AT THE STRUCTURAL

LEVEL.

UNFORTUNATELY ACTUALLY ONE

INTERESTING TOPICS I DID IN MY

Ph.D. THESIS WAS TO REDERIVE

ALL THESE PHYLOGENETIC TREES,

BECAUSE THESE ARE BASED ON

SEQUENCE WHICH DON'T HAVE TO DO

WITH LIGAND INTERACTION.

THE LIGAND INTERACTION IS

CREATED BY POCKET IN

NON-CONTIGUOUS AMINO ACID

SEQUENCES.

SO YOU CAN REDRAW THIS ON 3

DIMENSIONAL POCKETS TO INTERACT

WITH LIGANDS AN IT LOOK

COMPLETELY DIFFERENT AND YOU

BEGIN TO UNDERSTAND WHY CERTAIN

DRUGS HOP FROM ONE RECEPTOR TO

THE ANOTHER.

CLASSIFICATION BY PRIME SEQUENCE

DOESN'T MEAN THAT MUCH.

BUT WE CAN LOOK AT RECEPTOR IN A

GENERIC SENSE.

THIS IS THE CLASSICAL GPCR.

THE REASON I CALL IT A

NANOMACHINE IS WE KNOW SO MUCH

ABOUT -- GLYCOSYLATION IS

IMPORTANT FOR EXPRESSION OF THE

PROTEIN.

THIS LITTLE DRY MOTIF HERE,

EXTENSION, THIS IS ONE OF THE

IMPORTANT PROCESSES INVOLVED IN

ACTIVATION OF THE RECEPTOR, THAT

IS PROTONATED INSTANTLY AND THEN

THE RECEPTOR ACTIVATES.

IT ACTIVATES AND PUSHES DOWN THE

HELICAL EXTENSIONS OF TM-5 AND 6

AND THESE INTERACT WITH THE G

PROTEIN.

AND POST ACTIVATION YOU GET A

REGULATORY FEEDBACK

PHOSPHORYLATION OF THIRD LOOP OR

CARBOXY TERMINUS.

THIS IS GENERIC.

THERE'S LOTS OF VARIATION ON

THIS THEME BUT MAJORITY

RECEPTORS IN THE RHODOPSIN CLASS

SHARE CLASSICAL CHARACTERISTICS.

SO YOU CAN THINK OF THIS AS A

MOLECULAR MACHINE WHICH LITTLE

COMPONENTS HAS BEEN TRANSPOSED

TO OTHER RECEPTORS AN RETAINED

IN OTHERS.

AND I SHOW YOU THE LIFETIME WE

USED TO THINK RECEPTORS USED TO

LIVE AND BREE. THIS IS LIKE 6

LIVES ENCOMPASSES LIKE 25 YEARS

WORTH OF RESERK BY THOUSANDS OF

PEOPLE.

-- RESEARCH BY THOUSANDS OF

PEOPLE.

SO IT LOOKS SIMPLE NOW BUT IT

WAS A TREMENDOUS BURDEN TO GET

KNOWLEDGE.

IN THE GPCR LIFETIME OF

ACTIVATION AN AGONIST WILL BIND

TO THE RECEPTOR, YOU'LL GET

TURN-OVER OF G PROTEINS,

EXCHANGE OF GDP DOWNSTREAM

EFFECTS AND THEN REGULATORY

PROCESS.

THIS IS BRUTALLY SIMPLE, THERE'S

FAR MORE THINGS INVOLVED IN

THIS.

THIS IS A SIMPLE CARTOON BASED

FORMAT.

AS I MENTIONED AFTER THE

ACTIVATION YOU GET REFLEXIVE

INHIBITORY FEEDBACK MECHANISM,

PHOSPHORYLATION OF THE RECEPTOR

AND ALSO NEIGHBORING RECEPTORS

AS WELL.

SO THE EFFECT OF THE AGONIST

SPREADS LATERALLY AND

VERTICALLY.

ONE OF THE PARADIGMS WE WORK

WITH A LOT IS THE INTERDICTION

OF SUCCESSIVE INTERACTION WITH G

PROTEINS BY THE BINDING OF THE

ARRESTIN TO THE HIGHLY

PHOSPHORYLATED FORMS OF

RECEPTOR.

THE ARRESTIN IS BASICALLY A

TERMINATETOR OF SIGNALS WHICH

BIOINTERACTIONS WITH CLATHRIN

AP-2 DRAG ACTIVE RECEPTOR WHICH

IS PHOSPHORYLATED TO A CLASS

WHERE YOU CAN INTERNALIZE IT.

THIS POPS THE AGONIST OFF,

DEPHOSPHORYLATE THEN YOU HAVE

THE RECENT BACK TO MEMBRANE TO

CYCLE RECEPTOR OR WITH EXCESSIVE

STIMULATION FROM THE EXTRA

CELLULAR GENERAL YOU GET

LYSESOMAL DEGRADATION AND DOWN

REGULATION SO ALL THAT RESEARCH

WAS 20 YEARS AND 6 LIVES.

FROM THAT WE UNDERSTAND HOW THE

ACTIVATION PROCESS WENT AND WE

WANT TO WORK FROM POINT OF VIEW

OF PHARMACODYNAMICS TO

UNDERSTAND THE RELATIONSHIP

BETWEEN TYPE OF LIGAND AND THE

ACTIVATION OF ALL THESE COMPLEX

DOWNSTREAM EVENTS.

THIS TELLS US MORE ABOUT THE

PHARMACODYNAMIC MECHANISMS WE

CAN USE.

THIS IS THE OLD TWO STATE MODEL

WE USED TO WORK ON.

INACTIVE RECEPTOR WHICH ISN'T

TURNING OVER G COUPLED PROTEIN

AND CLASSIC STATE.

YOU GET PRODUCTIVE COUPLING, AND

INVERSE AGONIST DISCOVERED WHEN

CREATED CONSTITUTIVE MUTANTS

PUSHES THE OTHER WAY.

ANTAGONISTS CLASSICALLY SHOW A

POOR BINDING AFFINITY

DISTINCTION.

SO THEY DON'T DO MUCH.

BUT REALLY THAT'S A FALLACY,

THERE'S NO SUCH THING AS

ANTAGONIST.

AGONIST, ANTAGONIST, IT'S THE

SAME THING, IT'S CONTEXTUAL.

VIRTUALLY IMPOSSIBLE FOR A

LIGAND TO BIND WITH HIGH

AFFINITY TO RECEPTOR AND DO

NOTHING.

EVEN IF IT'S DOING NOTHING IT'S

DOING SOMETHING IN THE NETWORK

OF ENTERACTOR IT IS RECEPTOR IS

INVOLVED WITH ANYWAY.

A CLICHE USED IN SOCCER IS THAT

PLAY PLAYERS ARE ALWAYS

INTERFERING -- IF YOU'RE ON THE

PITCH YOU'RE INTERFERING WITH

PLAY.

EVEN IF YOU'RE DOING NOTHING

SOMEONE IS WASHING YOU,

THEREFORE YOU'RE -- WATCHING

YOU.

EVEN IF NO WHATEVER YOU'RE

SOMETHING.

SO A PASSIVE LIGAND DOESN'T

EXIST.

THE REASON THEY CALL THEM

ANTAGONIST IS BASED ON

CONTEXTUAL PHARMACOLOGY.

IF YOU BIND THE G CUPPED PROTEIN

RECEPTOR THAT WAS CALLED AN

ANTAGONIST BUT IF YOU LOOK AT

DIFFERENT OUTPUT FROM THE

RECEPTOR YOU SEE IT'S AN AGONIST

FOR SOMETHING ELSE.

THAT'S WHAT I WANT TO SHOW YOU

LATER ON.

SO WE PUSHED IT FURTHER WITH

MORE UNDERSTANDING HOW THEY

INTERACT AS CHEMICAL ENTITIES SO

WE HAD AN EXTENDED COMPLEX

MODEL, WHERE YOU GET

INTERACTIONS OF MULTIPLE

AFFINITY STATES AND MULTIPLE G

PROTEINS.

THIS EXPANDS AND EXPANDS AND

EXPANDS.

THE PROBLEM IS THOUGH, A LOT OF

THESE THEORIES ARE BASED ON G

PROTEIN INTERACTIONS.

THAT'S WHAT THESE ARE, G COUPLED

RECEPTOR, WHICH IS UNFORTUNATELY

IT PAINTS US INTO A CORNER TO A

CERTAIN EXTENT.

THE FAVORITE TERM NOW IS HELICAL

RECEPTORS.

EPIDETERMINEAL GROWTH FACTOR

RECEPTOR.

A LOT OF SIGNALING CAN GO

THROUGH GPCR WHICH IS

INTERDEPENDENT.

WHAT ARE THEY THEN?

HEXA HELICAL IS PROBABLY A

BETTER TERM.

INTERESTINGLY, IT'S NOT G

PROTEIN FUNCTIONALLY INTERACT

WITH RECEPTORS WHICH IS 11, 12

YEARS OLD BUT BACK THEN IT WAS

JUST OH, THIS IS AN ARTIFACT,

CELL BASED SYSTEMS, BLAH BLAH

BLAH, IT'S NOT REAL BECAUSE

THESE ARE -- SO YOU HAVE TO TALK

TO G PROTEINS.

NOW THIS IS STATUS OF FACT AND

THE WORLD OF NON-G PROTEIN

INTERACTION WITH RECEPTORS IS

NOW CONSIDERED PERFECTLY NORMAL.

TO SHOW HOW QUICK THIS ADVANCED,

IN 2004 A REALLY NICE REVIEW

SHOWING FUNCTIONAL PROTEINS

WHICH HAVE A DIRECT INTERACTION

WITH GPCR OTHER THAN G PROTEINS.

IT'S WAY MORE COMPLICATED THAN

WE THOUGHT.

THIS WAS AN 8-YEAR-OLD PAPER

EXPANDED DRAMATICALLY SINCE

THEN.

SO IMPORTANT THING IS THESE

ACCESSORY PROTEIN WHICH IS BIND

GPCRs OR THAN G PROTEIN, IT

DRAMATICALLY EFFECTS THAT

FUNCTION.

AND IT GIVES TEXTURE TO THE

TRANSDUCED SIGNALS SO YOU CAN

LIKEN TO A WAY LOOKING AT A SUB

CONDUCTANCE STATE OF AN ION

CHANNEL, THERE ARE CERTAIN

DEGREES OF OFF AND CERTAIN

DEGREES OF ON.

THAT'S WHAT WE THINK THE

ACCESSORY PROTEINS DO WITH

RESPECT TO GPCR PHARMACOLOGY.

THE BINDING INTERACTION OF

PROTEINS CREATE WHAT WAS CALLED

RECEPTORSOME WHICH WE ARE

BEGINNING TO REALIZE EARLY ON

WHEN DOING RECEPTOR AMINO

PURIFICATION, A LOT OF

DOWNSTREAM SIGNALING CASCADE WAS

PRE-ASSEMBLED TO THE RECEPTOR.

THIS MADE SENSE EVENTUALLY

BECAUSE IF WE LOOK BACK TO THE

MOTION STYLE INTERACTION OF

PROTEINS DOWNSTREAM OF RECEPTOR,

WHERE IS THE SPECIFICITY GOING

TO COME FROM?

HOW DO YOU SAY THIS SIGNAL IS

GOING TO MEC 4 AND NOT 7 SO

PRE-ASSEMBLY IS ONE MOST

IMPORTANT CONCEPT HERE.

THESE ACCESSORY PROTEINS ARE ALL

VARIETIES OF TYPES OF PROTEINS

SO IT'S NOT A SPECIFIC GENERIC

FORM.

THERE SEEM TO BE LOTS OF TYPES

THAT INTERACT WITH THE RECEPTOR.

SO WHAT THIS ESSENTIALLY MEANS

IS THE HELIXAL CORE DISCOVERED

AND WE KNOW ABOUT IS NOT REALLY

WHERE THE ACTUAL SIGNALING COMES

FROM.

THEREFORE, IF THAT'S NOT WHERE

IT COMES FROM, WE HAVE TO

UNDERSTAND WHAT IS THE RECEPTOR

TO REALLY BE ABLE TO LEARN HOW

TO DESIGN DRUGS SPECIFICALLY TO

CONTROL DIFFERENT TYPES OF

PHARMACOLOGY.

SO THIS IS BASICALLY WHAT WE

ENDED UP WITH, THIS MODEL OF G

PROTEIN DEPENDENT PROCESS.

AND THIS HUGE HORRIBLE EXPANSION

OF OTHER PROTEINS THAT INTERACT

FUNCTIONALLY WITH THE RECEPTOR.

THIS IS A TINY NUMBER OF

EXAMPLES OF THESE.

AND THESE REALLY DO ADD EXTRA

TEXTURE TO THE ACTUAL

PHARMACOLOGY OF GPCRs SO THIS

WONDERFUL WRINKLE WE PUSHED

QUITE HEAVILY WITH THIS IDEA

THAT THERE'S DIVERGENCE BETWEEN

THE SIGNALING PARADIGM WHICH IS

CLASSICALLY THROUGH SOLUBLE

SECOND MESSENGERS AN RELATIVELY

QUICK AND WANES WHERE

ACCESSIBILITY PARADIGMS

CONTROLLED BY ARRESTIN AND

NERVES AND TYROSINE KINASES

TENDS PRIMARILY TRANSCRIPTIONAL

IN ITS TARGET AND DEPENDENT ON

ASSEMBLY OF MULTI-PROTEIN

COMPLEXES.

THIS GUY CAN LIVE AND BREATHE

WITH JUST G PROTEINS.

RATHER THAN TWO STATE, WE THINK

THERE ARE MULTIPLE STAPLE

STATES, AND ALSO MULTIPLE

INACTIVE STATES AS WELL.

YOU HAVE SEEN AS WELL THE

DIFFERENT FORMS THAT EXIST

CONTROLLED BY DIFFERENT

INTERACTION WITH ACCESSORY

PROTEINS.

TO LOOK AT A FREQUENCY STATE, SO

A SINGLE RECEPTOR AND COLOR AND

HEIGHT CAN REPRESENT THE TYPES

OF BAY6ad ZILLION STATE RECEPTORS.

IF YOU CHANGE THE INTERACTION OF

A PROTEIN BINDING THE RECEPTOR

YOU CAN PROMOTE THEN ITS TYPE OF

STATE, ANOTHER PROTEIN WILL

PRODUCE A DIFFERENT STATE

PROFILE SO YOU CAN SEE HOW THE

RECEPTOR PHARMACOLOGY CAN BE

ALTERED QUITE DRAMATICALLY BY

THE STABLE INTERACTION WITH

BINDING PROTEINS.

WHAT THIS ESSENTIALLY DOES IS

CREATE DIFFERENTIAL COMPLEX.

THE NUMBER AND TYPE OF THESE IS

THE BIG DEBATE.

HOW ARE THESE LINKED TO AGING IN

OUR CONTEXT OF DISEASE, LOCATION

IS REALLY IMPORTANT.

WHAT WE THINK IS THOUGH THE

PREFERENCE AT LEAST FOR SAY A

SPECIFIC TYPE OF RESPONSE FROM A

SPECIFIC TYPE OF RECEPTOR STABLE

ISOFORM.

THESE ARE NOT EXCLUSIVE.

I DON'T BELIEVE THEY'LL BE ANY

EXCLUSIVITY BUT I WILL SAY

SOMEBODY HAS TO BE A WINNER,

THERE HAS TO BE A NUMBER ONE.

SO FOR THIS LIGAND IT MIGHT

PREFER THIS STATE OVER THIS

STATE.

THEREFORE, THIS TYPE OF LIGAND

WILL GIVE A MORE RELIABLE ALPHA

RESPONSE COMPARED TO B WHICH

GIVE AS BETTER RESPONSE.

THIS IS A HUGE BREAK THROUGH IN

PHARMACOLOGY BUT WE NEED TO TRY

TO UNDERSTAND WHAT MAKES THESE

FORMS AND HOW IMPORTANT THEY

ARE.

SO ESSENTIALLY WE THINK THESE

RECEPTORS AND STRUCTURES CAN

CONTROL SPECIFICITY AND FELIX --

HELIXIVITY.

BASED ON PREFERENCIAL

INTERACTION WITH PROTEINS.

I THINK THAT MY LAB IS

INTERESTED IN LOOKING AT

COMPOSITIONAL CHANGES AND

PATHOPHYSIOLOGY OF DISEASE TO

THE RECEPTOR SENSE.

A LARGE PART OF O THE

LONGITUDINAL DISEASE PROCESS IS

PROGRESSIVE ALTERATION OF THE

PHARMACOLOGICAL RELATIONSHIP

BETWEEN ENDOGENOUS COMPOUNDS AND

RECEPTORS THEY ARE SUPPOSED TO

INTERACT WITH.

I THINK THERE WILL BE PROMISE

SKEWTY AND ALTERATION OF

INTERACTION AND ALSO CHANGING OF

THE EFFICIENCY AND QUALITY OF

THE OUTPUT SIGNAL.

THOSE THINGS WE DON'T FACTOR

INTO APPLYING AS A TOOL TO

CREATE NEW DRUGS.

WE MODEL ON CLASSICS STATIC

SYSTEMS SO WE TRY TO CREATE NEW

MODELS FOR ADVANCED SYSTEMS IN

OUR LAB AND IT'S BEEN QUITE A

REVELATION THAT WE'VE SEEN.

THIS IS WORK I CARRIED ON BUTi/Y

ALSO IN THE PREEF YAWS LAB TO

SHOW AS A NICE EXAMPLE FOR

LOOKING AT THIS UNDERSTANDING OF

THESE RECEPTOR STRUCTURES AND

HOW IT CAN HELP UNDERSTANDING

DEVELOPMENT IN NEW DRUGS.

I WORK ON THIS RATHER

FASCINATING HORMONE, GNRH,

PRODUCED IN THE HYPOTHALAMUS,

IMPACTS THE PITUITARY WHERE IT

BINDS TO ITS COGNATE RECEPTORS

ON PITUITARY GONADOTROPES.

THIS IS THE PRIMARY CONTROLLED

REPRODUCTION IN VIRTUALLY ALL

ORGANISMS ALL THE WAY BACK TO

EVEN ELEGANS WORMS AND FLIES.

SO IN MAMMALS THERE'S THREE

PERFORMS OF PEPTIDE, GX 1, 2 AND

1 TO 5.

IT CONTROLS THE ACTIVITY OF THE

MAJORITY OF REPRODUCTIVELY

IMPORTANT TISSUES.

PROSTATE, TESTES, OVARIES AND

MAMMARIES, IT CONTROLS GROWTH

AND DEVELOPMENT AND ALSO ITS

PRIMARY FUNCTION WHICH INDUCES

DEVELOPMENT OF ALL THESE IS

RELEASE OF THESE GONADOTROPIN

FROM THE PITUITARY.

IT HAS SIGNIFICANT EFFECTS IN

THE BRAIN AS WELL AND ALSO LOCAL

EFFECTS AT ALL OF THESE TISSUES.

THIS WAS A INTERESTING POINT

WHEN I GOT INTO THIS FIELD WAS

THERE SEEM TO BE A DIFFERENCE

BETWEEN HOW GNRH BOUND HERE AND

BOUND TO THE THE PERIPHERAL

SITES.

THE ACTIVITY O THAT GENERATED

THESE SITES SEEMS DISTINCT FROM

THAT IN THE BRAIN.

AND AT THAT TIME THE CLASSICAL

ANSWER FOR THAT IS OH IT'S A

DIFFERENT RECEPTOR.

THAT TIME WE ONLY HAVE ONE

GENERATOR RECEPTOR IN MAMMALS.

IT'S EXCITING.

THE RECEPTOR SYSTEM CLASSICAL

RHODOPSIN LIKE TQ COUPLED

RECEPTORS HEAVILY WORKED ON.

SO WE CATALOG 250 RECEPTOR

MUTATIONS OF THIS VERY SMALL

RECEPTORS.

THE INTERESTING THING HERE IS

THE MAMMALIAN RECEPTOR GPCI

DOESN'T HAVE THE CARBOXY

TERMINUS WHICH WE SAW AS A

REGULATORY POINT IT MAKES

UNSTABLE AND DONE THAT TO

FACILITATE THE MAMMALIAN

REPRODUCTIVE SYSTEM SO BEFORE

YOU OVULATE YOU NEED A BIG SURGE

OF LH.

SO THE RECEPTOR WAS LOST ITS

AUTOREGULATOR CAPACITY TO ALLOW

IT TO RECEIVE CONSTANT PULSES OF

GNRH TO PRODUCE THESE

PRE-OVULATION SURGE.

UNITE A UNIQUE RECEPTOR.

THE REASON WE STUDIED IT IS

BECAUSE THAT LOSS OF CARBOXY

TERMINUS INDUCES A LOT OF EXTRA

INTERACTIONS AND HELICAL

REGIONS.

SO WE CAN SEE THE IMPORTANT

COMPONENTS OF HELICAL

INTERACTION RECEPTOR ACTIVATION.

FROM THIS M SYSTEM.

THE EXCITING THING IS PRESENTLY

THOUGH 2000 DERIVATIVES OF THIS

HORMONE PRODUCED SO SMALL

HORMONE BUT 2000 VERSIONS OF

THIS.

TO THINK SO WE HAVEN'T --

BECAUSE WE THOUGHT THERE'S ONE

RECEPTOR TO THIS GUY.

THIS IS WHAT WE TRY TO STUDY.

IN A CLASSICAL MAMMALIAN TYPE IS

THAT WE HAVE ONE TYPE OF

RECEPTOR, IT DOESN'T HAVE THIS

CARBOXY TERMINUS.

IN LOWER LIFE FORMS WE HAVE TWO

AND THREE TYPES OF GENERATOR

RECEPTORS.

IN HUMANS WE HAVE ONE.

THIS IS ESSENTIALLY HOW IT

CONTROLS THE REPRODUCTIVE

SYSTEM, RESPONSIVE TO GNRH BUT

THE TRUNCATION IS IMPORTANT FOR

THE PRODUCTION OF US MAMMALS.

SO IT'S PRIMARY USES AS A

CLINICAL DRUG ARE FOR FERTILITY,

CONTRACEPTION AND THERAPEUTIC

EFFECTS ARE BASICALLY

CONTROLLING THE HORMONE FUNCTION

ESSENTIALLY.

SO ALL THESEN CONDITIONS ARE

CONTROLLED BY THE CIRCULATING

STEROIDAL HORMONES.

THIS WAS THE PROBLEM WE WANTED

TO TRY AND SOLVE IN THIS CASE.

HOW COME THE PITUITARY EFFECTS

OF GNRH ARE DIFFERENT FROM THE

PERIPHERAL EFFECTS OF

REPRODUCTIVE TISSUES SO IF THE

PITUITARY WE HAVE HIGH AFFINITY

BIEPING OF GNRH, YOU HAVE MAP

KINASE.

CAUSES LH SECRETION.

HERE CLASSICAL AGONIST CALLED

STIMULATION OF THIS PATHWAY AND

CLASSICAL ANTAGONIST BLOCK THIS

PATHWAY BUT IN THE PERIPHERY

TISSUES LIKE PROSTATE OVARY AND

MAMMARY, SLIGHTLY LOWER AFFINITY

INTERACTION, DOESN'T GO THROUGH

GQ BUT GI, INHIBITS THE CYCLASE

AN ACTIVATES THE STRESS

ACTIVATING PROTEIN KINASE

FAMILIES.

ACTION IS ANTI-PROLIFERATIVE AND

THE INTERESTING THING IS, THESE

ANTAGONIST MOLECULES HERE WHICH

BLOCK IN THE ANTERIOR PITUITARY

ARE STIMULATORY HERE IN THE

PERIPHERY.

THIS IS WHAT CLASSICALLY WE

THOUGHT OF BEING A DISTINCT TYPE

OF SIGNALING EVENT.

A DISTINCT PHARMACOLOGY.Co:

SO CLASSICALLY YOU THINK THEY'RE

A DIFFERENT RECEPTOR.

WE HAVE A NICE COLONY OF WE HAVE

A VERY NICE COLONY THERE AND WE

DISCOVERED A TYPE 2 GNRH

RECEPTOR FROM THE PERIPHERAL

TISSUE.

AS YOU CAN SEE HERE WE DONE HAVE

THE CARBOXY TERMINUS IN

CLASSICAL RHODOPSIN LIKE

RECEPTOR.

THIS WAS ASTONISHING TO US, THIS

RECEPTOR CARBOXY TERMINUS.

THIS IS A MAMMAL.

SO THIS WAS LIKE QUITE SHOCKING

FOR US.

THIS SHOULD NOT HAPPEN IN A

MAMMAL.

BUT IT DID.

THE INTERESTING THING WAS WHEN

WE LOOKED IN HUMANS WE COULDN'T

FIND THIS PROTEIN, IT DOESN'T

EXIST IN HUMANS.

IT WAS RATHER UPSETTING FOR A

WHILE.

THE QUESTION WAS IF THIS DOESN'T

EXIST IN HUMANS WE HAVE A STOP

CO-DON TO TRY TO RESCUE BUT IT'S

JUST NOT THERE.

IF IT'S NO TYPE TWO RECEPTOR IN

HUMANS HOW DID WE GET THAT FUNKY

PHARMACOLOGY?

SO THAT'S WHAT WE TRIED TO LOOK

AT AND WE TRY TO SEE HOW THE

FUNCTIONALITY WE CAN TEASE OUT

BY USING LIGANDS INSTEAD.

SO HERE YOU CAN SEE THIS TYPE

TWO GENERATOR RECEPTOR, THE

CLASSICAL HALLMARK OF THIS AND

IF YOU LOOK AT PHOSPHATE

GENERATION WHICH IS DOWNSTREAM

ACTIVITY OF G ALPHA Q ACTIVATOR

YOU CAN SEE THE TYPE 2 LIGAND IS

MORE POTENT AND INTERESTINGLY

ENOUGH IN THIS MARM SET TYPE 2

THAN TAGNIST, THIS IS WHAT WE

SEE IN PERIPHERAL TISSUE, THIS

AGONISTIC PROPERTY, PHOSPHATE

TURN OVER, THIS IS FUNKY.

WHEREAS WE DIDN'T SEE IT ON THE

HUMAN TYPE 1.

THIS MARM SET TYPE 2 IS CREATING

A PHARMACOLOGY A LITTLE LIKE THE

PERIPHERAL PHARMACOLOGY WE

EXPECTED TO SEE FROM POTENTIAL

HUMAN TYPE 2.

AND YOU CAN SEE THIS HERE, WHERE

WE HAVE THE HUMAN TYPE 1,

PHOSPHATE TURN-OVER, FOR THESE

TWO AGENTS.

135 SEEMS ANING A TAGNIST AND WE

CHOSE ANOTHER STRUCTURAL AGENT

MOLECULE TO IT TO CHECK AS WELL.

THIS WAS THOUGHT A NEGATIVE

CONTROL BECAUSE HERE ON THE TYPE

2 THIS ANTAGONIST WAS

STIMULATORY AND THIS ANTAGONIST

AS WELL WAS INACTIVE.

SO WE ACTUALLY HAVE POTENTIALLY

WITH THIS COMPOUND A TOOL WHICH

WE CAN TRY TO LOOK TO SEE IF

THIS COULD CREATE OR THIS COUlNzÖ

REVEAL PRESENCE OF POTENTIAL

TYPE 2 IN HUMANS.

SO AS I MENTIONED, THE EFFECT OF

THESE COMPOUNDS ON PERIPHERAL

TISSUE IS ANTI-PROLIFERATIVE.

THIS IS POTENTIALLY IMPORTANT

FOR CANCER DRUG THERAPY.

SO WE USED A HUMAN CARCINOMA

CELL CALLED JAG 3 AND WITH

CONTINUOUS EXPOSURE TO THE THE

GENERATED LIGANDS YOU GET

REDUCTION OF CELL VIABILITY.

SO BASICALLY WE'RE KILLING OFF

THESE CHORE OWE CARCINOMA CELLS

WITH INCREASING CONCENTRATION.

BUT THE ANNOYING THING WAS THIS

ANTAGONIST WE THOUGHT WOULD BE

ABLE TO MIMIC THE TYPE 2

PHARMACOLOGY WAS THE WEAKEST AND

THIS ONE WHICH SEEMED COMPLETELY

INACTIVE, ANTAGONIST 135 WAS AS

POTENT AS GENERATOR 2.

THESE ARE HUMAN CELLS BY THE

WAY.

SO WE TRIED ANOTHER CELL LINE.

THIS IS THE BENIGN PROSTATE

CELL.

ANOTHER HUMAN LINE AS WELL.

WHEN YOU LOOK AT THE VIABILITY,

WHICH ACTIVATED THE TYPE 2 MARM

SET RECEPTOR, SEEMED TO BE

VIRTUALLY INEFFECTIVE WHEREAS

THE ANTAGONIST HERE LOOKING

INACTIVE, ANTAGONIST 135 IS VERY

GOOD INHIBITING ACTUAL CELL

GROWTH.

JUST TO MAKE SURE THESE EFFECTS

WERE THROUGH WHAT WE DIP WANT

THE SEE, THE HUMAN 1 RECEPTOR

WHICH WAS THE ONLY THING THERE.

WE TOOK 3 CELLS AN OVEREXPRESSED

THE HUMAN TYPE 1, AND SAW THE

SAME PHARMACOLOGY.

THIS PERIPHERAL FUNKY

PHARMACOLOGY WAS DEFINITELY ALL

VERY STRONGLY CONTROLLED BY THE

HUMAN TYPE 1 RECEPTOR AND NOT A

POTENTIAL TYPE 2.

THIS PHARMACOLOGICAL EVIDENCE

AND LACK OF GENE EXPRESSION MADE

US REALIZE IT'S TYPE 1.

TYPE 1 IN DIFFERENT CELL CONTEXT

THAT RESPONDS COMPLETELY

DIFFERENTLY, BINDS LIGANDS

DIFFERENTLY AND ACTIVATES A

DOWNSTREAM PATHWAY COMPLETELY

DIFFERENTLY.

SO THIS WAS OUR FIRST EXAMPLE OF

THIS REAL TRANSITION OF

PHARMACOLOGY.

WE CAN SEE THIS NICELY WITH

RESPECT IF WE LOOK AT SAY THE

ABILITY TO ACTIVATE G ALPHA I,

THIS PERIPHERAL DOWNSTREAM

EFFECT THAT WE NORMALLY SEE IN

PERIPHERAL TISSUES SO HERE ARE

THE JAG 3 CELLS AND BPH-1 CELLS.

IF YOU INCREASE CYCLIC K WITH

PHOSPHOLIN WE SEE THE CLASSICAL

ANTAGONIST HERE AND THIS GUY

WHICH HAD NO EFFECT SO WE SEE IT

IN BOTH CELL LINES.

WE ALSO SEE THIS ANTAGONIST ALSO

CAUSING APOPTOSIS OR PRO

APOPTOSIS IN THESE CELLS, WHICH

RECOGNIZES THE TRANSLOCATED

ACETYL CO-CLEAN TO THE OUTSIDE

OF THE CELL AND YOU CAN --

CHOLINE WITH THE CELL AND YOU

CAN P-38 AND JUNK, THE STRESS

ACTIVATED PROTEIN KINASES.

WE DO SEE THIS CLASSICAL

PHARMACOLOGY IN THESE CELL LINES

THAT WE SEE IN TISSUES.

AND YOU CAN SEE THESE DRUGS

THEMSELVES HAVE QUITE DISTINCT

EFFECTS.

YOU CAN SEE IT ON THE WESTERN

BLOTS HERE, SO THIS ANTAGONIST

LOOKS EXACTLY LIKE AB AGONIST.

WHEREAS THIS GUY HAS NO EFFECT

AT ALL.

AND THIS DIDN'T BLOCK CELL

GROWTH WHEREAS THIS ONE DID.

AND THE JAG 3 AND BPH.

THE EXCITING THING FOR US WAS WE

ALREADY HAVE THIS KNOWN ALREADY

POINT OF VIEW OF DOING THE TALK

IS THAT THESE TWO ANTAGONISTS WE

DID MORE THAN THIS.

BUT ONLY DISTINCT FROM ONE AMINO

ACID, HIGHLY DIGITIZED FORMS OF

GNRH BUT AFTER LISTENING TO,

GAVE US ALL THAT RECEPTOR

PHARMACOLOGY DIFFERENCE SO QUITE

A DISTINCTION OF STIFT ACTIVITY

AND QUITE A DISTINCTION OF ALPHA

I COUPLING SO WE HAVE A

SELECTIVE LIGAND PAIR TO CAUSE

AN EFFECT IN THE SPECIFIC TYPE

OF TISSUE.

AND WHAT WE DID WAS THEN BECAUSE

WE KNEW THE AMINO ACID CHANGES

CAUSE PHARMACOLOGY AND BINDING

DOWNSTREAM ACTIVATION, WE

STARTED TO TAKE ALL TYPES OF

GENERATORS WE HAVE TO SEE IF WE

CAN GET A PATENT INHIBITION OF

CELL GROWTH COMPARED TO IP

GENERATION HERE SO THIS BANDS

WITH GI EFFECT AND THIS IS THE

GQ EFFECT.

SWOAER LOOKING AT RARE YANS OF

AMINO ACID COMPOSITION OF THESE

GNRH ANALOGS TO THE TWO TYPES OF

PHARMACOLOGY.

SO YOU CAN SEE GROUPS

ESSENTIALLY OF DIFFERENT PATENTS

WHERE YOU -- THE LIGAND

ALTERATION CAUSES A SHIFT

TOWARDS ONE PATHWAY OR THE OTHER

O.

HUMAN TYPE 1 SITS IN THE MIDDLE.

THIS IS THE POTENT LIGAND.

THE LIGAND THAT DOES EVERYTHING

EVERYWHERE.

ALL THE OTHER ONES ARE VARIANTS.

SO WHAT WE'RE TRYING TO DO

EVENTUALLY IS FIND GUYS TO DO

THIS AND NOT THIS.

SO SUBSEQUENTLY IN RECENT YEARS

THIS IS LIKE THE OLD SCHOOL TWO

DIMENSIONAL FORMAT.

IMAGINE IN DIMENSIONS.

THAT'S WHAT WE'RE LOOKING FOR IN

THE FUTURE, BECAUSE THIS IS TWO

EFFECTS OF A RECEPTOR.

WE'RE NOW CURRENTLY SCREENING IN

OUR LAB ABOUT 200 INDICES PER

RECEPTOR ACTIVATION USINGkd

PHOSPHOPROTEIN ANALYSIS OF

RECEPTOR STIMULATION.

SO YOU GET MORE DOWNSTREAM

INVOLVEMENT OF O SIGNALING

PATHWAYS RATHER THAN CLASSICAL G

PROTEINS.

IT'S QUITE SCARY TO ROOK AT WHAT

A SINGLE LIGAND ON RECEPTOR TYPE

DOES IN A CELL.

WAY MORE COMPLICATED THAN WE

THOUGHT.

INTERESTING THING FOR US HOW

DOES THE PHARMACOLOGY FROM THE

RECEPTOR, HOW IS IT OCCURRING

AND DIFFERENT TISSUES IN THE

BODY, THIS IS MARM SET WE CLONED

AND GOT EXCITED ABOUT AND HAVE

THE CARBOXY TERMINUS, IT WAS

GROUND BREAKING BUTLY

MEANINGLESS IN HUMAN PHYSIOLOGY,

IT'S NOT PRESENT ENOUGH.

INTERESTINGLY WE SAW AS FAR AS

PROTEIN PROTEIN INTERACTIONS GO,

A NICE CARBOXY TERMINAL PDZ

DOMAIN.

THAT'S INTERESTING, TO CERTAIN

TYPES OF PROTEINS, AND

COLOCALLIZE IT IN CERTAIN AREAS.

IF THE MARM SET GOT THIS, WE

HAVE ONE RECEPTOR, SIMILAR TO

MARM SET FUNCTIONALLY, SO SURELY

THE TYPE ONE RECEPTOR HAVE A PDZ

DOMAIN AS WELL AND SIMILAR

FUNCTIONALITY.

BUT IT HASN'T GOT THE CARBOXY

TERMINUS.

THE ONLY PLACE TO CUT SEQUENCE

OF HUMAN TYPE 1 RECEPTOR TO KEEP

AN EXTENT PDZ DOMAIN IS RIGHT

WHERE IT'S CUT.

FUNKY THING HERE IS THIS IS

MEMBRANE SO THIS CAN'T BE A PDZ

DOMAIN.

BUT REMEMBER WHEN I WAS TELLING

YOU ABOUT THE 7TH HELIX FUNKY IN

GPCRs, THERE'S WAYS TO ALTER

THE PROPER ALPHA HELICAL PACKING

STRUCTURE, YOU CAN DO IT WITH

LARGE AMINO ACID LIKE TRIP AND

FEE.

YOU CAN HAVE PROLIEN DISRUPTION

OF VERTICAL HYDROGEN BONDING,

THIS IS AMINO ACID AND NON-AMINO

ACID AND SMALL AMINO ACIDS TO

INCREASE THE FLEXIBILITY.

LET'S LOOK AT THE HELIX OF HUMAN

TYPE 1 GENERATED RECEPTOR.

SO AS YOU CAN SEE HERE WHAT I'M

SHOWING AGAIN IS THE CLASSICAL B

TO 2 PLOT, THE CLASSICAL.

(OFF MIC)

>> SORRY.

IT'S AN INTERACTION MOTIF FOR

PDZ DOMAIN PROTEINS.

SO JUST A QUARTET.

WE CALL IT THE PDZ DOMAIN IS

LIKE 130 AMINO ACID PEPTIDE

ESSENTIALLY PRESENT IN A LOT OF

POST SYNAPTIC TRANSMEMBRANE

PROTEINS BUT THIS IS -- THAT'S A

BINDING MOTIF.

SAYING THE AMINO ACIDS IS

EVERYTHING IS NOT THE CAIRKS I

OOH TES PRESENTATION OF THAT 4

AMINO ACID.

YOU CAN SCREW IT BY ALTERING

AMINO ACIDS IN THERE BUT IT'S

JUST LIKE A LITTLE KEY.

SO ON THE CLASSICAL B TO 2 YOU

CAN SEE IT'S A PRETTY WATERY

TRANSMEMBRANE REGION.

SO THIS IS THE ACTUAL ALPHA

HELIX FROM THE HUMAN TYPE 1

RECEPTOR AND YOU CAN ACTUALLY

SEE TWO PROLIENS VERTICAL AND

THEN THERE'S A GLYCENE BENEATH

IT SO POTENTIALLY THE PROTEINS

WILL NOT SUPPORT THE ALPHA HELIX

AND THEN WE HAVE THE GLYCENE

HERE.

THESE AMINO ACIDS THAT I JUST

SHOWED IN GREEN, P, D, Y THESE

CHANGE SHAPE DRAMATICALLY WITH

THE ACTIVATION OF THE RECEPTOR.

SO WHAT SEEMS TO BE OCCURRING IS

UPON ACTIVATION THIS HELICAL

REGION IS DISTORTED, THIS

GLYCENE IS A PIVOT, POTENTIALLY

THE YSL MOTIF TO BE EXPOSED TO

THE REGION.

SO POTENTIALLY ALLOW THIS HELIX

TO ACT AS A SURROGATE CARBOXY

TERMINUS.

THIS IS THE LITTLE MOTIF DOWN

HERE AND DESTROY THE INTERACTION

OF THAT MOTIF TO MAKE THE

PROTEINS YOU CAN DO A SIMPLE

ALANINE MUTATION.

WHAT WE FOUND WAS THIS L-328

RECEPTOR EXPRESSED IN COX 7

CELLS DIDN'T SUPPORT YOU CAN

ACTIVATE WITH GNRH BUT IT ONE

HAVE THE INHIBITOR EFFECT ON

CELL GROWTH THE WILE TYPE

RECEPTOR DID.

YET, IT SUPPORTED PHOSPHATE

PRODUCTION SO G-2 COUPLED

RECEPTOR.

THIS DIDN'T SEEM GI COUPLED THAT

WELL.

THIS IS A TINY BIT OF STONE AGE

PROTEOMICS I'LL SHOW YOU TODAY.

WE WERE INTERESTED TO TRY TO

FIND USING THE RECEPTOR MUTANT.

WHICH PROTEINS WAS IT NOW

BINDING OR NOT BINDING TO AFTER

THE MUTATION, A VERY SIMPLE

PROCESS OF OVEREXPRESSING AN

ISSUE WITH RECEPTOR PHARMACOLOGY

TO GET ENOUGH TACT TO PULL DOWN

TO IDENTIFY WE HAVE TO PUSH

BEYOND PHYSIOLOGICAL PARADIGMS.

AN ISSUE WE HAVEN'T GOT BEYOND.

IDENTIFYING AND GO FROM THERE.

PARDON THE TERRIBLE GEL, THIS

WAS QUITE A WHILE AGO.

WE DISCOVERED THERE WERE

PROTEINS LOST, THAT WERE DOWN

REGULATEED FROM THE WILD TYPE

AND SOME WE ACTUALLY GAINED.

SO WE CUT OUT ONE OF THESE WHICH

WAS LOST FROM THE TYPE 1

RECEPTORSOME AFTER THIS

MUTATION, IT TURNED OUT TO BE A

MULTIPLE PDZ DOMAIN PROTEIN MAP

1.

HIGHLIGHTED YELLOWS WITH

PEPTIDES TO BE PICKED UP SO WE

GOT A REASONABLE COVERAGE OF

THIS QUITE BIG PROTEIN.

THIS MAP 1 IS A MEMBRANE PROTEIN

INVOLVED IN SCAFFOLDING VARIOUS

TYPES OF RECEPTORS.

WE OBTAINED A CLONE OF THIS MAP

1 AND SHOWEDDED BY IP THAT WE

CAN SEE HA TYPE 1 HERE, A TIME

DEPENDENT DRUG DEPENDENT

INTERACTION OF MAP 1 WITH

RECEPTOR WHICH IS FLAG TAGGED.

WE DON'T SEE THIS WITH THE A

RECEPTOR WHEN IT BINDS TO MAP

ONE.

THE INTERESTING THING IS WITH

RESPECT TO THE PHARMACOLOGY WE

SAW EXPRESSION OF ENDOGENOUS MAP

1 LOW IN THE PITUITARY WHERE WE

SEE CLASSICAL GENERATOR TYPE 1

PHARMACOLOGY BUT HIGHER IN THESE

PERIPHERAL REPRODUCTIVE ORGANS

THAT IS A FACT OR OF THE

PHARMACOLOGICAL SHIFT.

WHEN WE TAKE CELLS AN

OVEREXPRESS MAP 1 AND TRANSFORCE

THE SITUATION TO MAKE A NORMAL

PHYSIOLOGY BECOME THIS

PERIPHERAL PHARMACOLOGY WE CAN

OVEREXPRESS MAP 1 AND GET

INCREASE OF THE ACTIVATION OF

THIS JUNK PATHWAY WHICH IS THE

STRESS ACTIVATEDDED PATHWAY AS

THE ANTI-PROLIFERATIVE EFFECT IN

CELLS.

SO WE ALSO WANTED TO SHOW THIS

MAP 1 INTERACTION OR THE

OVEREXPRESSION OF IT ALSO

AFFECTED THE GI COUPLING WHICH

IS PERIPHERAL COUPLING THAT WE

SAW.

WE TAKE THE L-328A RECEPTOR AND

OVEREXPRESS MAP 1 YOU DON'T SEE

BIG INCREASE OF GI COUPLING.

THIS IS A CELL LINE WHICH

DOESN'T HAVE THAT MAXIMUM,

WHEREAS IF YOU'RE OVEREXPRESSING

MAP 1 WITH WILD TYPE RECEPTOR

YOU'RE ALLOWED TO INCREASE THE

ABILITY OF GNRH TO CAUSE THIS

iCOUPLING.

THIS SEEMS TO COMPARTMENTALIZE

AND PUSH THE RECEPTOR INTO A

COMPARTMENT OF THE CELL WHERE

THERE'S PREFERENCIAL INTERACTION

OF THE GI.

NOT THE PERIPHERAL TISSUES THAT

CAN'T COUPLE THE LIGANDS CAN'T

COUPLE 2GQ.

THERE'S OTHER RECEPTORS THAT

CAN.

IT'S JUST THIS RECEPTOR

POTENTIALLY THROUGH INTERACTION

WITH MAP 1 IS TAKE AIN WAY FROM

REGIONS WHERE G ALPHA Q IS KEPT.

THIS INTERACTION AND ALTER OF

PHARMACOLOGY BY ACCESSORY

PROTEINS WHICH COULD BE SPECIFIC

TO DIFFERENT TYPES OF CELLS OR

SITUATIONS REALLY MADE ME THINK

RECEPTORS BEING SYMMETRICAL

ENTITY.

IF YOU LOOK AT IT THE RECEPTOR

FROM TIN SIDE OR OUTSIDE LOOKS

EXACTLY THE SAME.

YOU HAVE THIS MOTIF, YOU HAVE

THIS MOTIF.

YOU HAVE INTRACELLULAR AND EXTRA

CELLULAR LOOPS.

THIS IS THE WAY WE THINK OF

LIGAND TRANSFER OF SIGNAL FROM

THE OUTSIDE, LIGAND BINDS,

ATTARS THE STRUCTURE AND YOU GET

TRANSMISSION.

LET'S THINK ABOUT IT FROM THE

OTHER WAY AROUND.

LET'S THINK IF YOU HAVE A LIGAND

IN THIS CASE WHICH IS A BINDING

PROTEIN INTERACTS WITH A

RECEPTOR ON THIS SIDE, WHY DONE

WE THINK THERE WILL BE

ALTERATION OF STRUCTURE ON THE

OTHER SIDE.

PEOPLE THINK WELL, WHAT'S THE

TRANSMISSION SIGNAL HERE?

THERE ISN'T ONE.

THE LIGAND INTERACTION MAYBE

FLEETING BUT RECEPTORS CAN

CHOOSE WHICH LIGAND THEY BIND.

THE TYPE 1 GENERATOR RECEPTOR IS

ALSO OXYTOCIN RECEPTOR AND ALL

THOSE LIGANDS BIND.

RECEPTORS DON'T KNOW.

THAT'S WHY I MENTIONED AT THE

START WHEN WE DID WRITING MY

Ph.D. WE DID THE BINDING

DOMAIN AS DISCRIMINATOR OF

PHARMACOLOGICAL FUNCTION, NOT

THE TRANSMEMBRANE AMINO ACID

SEQUENCE.

A LOT OF SEQUENCE IS REDUNDANT,

YOU CAN GET RID OF A LOT OF

CHUNGS BUT STILL KEEP THE LIGAND

BINDING DOMAIN.

IT'S A CONDUIT FOR A LIGAND.

LIGANDS CHANGE WITH

CONCENTRATION WITH AGE, THEY CAN

CHANGE POST TRANSLATIONALLY WITH

MODIFICATION OF DISEASE,

BASICALLY THIS CHOICE IS

EFFECTED DRAMATICALLY BY

INTRACELLULAR PROTEINS.

SO DEPENDING ON ALTERATION OF

LIGAND OR PRESENCE OF DIFFERENT

BINDING LIGANDS YOU CAN GET A

ACCESSORY PROTEIN SIGNAL

TRANSFER EVENT.

THIS IS REALLY IMPORTANT BECAUSE

IT CONTROLS THE FIDELITY OF

RECEPTOR LIGAND INTERACTIONS AND

ALSO WHAT WE'RE SEEING IS THIS

CHANGES DRAMATICALLY WITH AGE

AND DISEASE.

WHAT THIS IS GOING TO HOPEFULLY

LEAD US TOWARDS IS THE CAPACITY

TO DERIVE CONTEXTUALLY

PREFERENTIALLY DRUGS SO IN THIS

CASE WE HAVE A DRUG WHICH ACTS

ASD

AND AGONIST IN THE PERIPHERY.

BECAUSE IT CAN INTERACT WITH

POTENTIALLY DIFFERENTIAL FORMS

OF THE SAME RECEPTOR.

BASICALLY IMPORTANCE OF THIS

WORK IS TO REALIZE THAT THERE

ARE FAR MORE ACCESSORY SIGNALING

FACTORS TO A GPCR THAN WE REALLY

THOUGHT AND THESE INCREASED

RECEPTIVITY GIVE A HUGE VARIETY

OF RECEPTOR FUNCTION AT CELL

LEVEL, TISSUE LEVEL AND ALSO IN

OTHER FORMS OF CONTEXT, AGE OR

DISEASE.

THE QUESTION, CAN WE EFFECTIVELY

TARGET THESE DIFFERENT RECEPTOR

SYSTEMS.

SO THIS IS WHAT WE HAVE GONE

FROM IS THE SIMPLE TRANSDUCTION

MODEL OF TRADITIONAL RECEPTOR

WHICH INVOLVED THE TRANSIENT

INTERACTION OF G PROTEINS TO

THIS RECEPTOR MORTAL AS WELL

WHICH ALSO EXISTS ALONG WITH

THIS GUY.

THESE AREN'T EXCLUSIVE OF EACH

OTHER.

BUT THESE ARE STABLE

INTERACTIONS.

AS I SAID BEFORE, THESE TEND TO

PROMOTE OR SHOW STRONG

CONNECTION MORE TRANSCRIPTIONAL

LONG TERM EFFECTS, WHERE THESE

ARE WHAT WE CALL INTERMEDIARY

CELL METABOLISM, THINGS THAT

COME AND GO WITHIN 20 OR 30

MINUTES.

SO BASICALLY PRE-ASSEMBLY WITH

ACCEPTANCE TO PHARMACOLOGY LIMIT

THE INITIAL PROMISCUITY OF

SIGNAL TRANSDUCTION WE THOUGHT

OCCURRED WITH ONE SIGNAL

TRANSDUCTION SYSTEM THAT

EXHAUSTS AND GO TO THE NEXT AND

TO THE NEXT.

IF YOU HAVE ALL THESE PROTEINS

BIND THE ABILITY FOR THIS TO

TRANSFER TO ANOTHER SIGNALING

RECEPTOR BECOMES LESS AND LESS.

THEREFORE YOU CREATE DE FACTO

DIFFERENT RECEPTOR ISOFORMS

BASED ON THE SAME HEXA HELICAL

CORE.

THESE ARE LINKED TO

COMPARTMENTALIZATION REGIONS

DEPENDING ON TYPES OF PROTEINS

HERE AND BECAUSE THEY WILL THEN

SPECIFY AND HAVE A DIFFERENCE

FOR DIFFERENT TYPES OF

SIGNALING, YOU GET A UNIQUE TYPE

OF SIGNALING PROFILE FROM THESE

RECEPTORS.

SO THEREFORE, LIKELY A TEMPORAL,

PATHOLOGICAL AND TISSUE

VARIATION OF STRUCTURE BASED ON

UNIQUE EXPRESSION PROFILE OF

BINDING PROTEINS, WHERE

RECEPTORS ARE ON THE CELL AND

HOW IT'S AFFECTED BY PATHOLOGY

AND DISEASE.

SO BASICALLY WE WILL SEE GOING

FROM ONE SECTOR WITH ONE TYPE OF

SIGNAL THROUGH AGING, DISEASE,

OR TISSUE LOCATION, WE CAN GET A

TRANS-MODIFICATION OF RECEPTOR

FUNCTIONALITY BASED ON

ALTERATION OF THESE BINDING

PROTEINS.

THIS WE REALLY HAVEN'T FACTORED

INTO DRUG DESIGN.

WE OFTEN STICK WITH A STATIC

MODEL.

IT MAKES THINGS eSI BUT ALSO

MAKES THINGS FALL DOWN WHEN YOU

GO INTO THE COMPLEX WORLD OF

ACTUAL PHYSIOLOGY.

SO THIS IS WHAT I WANT TO SORT

OF END ON TODAY WITH THE IDEA WE

ARE AT THE TIP OF A

PHARMACOLOGICAL ICEBERG THOUGH

50% OF CURRENT DRUGS TARGETED

TOWARDS THESE PROTEIN, WE KNOW A

LOT ABOUT THEM, WE STILL DON'T

KNOW EVERYTHING.

WHAT WE'RE LOOKING AT IS THIS IS

WHAT WE HAVE DONE SO FAR AND WE

HAVE DONE WELL TARGETING G

PROTEINS WHICH IS ASTONISHING.

BUT ALWAYS BELOW THE WATER.

THERE ARE POTENTIAL AGONISTS FOR

THE NON-G PRESENT COUPLED FORM

OF THE RECEPTOR AND WE'RE DOING

WORK ON THAT AT THE MOMENT.

THERE ARE HETERO COMPLEX TARGETS

OF GPCR WHERE THEY

HETERODIMERRIZE WITH OTHER FORMS

AND UNIQUE LIGAND POCKETS.

IN MY FIELD IN NEURODEGENERATION

THE FIELD OF ALLOSTERIC

MODULATORS WHICH BIND TO

ALLOSTERIC REGIONS HERE OUTSIDE

THE ALLOSTERIC LIGAND BINDING

POCKET WHICH ARE IMPORTANT FOR

NEUROLOGICAL DISORDERS BASED ON

A NETWORK FUNCTION INTERACTION

OF NEURONAL SIGNALS BECAUSE THEY

DON'T DIRECTLY ACT INVESTIGATE

THIS SYSTEM THEY CAN PARTIALLY

MODIFY AND ENHANCE OR REDUCE

CERTAIN TYPES OF DOWNSTREAM

SIGNAL.

WE CAN ALSO AT THE MOMENT, THIS

IS WHAT WE HAVE DONE ALREADY IS

PRODUCE AGONISTS WITH A G

PROTEIN SELECTIVITY AND WE CAN

TRANSHELIX SPECIFIC TYPES OF

EFFECTS AND FROM THESE WE CAN

FIND TISSUE SELECTIVE AGONISTS

THAT WE CAN PRODUCE.

WHAT WE'RE TRYING TO DO IN MY

LAB IS TRYING TO PRODUCE WHAT'S

CALLED A CONTEXT-SPECIFIC

AGONIST.

SO WE'RE SPECIFICALLY LOOKING

FOR LIGANDS WHICH RETAIN A HIGH

FUNCTIONAL EFFICACY WITH RESPECT

TO NEUROPROTECTIVE PARADIGMS IN

TISSUES OF A NEURONAL ORIGIN

THAT WE AGE ARTIFICIALLY.

WORKING ON AGE NEURONS IN A CELL

CULTURE MODEL IS VIRTUALLY

IMPOSSIBLE.

ESPECIALLY FROM THE CENTRAL

NERVOUS SYSTEM.

THE CELLS JUST DIE.

THEY JUST REALLY HATE IT.

BUT WHAT WE TRIED TO DO IS TRY

TO CREATE A DISEASE CELL MODEL

THAT DOES MIMIC THE PHARMACOLOGY

AS FAR AS THE PROTEOMIC

EXPRESSION OF CNS TO FIND A

COMPOUND WHICH CONTAINS EFFICACY

IN THAT DISEASE STATE AND SEE IF

THAT HAS A GREATER FUNCTIONALITY

WITH RESPECT TO NEUROPROTECTION

IN AN OLD MODEL.

FINALLY ONE THING THAT I HAVE

TRIED TO PUSH A LOT IS THIS IDEA

OF LIGAND DIAGNOSTICS.8

SO IF WE APPRECIATE THE

FUNCTIONALITY LIGAND BINDING AND

PHARMACOLOGY OF GPCR IS

CONTEXTUALLY SENSITIVE TO THE

MILE EWE, THE PROTEINS THAT ARE

PRESENT, THEN TECHNICALLY YOU

HAVE A LIGAND WHICH CAN BIND AND

INTERACT WITH A CELL ENTIRELY

BASED ON WHAT PROTEINS ARE

INSIDE.

SO WE'RE TRYING TO DO THIS WITH

A CANCER CELL TYPE MODEL

OVEREXPRESSING A P-53 MUTANT.

NOW, P-53 BINDS MDM-2, SO MDM-2

IS E-3 UBIQUITIN LIGASE.

MDM-2 BINDS ARRESTIN.

THESE ARRESTIN BINDS THE

RECEPTOR.

SO P-53 CAN DIRECTLY SENSE AND

IMPACT THE STRUCTURAL

INTERACTION OF THE RECEPTOR

ITSELF SO ALTERATIONS IN P-53

FUNCTION FUNNEL BACK THROUGH

THREE PROTEINS TO AFFECT THE

RECEPTOR.

SO WHAT WE'RE TRYING TO DO IS

FIND LIGANDS WHICH BIND

SPECIFICALLY TO P-53 MUTANT

LINES AND NOT WILD TYPE P-53

LINES SO WE'RE LOOKING FOR

LIGANDS TO PUT A PET LABEL ON

AND INJECT PATIENTS AND SEE

WHICH REGIONS DOES IT LIGHT UP.

THIS IS A NEW FIELD WE USE TO

THE TRY TO EXPLOIT THIS REVERSE

SIGNAL TRANSFER 3 RECEPTORS.

AT THE MOMENT WE'RE HAVING QUITE

GOOD SUCCESS WITH THIS.

EXCLUSIVITY, IT'S HARD TO GET.

BUT WHAT WE'RE TRYING TO DO IS

FIND ONES WITH THE HIGHEST

AFFINITY SO IF YOU GET THE DOSE

RIGHT YOU CAN GET A REAL

DISCRIMINATION BETWEEN DOES IT

BIND TO A CELL WITH MUTANT P-53

OR WILD TYPE P-53.

BECAUSE PROTEIN COMPLEXS WE

DON'T PROPERLY UNDERSTAND HOW

THEY INTERACT WITH RECEPTOR,

BECAUSE THEY CAN BE SO LARGE,

THEY CAN VIRTUALLY ENCOMPASS ANY

PHARMACOLOGY YOU'RE INTERESTED

IN.

CANCER IS FIRST POINT BECAUSE

THERE'S A SIMPLE TRACE BACK

PROCESS TO IS RECEPTOR, FOR ANY

FORMS OF PATHOPHYSIOLOGY, WHICH

IS AFFECTED OR CHANGEDDED THAT

WILL AT SOME POINT FEEDBACK TO

GPCR.

YOU CAN FIND WHICH ONE IS THE

MOST IMPORTANT.

NO EXCLUSIVE LINKAGE BETWEEN

INTRA CELLULAR PROTEIN AN

RECEPTOR BUT SOMEBODY HAS TO BE

THE BEST ONE.

SO WE'RE TRYING TO USE THIS NOW

TO CONTROL INTRACELLULAR PROTEIN

IT IS OTHER WAY AROUND.

SO WE HAVE A REALLY GREAT TARGET

MOLECULE FOR AGING.

AND WE'RE TRYING TO CONTROL THAT

SPECIFICALLY.

WHICH IS A NEW FORM OF

PHARMACOLOGY ESSENTIALLY.

WE'RE NOT TRYING TO CONTROL A

KINASE OR BLOCK ION CHANNEL BUT

CONTROL THE EXPRESSION OF

SCAFFOLD PROTEIN BECAUSE WE KNOW

THAT EXPRESSION THAT SCAFFOLD

PROTEIN CONTROLS THE AGING

PROCESS.

WE JUST DON'T KNOW WHICH

RECEPTOR IS PREFERENTIALLY

COUPLED TO THAT PROTEIN.

BUT ONE WILL SHOW THE COUPLING

SO WE'RE TRYING TO FIND THE

TALLEST SKY IN THE VILLAGE

ESSENTIALLY.

-- GUY IN THE VILLAGE

ESSENTIALLY.

AS A CONCLUSION, COMING RIGHT

TOWARDS THE END, THE INTERESTING

THING IS WHEN I WAS POST-DOC

LOOK AT -- WORKING WITH BOB, WE

REALLY THOUGHT WE KNEW

EVERYTHING ABOUT GPR.

WE HAVE DONE EVERYTHING, BLAH,

BLAH BLARKS.

IT JUST KEEPS EXPANDING

EXPONENTIALLY EVERY YEAR.

SO THE SIGNALING UNIT ISN'T WHAT

WE WAS, IT'S WAY MORE

COMPLICATED.

THE STABLE PROTEIN PROTEIN

INTERACTIONS WHICH -- THIS IS

THE REASON WHY I CAME TO TALK TO

YOU TODAY, THIS IS THE PROBLEM.

THIS IS WHERE WE BREAK DOWN.

IT'S THE ABILITY TO IDENTIFY AND

CAPTURE THESE COMPLEXES.

THIS IS WHERE IT'S OCCURRING,

NOT THE GENOMIC LEVEL OR

ANYTHING LIKE THAT BUT THIS

FLEETING INTERACTION.

HOW CAN WE IDENTIFY THE

DIFFERENT GROUPS OF RECEPTOR

PROTEIN COMPLEX SMS THIS IS

REALLY HARD TO DO STILL.

IT TOOK ALMOST 30 YEARS TO GET A

CRYSTAL.

THAT'S BRUTAL.

HERE WE'RE LOOKING AT REAL TIME

PROTEIN PROTEIN INTERACTION

CHANGES.

AND THE PLASMA MEMBRANE POORLY

EXPRESSED PROTEIN.

THIS IS WHY WE NEED TO PUSH

FORWARD, THE ABILITY TO EXTRACT

AND THIS IS WHAT I WAS TALKING

WHEN I MET RECENTLY AT THE

BALTIMORE, WASHINGTON,

DISCRETION GROUP TO INCREASE

CAPACITY TO IDENTIFY

TRANSMEMBRANE HYDROPHOBIC

PROTEINS, IT'S STILL ONE OF THE

BIG ISSUES SO THIS IS WHAT WE

NEED TO PUSH FOR IN THE FUTURE

FROM A REALLY FROM A PROTEOMICS

STANDPOINT.

AND THESE DIFFERENT SUB STATES,

THESE ISOFORMS THAT ARE CREATED

BEYOND THE HEXA HELICAL CORE,

THESE ARE THE PHARMACOLOGICAL

TARGETS.

WE JUST DONE HAVE A WAY OF

SPECIFYING OR CREATING OR

DISCREETLY STUDYING HOW THEY

OCCUR.

SO WE HAVE ACTUALLY TRIED TO GET

AROUND THIS ISSUE BY A BLACK BOX

APPROACH.

WE -- I WON'T WAIT FOR THE

TECHNOLOGY TO EXIST WHICH CAN DO

THIS.

WHICH WE CAN PLUCK THIS OUT FROM

THE SOWN AND SAY THIS COMPLEX

NUMBER THAT COMPLEX, IT'S TOO

DIFFICULT.

BUT IF YOU CAN DECONVOLUTE THE

DOWNSTREAM SIGNALING ACTIONS OF

A RECEPTOR YOU CAN START TO SEE

A DISCONTINUITY OF FUNCTION,

EVEN ACROSS A DEES CURVE.

IF YOU TAKE A DOSE CURVE OF A

COMPOUND YOU SEE DISTINCT

FUNCTIONAL PATENTS OCCURRING AT

DISTINCT DOSES.

THOSE DISTINCT PATENTS ARE

STRONGLY CORRELATED TO DISTAL

DOWNSTREAM PHYSIOLOGICAL EVENTS

LIKE SAY CHANGE IN BONE MASS.

YOU CAN REALLY TRACK BACK TWO OR

THREE MONTHS IN AN ANIMAL THAT

YOU HAVE GIVEN A COMPOUND

INFUSED WITH A PUMP AND YOU HAVE

A CERTAIN TYPE OF BONE

PHYSIOLOGY.

YOU CAN TRACK ALL THE WAY BACK

AND YOU CAN TELL THE DIFFERENCE

BETWEEN A LIGAND WHICH BINDS ONE

RECEPTOR AND A LIGAND THAT BINDS

ANOTHER RECEPTOR AND YOU CAN

ACTUALLY PHYSICALLY ASSOCIATE

THOSE TWO.

SO WE'RE TRYING TO DO PHENOTYPIC

DRUG SCREENING ESSENTIALLY.

THAT'S WHAT WILL ALLOW US TO

POTENTIALLY SAY THERE ARE

DISTINCT RECEPTOR SUB STATES --

SUBSTRATES.

BUT WE HAVE TO DEVISE A SERIES

OF PROGRAMS TO DO THAT BECAUSE

IT IS REALLY THAT DIFFICULT.

WE'RE ACTUALLY ANALYZING DRUG

EFFECTS NOW, USING 20 TO 30,000

POINTS.

YOU NEED THAT DEGREE OF GENOMIC

ACTIVITY OR PROTEOMIC

ALTERATIONS TO SEE SUBTLE

DIFFERENCES BETWEEN DIFFERENT

TYPES OF RECEPTOR EVENTS BECAUSE

RECEPTOR INTERACTIONS ARE

RELATIVELY STABLE IT'S LIKELY

THOSE DISTINCT DOWNSTREAM EVENTS

ORIGINATED FROM DISTINCT SUB

STATE OF THE RECEPTOR ITSELF.

THAT'S THE THEORY.

EVERYTHING SO FAR SUGGESTIONS

THAT'S THE CASE.

AS FAR AS THE LIFETIME RECEPTOR

GOES, IF YOU TOOK A 24 HOUR

PERIOD, THE TIME SPENDS

ENTERACTING WITH G PROTEINS IS

TWO OR THREE MINUTES OF THAT 24

HOUR PERIOD.

REST OF THE TIME IS BINDING

THESE OTHER STRUCTURES.

SO THE GPCR IS NOT REALLY A

GPCR.

THE MAJORITY OF THE TIME

CONDITIONING TRANSCRIPTIONAL

RESPONSES I THINK.

THAT'S ONE OF THE MOST IMPORTANT

JOBS.

WE HAVE NEVER SEEN THAT DRUG

TARGET BECAUSE IT'S TOO FAR

REMOVED FROM THE INITIAL

ACTIVATION EVENT.

SO THAT'S THE WAY TO GET AROUND

THIS ISSUE IS TO DO IT

AUTOMATICALLY.

SO ON TIME AMAZINGLY.

I THOUGHT I WOULD GO OVER.

THANKS FOR YOUR ATTENTION.

I HOPE -- THIS WAS SORT OF

OLDISH DATA BUT IT TELLS A STORY

AND I HOPE YOU HAVE BEEN

INTERESTED.

SO THANKS FOR COMING.

[APPLAUSE]

>> THANK YOU VERY MUCH.

ARE THERE ANY QUESTIONS?

>> IT'S A LITTLE DISCONCERTING

AS FAR AS YOUR PERCEPTION OF THE

ANALYTICAL CHEMISTRY AND WE TRY

TO RESPECT THAT YOU WILL PREFER

TO TREAT THAT AS A BLACK BOX AND

GO AROUND I WONDER ARE THERE

SIMPLE SYSTEMS REFERRING BACK TO

RHODOPSIN MODELS.

CONTEXTUAL CHANGES TAKING PLACE

AND WHERE YOU ENCOURAGE

INVESTIGATORS TO GIVE LOOKING AT

THE ASSISTANT?

>> TO PARAPHRASE THE QUESTION

WITH RATHER THAN AVOIDING THE

ISSUE OF INVESTIGATING THE

COMPLEXITY OF RECEPTOR

ACTIVATION TO FUNCTION, IS THERE

A WAY OF CRUNCHING DOWN TO EASY

TO USE MODEL.

SO THAT'S EXACTLY WHAT WE TRIED

TO DO.

WE HAVE FOCUSED ON ONE SYSTEM.

THE WAY I THOUGHT OF DOING IT IS

REVERSE SLIGHTLY WHAT WE'RE

DOING.

I HAVE GONE THE INSIDE OUT ROOT.

RATHER THAN TO ISOLATE THIS GUY

I THOUGHT THAT'S REALLY HARD.

HE'S FLOPPY, VERY HARD TO

HONESTLY PULL HIM DOWN AND

ISOLATE HIM IN AN IMPARTIAL

MANNER BECAUSE HIS REGIONS ARE

SO FLEXIBLE AND IS GOING TO MAKE

SOME CHOICE WHICH I DON'T

UNDERSTAND.

WE'RE GOING THE INTERNAL ROUTE

SO RATHER THAN SPECIFYING BASED

ON RECEPTOR WE'RE LOOKING AT

SIGNALING SCAFFOLDING FACTORS TO

SEE HOW THEY ENTERACT WITH GPCR

THE BACK WAY IN.

SO THAT'S WHAT WE'RE DOING NOW,

THAT WAS AN EASY OPTION FOR ME

WAS SO WE PUBLISHED A FEW PAPERS

SHOWING OXIDATIVE STRESS AND

AGING.

SO WE FOUND A WHOLE SERIES OF

PROTEINS THAT INTERACT WITH

RECEPTOR WHICH ARE SPECIFICALLY

UPe9p REGULATED WITH OX DAY ACTIVE

EXPOSURE.

WE FOUND ONE IS VERY -- VERY

HIGH AFFINITY INTERACTION WITH

GPCRs AB WE'RE NOW

PRIORITIZING WHICH RECEPTORS

DOES IT BIND IN OXIDATIVE STRESS

SENSITIVE MANNER.

WHAT WE'RE FINDING VERY

INTERESTINGLY, WHICH IS A WHOLE

OTHER PARADIGM POTENTIALLY GPCR

FUNCTION IS A LOT HAVE A

EXCESSIVELY HIGH COUNT OF

INTRAMEMBRANE SISTINE RESIDUES

SO I THINK THERE'S A GROUP OF

RECEPTORS THAT DON'T ACT ON

EXTRA CELLULAR PLASMA MEMBRANE

BUT INSIDE THE CELL AS OXIDATIVE

STRESS SENSES THAT ACTUALLY WHEN

YOU GET THE INSIDE OF A CELL IS

NORMALLY REDUCING ENVIRONMENT

BUT AS YOU GET OLDER AND

OXIDATIVELY STRESSED THAT

BECOMES PARTIALLY OXIDIZING AND

CAUSES ALTERATION OF INTERACTION

OF SCAFFOLDING PROTEIN WITH

RECEPTOR.

SO I THINK THAT'S THE WAY

FORWARD.

THAT DOES WORK NICELY BECAUSE

YOU ARE GRABBING HOLD OF EASILY

BINDING TARGET HIGHLY EXPRESS

BUT ALSO PULL DOWN A RECEPTOR IN

AGONIST MANNER.

GOING THE OTHER WAY, IF YOU HAVE

BEEN REALLY HONEST ABOUT IT YOU

GET INTO TROUBLE TWO WAYS.

YOU OVER EXPRESS TAG RECEPTOR

PUSHED TOWARDS THE

NON-PHYSIOLOGICAL PARADIGM OR

YOU HAVE TO USE AN ANNSERA MORE

MORE CLASSICALLY NOW ANTIBODIES

WHICH IS WHAT (INDISCERNIBLE)

USED THE SMALL PEPTIDES WHICH

BIND USING THOSE TO TRY TO

INTERACT.

BUT I SAID AT THE START ALL

THOSE ARE CONTEXTUALLY

SENSITIVE.

SO THE CORE IS DEPENDENT ON

WHAT'S BINDING HERE.

MORE SO FOR A PEPTIDEERGIC

RECEPTOR WHERE THE PEPTIDE IS ON

THE OUTSIDE.

YOU THINK OH WELL THEY'RE NOT

AFFECTED BY THE ACTUAL PROTEIN

STRUCTURE.

BUT THEY REALLY ARE.

IT'S JUST THAT WE HAVE ALWAYS

LOOKED AT RECEPTORS FROM HECK

293 CELLS OVEREXPRESSED AND

THAT'S IT.

SO THIS THAT'S MY WAY TO GO

AROUND THAT.

SO YOU CAN DO IT NICELY.

(OFF MIC)

I HEARD THE SAME THING A FIELD

IN WAY CLOSING DOWN, I SEE

ANOTHER ONE USUALLY (INAUDIBLE)

MY QUESTION IS RELATES TO A

COMMENT THAT WAS MORE

(INAUDIBLE) AT LEAST FOUR TIMES

TALKING ESSENTIALLY (INAUDIBLE).

MY QUESTION IS WHAT IF THE

BETTER METAPHOR IS THAT'S PAPERS

AND THERE'S ROCK.

(INAUDIBLE) MUTUALLY EXCLUSIVE.

FAR FROM IT.

IF YOU'RE DEALING WITH A

RECEPTOR (INAUDIBLE) YOU WILL

FIND IN MOST CELLULAR CONTEXT

PERFECTLY SO THAT CAN MEAN ONE

OF TWO THINGS.

(INAUDIBLE) IN OTHER WORDS YOU

SHOULDN'T BE TESTING DRUGS

(INAUDIBLE) SO THAT MAKES MANY

THINK AS PHARMACOLOGIST THAT

MAYBE WHAT WE NEED TO BE LOOKING

AT EXTREMELY CAREFULLY EXTREMELY

CAREFULLY (INAUDIBLE) WHATEVER

DOSE WE FEEL LIKE, WHAT THE

ACTUAL DOSAGE OF LIGAND ON THESE

CELLS ARE, BECAUSE IF YOU'RE --

YOUR ARGUMENT FOR (INAUDIBLE)

TRANSCRIPTIONAL REGULATION IS

GENERALLY TRUE FROM PHYSIOLOGY

AND PATHOPHYSIOLOGY THEN THE

DRUGS ARE (INAUDIBLE) COMPLETELY

DIFFERENT PICTURE (INAUDIBLE)

ARGUING FOR IN THE FIRST PLACE.

SO WHAT -- IF YOU WERE A

(INAUDIBLE) HIGH THROUGH PUT

SCREENERS, HOW THEY SHOULD

DEVELOP DOSE RESPONSE CURVES AND

WHAT THEY SHOULD BE LOOKING AT

BASED ON WHAT YOU SAID HERE

TODAY, WHAT SORT OF THINGS MIGHT

YOU SAY.[gv

>> SO I'LL PARAPHRASE FOR THE

CAMERAS.

SO IN A CHANGING CONTEXT OF A

CELL MODEL FOR LIKE A DRUG

SCREENING PROCESS HOW DO WE COME

AT SOME FORMALIZATION OR

STANDARDIZATION OF LIGAND DOSE

AND ANALYSIS.

THAT IS A REALLY -- I ALWAYS

TELL MY STUDENTS, WE DON'T KNOW

WHAT DOSE IS THERE.

IN THE MICROENVIRONMENT, IN THE

SYNAPSE YOU HAVE LIKE 20

MICROMOLAR ACETYL CHO LIEN FOR A

FEW MICROSECONDS.

THAT'S LUDICROUS.

WE WOULD NEVER USE THAT IN A

BENCH SITUATION.

THAT'S WHAT REALLY HAPPENS.

SO THAT'S AN ENORMOUS QUESTION.

AND ENORMOUS QUESTION.

WHAT WE TRY TO DO WITH THE MUP

MODEL IS SHOW REPRODUCTION OF

BIOLOGY.

THAT'S THE MOST IMPORTANT THING

TO TRY AND DO, REPRODUCE SOME

FACET OF PHYSIOLOGY THAT WE

EXPERIENCE FROM MANY, MANY,

MANY, MANY TRIALS OF COMPOUNDS

IN A PERSON THAT WE SEE A

CERTAIN TYPE OF EFFECT.

CAN WE MIMIC THAT IN A CELL

MODEL.

HOWEVER ACCURATE OR INACCURATE

IT MAYBE BUT IMPART THROUGH SOME

EITHER -- WE USE OXIDATIVE

STRESS EXPOSURE, EXPRESSION OF

CERTAIN TYPE OF SCAFFOLDING

PROTEIN TO REPRODUCE A

PHYSIOLOGICAL EVENT.

PHARMACO LODGEICALLY YOU SEE

FUNCTIONAL DOMAINS ACTIVATED

ACROSS A DOSE RANGE OF A

COMPOUND, WE DON'T KNOW EXACTLY

WHAT THAT IS CAUSED BY N. MY

PAPERS I SAY DIFFERENT SUB SETS,

BLAH BLAH BLAH.

BUT WE PUBLISHED ONE RECENTLY

AND YOU CAN'T SAY THAT BECAUSE

YOU CAN'T ISOLATE DIFFERENT SUB

STATES.

IT'S IMPOSSIBLE AT THE MOMENT.

WE CAN'T DO IT.

IF YOU CAN SHOW -- AS YOU SAY,

THERE'S NOT A -- THIS DOES THIS

ALL THE WAY UP THE DOSE, IT WILL

JUST DO THIS FUNCTION AND THAT'S

IT.

THIS NEVER HAPPENS.

IF YOU CAN GET A BROAD ENOUGH

SEPARATION BETWEEN

PHARMACOLOGICAL DOSE AN

FUNCTIONAL EFFICACY, THAT'S THE

THING TO LOOK FOR.

WHAT YOU NEED TO DO IS SHOW THAT

YOUR CELL SYSTEM, SHOWS A

SPECIFIC RECAPITULATION OF

PHYSIOLOGICAL EVENT SO WE'RE

DOING THIS NOW AT CLINICAL

CENTER WITH PATIENTS WITH A

SYNDROME CALLED (INDISCERNIBLE)

SYNDROME.

WE HAVE A MODEL WE'RE WORKING ON

SO WE HAVE TO WAIT 3 OR 4

MONTHS.

WE HAVE A FEELING WE NO HOW THIS

WORKS, WE LOOK TO SEE WHICH

PROTEINS ENTERACTS WITH THE

TARGET RECEPTOR, WHICH ONES

ALTER WHEN OBESE.

WE FOUND ONE, NDRG-4, RECEPTOR

BINDING PROTEIN, AND

DISPOSITION.

IT WILL RECAPITULATE THE

PHYSIOLOGY WE SEE IN A CELL LINE

SYSTEM.

SO THAT WE HAVE HAD TO USE

ANIMAL MODELS AND ALSO THAT'S

WHAT HUMAN SITUATIONS -- THAT'S

A PHARMACO GENOMIC SWITCH WHICH

ALLOW IT IS COMPOUND TO WORK.

FIVE YEARS WORTH OF WORK TO GET

TO THAT POINT BUT NOW WE HAVE A

POINT OF ALTERATION THAT WE CAN

THEN COMPARE TO ANIMAL MODEL OR

HUMAN SYSTEM, WE FEEL THOUGH WE

DONE KNOW INs AND OUTS THE

IMPOSITION OF THIS MIMICS THE

PHARMACOLOGY, THAT IS A MORE

CREDIBLE TESTING SITUATION AND

PHARMACO GENOMIC TARGET.

THAT'S DIFFICULT.

THAT'S THE THING, WE HAVE GONE

FROM EASY MODELS TO REALIZING

THAT ACTUALLY JUST CREATING A

CELL SCREEN IS JUST AS HARD IN

THIS TIME CONSUMING AS LOOKING

AT THE HUMAN SITUATION.

BUT MORE WE CAN DRAG BACK FROM

PHYSIOLOGY AND CLINICAL DATA

BACK TO A CELL SYSTEM WE'LL HAVE

BETTER SCREENS.

(OFF MIC)

>> EXACTLY.

(OFF MIC)

YOU GET RID OF 2000 LIGANDS.

YOU GET RID OF ONE AND HOPE THAT

GUY WORKS IN ALL DIVERGENCE.

EVERYTHING AT THE GET GO.

EVERYTHING YOU CAN UP FRONT EVEN

IF IT ISN'T HIGH AFFINITY IT'S

STILL IMPORTANT.

YOU DON'T NEED -- IF YOU START

TO REALIZE DRUGS IMPACT A SERIES

OF NETWORKS OF INTERACTIONS.

YOU HIT THE RIGHT NODE YOU DON'T

NEED SUPER HIGH EFFICACY OR

SUPER HIGH AFFINITY.

IF YOU HAVE A SYSTEM LIKE THE

ENDOPLASMIC RETICULUM YOU CAN DO

A WORLD OF GOOD WITH A TINY

LITTLE PUSH.

IF YOU'RE PUSHING THE RIGHT

SYSTEM, I WENT TO ERIC SHAF'S

TALK A COUPLE OF YEARS AGO, IT'S

MIND BLOWING.

IT'S LIKE DON'T THINK PATHWAYS

THINK NETWORKS.

THAT'S WHAT WE'RE TRYING TO DO

NOW.

SAY OTHER TALLER GUYS IN THE

NODE NETWORK THAT WE CAN HIT.

AND CAN WE TARGET THOSE GUYS.

AND IF YOU CAN TARGET THOSE GUYS

WE DON'T NEED TO PUSH EFFICACY

AND AFFINITY HARD.

WE CAN HAVE MICROMOLAR LEADS, AS

LONG AS THEY'RE NOT HARMFUL

THEIR EFFECTS WILL BE B

REINFORCED.

BY USING ENDOGENOUS NETWORK.

AND THAT'S WHAT WE CAN DO IN

REVERSE TRANSLATIONAL FORMAT TO

APPLY MOLECULAR ANALYSIS TO

PHARMACOLOGICAL EVENTS OF

PATIENTS ALL THE WAY BACK TO

SCREENING PROCESS.