HELLO TO EVERYONE HERE IN
LIPSETT, AND TO EVERYONE JOINING
ONLINE.
IT'S MY DISTINCT PLEASURE TO
INTRODUCE DR. LEI WANG TODAY.
ONE QUICK NOTE BEFORE WE GET
STARTED THE CME CODE IS 57654.
IF YOU HAVE A QUESTION DURING
THE SEMINAR OR AFTER THE SEMINAR
THESE CAN BE SUBMITTED VIA THE
SUBMIT FEEDBACK BUTTON ONLINE.
AND FOR THOSE IN PERSON, THERE
WILL BE A RECEPTION THAT FOLLOWS
THE LECTURE OUTSIDE IN THE
SEMINAR HALL.
CURRENTLY PROFESSOR WANG IS A
PROFESSOR AT UCSF WHERE HE'S
BEEN SINCE 2014.
HE PERFORMED HIS BACHELOR'S
AND MASTERS STUDIES AT PEKING
UNIVERSITY BEFORE MOVING TO HIS
Ph.D. STUDIES IN THE LAB OF
PETER SCHULTZ WHERE HE PIONEERED
METHODS TO EXPAND GENETIC CODE
FOR TRANSFORMING AND
INCORPORATION OF NONAMINO ACIDS.
HE THEN PERFOR THE PURPOSED
HIS POST DOC STUDIES WITH
DR. CHEN AT UCSD, AND HE BEGAN
HIS INDEPENDENT CAREER AS AN
ASSISTANT PROFESSOR, AND THEN IN
2014 HE JOINED UCSF.
DR. WANG HAS RECEIVED SEVERAL
AWARDS INCLUDING THE KAISER
AWARD FROM THE PROTEIN SOCIETY
FOR SIGNIFICANT CONTRIBUTIONS TO
THE STUDY OF PROTEINS AS WELL AS
NIH DIRECTOR'S INNOVATION AWARD
AMONGST MANY OTHERS.
AS DR. WANG WILL TELL YOU TODAY,
HIS LAB DEVELOPS NEW METHODS
BASED ON GENETIC INCORPORATION
OF NONNATURAL AMINO ACIDS TO
INTRODUCE NOVEL FUNCTIONALITY
INTO PROTEINS TO INVESTIGATE
BIOLOGICAL PROCESSES, IN VIVO,
WHICH HE WILL COVER DURING HIS
TALK TODAY.
SO YOIN ME IN WELCOMING
DR. WANG.
[ APPLAUSE ]
>> THANK YOU VERY MUCH, ROSS,
FOR THE KIND INVITATION AND
GENEROUS INTRODUCTION.
I HAD A GREAT TIME MEETING WITH
THE OUTSTANDING PIs AND
TRAINEES HERE, REALLY ENJOYED
THE VISIT.
IT'S A GREAT HONOR AND PLEASURE
TO BE HERE.
MY TALK TODAY WILL BE
BIOSPECIFIC CHEMISTRY FOR
COVALENT LINKING OF
BIOMACROMOLECULES.
BEFORE WE PROCEED, I WOULD LIKE
TO DISCLOSE POTENTIAL CONFLICT
OF INTEREST EMPLOY I SERVE AS
SCIENTIFIC ADVISOR TO ANOTHER
THEOR PUTTICS BIOTECH COMPANY
FOCUSING ON DEVELOPING COVID AND
BIOLOGICS.
JUST TESTING MY CURSOR AS YOU
CAN SEE.
OKAY, GREAT.
IN NEARLY ALL LIFE FORMS PROTEIN
INTERACTING WITH PROTEINS AND
OTHER CLASSES OF BIOMOLECULES TO
PERFORM THE ESSENTIAL
BIOLOGICAL FUNCTIONS.
THIS INTERACTIONS INCLUDING
ELECTROSTATIC FORCES,
[INDISCERNIBLE] AND CARBOHYDRATE
ACTIONS ARE NONCOVALENT IN
NATURE.
AS A RESULT IT TENDS TO BE WEAK,
TRANSIENT AND REVERSIBLE.
ALTHOUGH IT'S TRYING TO ENGINEER
SUCH NONCOVALENT INTERACTIONS
INTO COVALENT BONDS CREATING
STRONG, STABLE AND IRREVERSIBLE
CONNECTICUT NEKSS FOR
BIOMOLECULES TO STUDY SPECIAL
HARNESS BIOLOGICAL ACTIVITIES.
PROTEINS HAVE A LIMITED ABILITY
TO FORM COVALENT BOUNDS, A KEY
EXAMPLE IS THE [INDISCERNIBLE]
FORMED BETWEEN 2 CYSTINE
RESIDUES SPONTANEOUSLY.
CONCRUCIAL FOR PROTEIN STRUCTURE
AND FUNCTION, THE DISULPHIDE
BOND IS IS REVERSIBLE AND NOW A
ANOTHER MAJOR FACTOR IS CATTAL
SIS, OR THE ATTACHMENT OF
UBIQUITIN THROUGH OTHER PROTEINS
THROUGH THE ENZYME MEDIATED
CATTAL SIS.
AS YOU CAN SEE THIS WILL REQUIRE
MULTIPLE RESIDUES IN A SPECIAL
MICROENVIRONMENT.
AND HOWEVER RATIONALLY DESIGNING
THIS BOUNDS REMAINS CHALLENGING
DUE TO THE COMPLEXISITY OF THE
PROCESSES.
THE CHEMISTRY HAS REVOLUTIONED
THE BIOMARK ROUGH ATOM
MOLECULES, TO THIS RESULT, 2
GROUPS ARE SEPARATED AND
INTRODUCED TO A SMALL COLKIEWL,
WHICH THEN LINK TO FORM THE
CONNECTION.
BECAUSE THE TALKATIVE
BIOMOLECULE MUST BE MODIFIED
WITH A BIOORTHOGONAL FUNCTIONAL
GROUP, IMPLEMENTING THIS
APPROACH IN VIVO IS CHALLENGING
IN PARTICULAR ESPECIALLY FOR
THERAPEUTIC APPLICATIONS WHERE
THE TARGETED MOLECULE ARE VERY
DIFFICULT TO BE ALTERED.
TO ADDRESS THIS, WE PROPOSE
BIOSPECIFIC CHEMISTRY, THIS
APPROACH INTRODUCED A SINGLE
BIOREACTIVE GROUP INTO A
BIOMOLECULE WHICH THEN REACTED
WITH ANOTHER BIOMOLECULE WITHOUT
THE NEED TO MODIFY THE LATTER.
THIS ABLE TO COVALENTLY MODIFY
BIOMOLECULES SHOULD MAKE BI
SPECIFIC CHEMISTRY IDEAL FOR IN
VIVO STUDIES IN THERAPEUTIC
APPLICATIONS.
WE BEGAN EXPLORING BIOSPECIFIC
CHEMISTRY TO ENABLE PROTEINS TO
COVID AND TARGET ANOTHER PROTEIN
BEYOND THE DISULPHIDE BOUNDS WE
ENVISIONED WHETHER ADDITIONAL
COVALENT LINKAGES COULD FORM
BETWEEN THE UNNATURAL AND
NATURAL RESIDUE.
THIS BIOREACTIVE ACID IF
SUCCESSFUL WOULD BREAK THE
NATURAL BARRIER INTRODUCING NEW
COVID AND ABOUNDING CAPABILITIES
INTO PROTEINS.
UNNATURAL ANALYSIS CAN BE
SPECIFICALLY INTRODUCED THROUGH
PROTEINS THROUGH GENETIC CODE
EXPANSION USING A ORTHOGONAL
[INDISCERNIBLE] PAIR TO SUPPRESS
A STOP CODON INTRODUCED
ARTIFICIALLY INTO THE RNA.
HOWEVER THE INCORPORATION OF THE
PROTEIN INTO THE CELLS HAS LONG
BEEN CONSIDERED INFEASIBLE.
FOR A BIOREACTIVE AUTOIMMUNE
REACTIVE TO FUNCTION, IT MUST
REACT WITH BIOMOLECULES INSIDE
THE CELLS, BUT IS SUCH REACTION
RISK CYTOTOXICITY AND COULD
STORE TRANSLATION IF IT WILL
REACT WITH THE TRANSLATION
MACHINERY.
ADDITIONALLY THE ABUNDANCE OF
THE TERMINUS IS IN PROTEIN ANDS
IN CELLS POSE A CHALLENGE FOR
NONSELECTIVE REACTIONS WHICH CAN
COMPROMISE SPECIFICITY.
TO ADDRESS THESE CHALLENGES WE
DEVELOP A STRATEGY CALLED
PROXIMITY ENABLED BIOREACTIVITY.
IN THIS APPROACH THE UAA, NOW
REFER NATURAL [INDISCERNIBLE], I
WILL NOW REFER TO THIS AS UAA,
THE U,A APAYS IS DESIGNED TO BE
LATENT INSIDE THE CELLS
PREVENTING UNWANTED REACTIONS
WITH AN INTRACELLULAR MOLECULES
AND THEN ALLOWING FOR GENETIC
INCORPORATION.
ONCE INCORPORATING TO PROTEIN
AND THE BROADER COAST TO THE
TARGETED RESIDUE, THE
UAA ACTIVITY IS TRIGGERED BY THE
PROXIMITY EFFECT WHICH REDUCES
THE ENTROPY OF ACTIVATION AND
INCREASE THE EFFECTIVE
CONCENTRATION ENABLING A
[INDISCERNIBLE] BOND TO FORM
SELECTIVELY BETWEEN THE
[INDISCERNIBLE] AND THE TARGETED
RESIDUE.
THE REACTIONS WE AIM TO DEVELOP
MUST BOTH BE BIOCOMPATIBLE AND
EFFICIENT UNDER PHYSIOLOGICAL
CONDITIONS WHICH IS A CHALLENGE
IN VALENCE TO,A CHIEF.
THE FOCUSED INITIALLY ON
TARGETED ASSISTIVE RESIDUE DUE
TO HIGH FOLLICULAR
[INDISCERNIBLE].
OUR HYPOTHESIS WAS THAT
[INDISCERNIBLE] COULD STAY INERT
IN ITSELF, BUT EFFECTIVELY ASHES
CYSTING WHEN BROUGHT INTO CLOSE
PROXIMITY.
TO TEST THIS WE CHOOSE A FLORAL
METHOD [INDISCERNIBLE] AND THE
DESIGN OF THE UNNATURAL TERMINAL
[INDISCERNIBLE] AFFECT.
WE INCORPORATE FFACT INTO A
[INDISCERNIBLE] AND EXPRESS THIS
IN E.COLI.
AFTER PURIFICATION MASS PECK
VERIFIED IN COOPERATION WITH
THIS AMINO ACID AND THAT WE
DON'T SEE ANY ADDITIONAL
MODIFICATION TO THE FFACT.
INCLUDING OUR INTENDED REACTIONS
WITH INTRACELLULAR ASSISTINE
GLUTEA THIGH OWN.
THIS INDICATING IT STAYS
INACTIVE DURING THE BIOSYNTHESIS
AND PURIFICATION.
TO TEST WHETHER IF EFFECT CAN
FORM A COVID AND BOUND WITH THE
CYSTINE IN PROTEINS, WE
INCORPORATE FFACT INTO A BODY
AND PLACE ASSISTING RESIDUE INTO
THE PROTEINS AND BINDING AT THE
INTERFACE AND THESE 2 PROTEINS
WILL PURIFY AND INCUBATE IT AND
WE THEN RUN AN SDS PAGE UNDER
THE NATURE AND CONDITIONS TO
TEST WHETHER THIS TOO ACTIVATES
EACH OTHER.
IT SHOWS A COMPLEX OF
[INDISCERNIBLE] Z WAS FORMED.
HOWEVER IF WE REPLACE THE FFACT,
WITH THE NON[INDISCERNIBLE]
FFACT, OR PLACE THE CYSTINE WITH
OTHER AMIN ACID RESIDUES OR PUT
THE CYSTINE INTO DIFFERENT
POSITIONS, NO COVID COMPLEX
FORMED SO THIS RESULT I HAD
BEENICATING THAT THE COVALENT
BOUNDING FORMATION BETWEEN THESE
2 PROTEINS DEPENDS ON THE
PROXIMITY OF THE FFACT AND
CYSTINE.
NEXT WE TESTED THE INTRA
MOLECULAR REACTIVITY IN THE
FLUORESCENT PROTEIN.
WE PLACED THE [INDISCERNIBLE] 67
OF THE FLORA FORWITH THE FFACT
AND REPLACED SERIES POINTSINE
WITH CYSTINE.
WE THEN EXPRESS IN MUTANT
PROTEIN IN E.COLI, PURIFIED IT
AND MASS SPEC CONFIRMED NEARLY A
HUNDRED PERCENT EFFICIENT IN THE
SINGLE FORMATION AND A SINGLE
ACCOUNT OF THIS PROTEIN SHOWED
THAT THERAY A SINGLE COVALENT
BOND DOUBLED THE LIFE SPAN AND
[INDISCERNIBLE] OF THIS MUTANT
FOR US IN PROTEIN.
THIS BENEFITS THAT ARE VERY
DIFFICULT TO ACHIEVE USING ONLY
NATURAL AMINO ACIDS.
CONTINUING ON THIS BREAK
THROUGH, WE AND SEVERAL OTHER
RESEARCH GROUPS HAVE SINCE
DESIGNED AND GENETICALLY
INCORPORATED A VARIETY OF LATENT
BIOREACTIVE TERMINAL ASSETS TO
TARGET DIFFERENT AMINO ACID
RESIDUES.
HERE ARE SOME REPRESENTED
EXAMPLES.
AS YOU CAN SEE BESIDES CYSTIN,
WE'RE ABLE TO COAST AND TARGET
SEVERAL OTHER RESIDUES.
THROUGH THIS [INDISCERNIBLE]
MECHANISM, HIGHLIGHTING THE
GENERALITY OF THIS APPROACH.
ONE VERSATILE UAA, WE HAVE
DEVELOPED THIS FSY FOR ITS
EXCEPTIONAL BIOCOMPATIBILITY AND
MULTITALKATIVE AFFINITY WITHIN
LIVE CELLS.
IN SPITE OF THE PIONEERING WORK
OF BARRY SHARPLESS ON THE
CHEMISTRY WE DESIGNED THIS
[INDISCERNIBLE] TO FORM A
FUNCTIONAL GROUP.
WE INCORPORATE FSY INTO THE
BINDING FACE OF F-BODY Z AND
INTRODUCE DIFFERENT NUCLEAPHILIC
RESIDUES TO TEST WEATHER FS1 CAN
REACT WITH THEM THROUGH THE
SUFEX CHEMISTRY.
AND WE EXPRESS THESE PROTEINS
DWECT DREKING E.COLI WITHOUT
PURIFYING THEM WITH THE CELLS
WHERE THEY ANALYZE THE CELL
LIAISON SAIT.
AS YOU CAN SEE HERE, BOTH ON THE
WESTERN BLOT AND SDS PAGE TO
RESULT SHOWED THAT THE FSY WAS
ABLE TO REACTIVATE WITH LYSINE,
HISTODINE AND TYROSEEN.
AND THIS REACTION SPECIFICITY
WAS FURTHER VERIFIED BY MASS
SPEC.
SINCE THESE ARE ABUNDANT AT MANY
PROTEIN INTERACTION PHASES, SO
FSC HAS THE POTENTIAL TO TARGET
VIRTUALLY ANY PROTEIN.
ANOTHER VERSE TIE REACTION IS
PFEX, THE PHOSPHOROUS FLUORIDE
EXTEND TO REACTION, WHILE PFEX
TYPICALLY REQUIRES A CATALYST OF
FULL REACTIONS BETWEEN SMALL
MOLECULES, WE DISCOVERED THROUGH
GENETIC INCORPORATION OF THIS,
TOO, END TERMINAL SIS INTO
PROTEINS, WE FOUND IN THE
PROTEIN ENVIRONMENT, THE PFEX
REACTION CAN NOW BE ACTIVATED
SIMPLY BY THE PROXIMITY EFFECT.
AND THIS ALLOWS, WE USE PFEX TO
TARGET HISTIDINE, LIESINE AND
ADDITIONAL CELLS.
ADDITIONALLY IT'S REALLY
INTERESTING, WHEN WE REACTS WITH
EFTHIMIOS DINE IT DISPLACES
THERMAL ACTIVITY AS WELL.
WHAT CAN WE ACHIEVE WITH THIS
NEW COVALENT BONDING ABILITIES?
FIRST I WOULD LIKE TO INTRODUCE
GENETIC ENCODED CHEMICAL CROSS
LINKING OR GECX TO IDENTIFY
PROTEIN-PROTEIN INTERACTIONS.
ONE APPLICATION WE DEVELOPED IS
TRYING TO USE THIS TO PROBE
LIGAND RECEPTOR INTERACTIONOT
[INDISCERNIBLE] CELL SURFACE.
WHEN WE EXPERIENCE STRESS, THE
HYPOTHALAMUS WILL RECEASE
CORTICALE RELEASE FACTOR CRF, A
PEPTIDE HORMONE THAT BINDS TO
CLASS TPC R, [INDISCERNIBLE]
TRIGGERING A CASCADE OF BRAIN
AND MUSCLE RESULT NOTHING STRESS
RESPONSE.
HOWEVER CHRONIC STRESS CAN LEAD
TO ANXIOUS AND DEPRESSION.
AND BLOCKING THE CRF RECEPTOR
INTERACTION COULD POTENTIALLY
TREAT THIS CONDITIONS BUT AT THE
TIME IT WAS UNCLEAR HOW THE CRF
PEPTIDE HORMONE BINDS AND
ACTIVATES THE RECEPTOR.
THEN AS ASSISTANT PROFESSOR
UNDER THE PRESSURE OF THE NIH
STUDY PANELS, SEEKING PROMOTION
IN THE PUBLISHING, I WAS
IMMEDIATELY INTRIGUED BY THE
POSSIBILITY OF CREATING A
HAPPILY EVER AFTER PILL BY
BLOCKING THIS INTERACTION.
SO MY GROUP STARTED MAPPING
BINDING SIZEOT GPCR USING THE
CROSS LINKING U,A,A, WE USED THE
[INDISCERNIBLE] CALLED AZI, WE
CAN INCORPORATE AZI INTO VARIOUS
DIFFERENT POSITIONS INTO THE TPC
R, WHICH WAS EXPRESSED THERE
FROM THE MAMMALIAN CELLS AND
THEN WE THROW IN THE PEPTIDE
HORMONE, PERFORM THE CROSS
LINKING ON THE MAMMALIAN CELL
SURFACE, AFTER WHICH WE JUST
ANALYZE THE CELL LIAISON SAIT TO
DETECT WHETHER LIGAND IS CROSS
LINKED WITH THE GBCR.
AS YOU CAN SEE IN THIS PANEL A,
THEY INCORPORATE ACI INTO
SPECIFIC SIZE THAT IT CAN
CAPTURE THE LIAISON GAPPED AND
COMPLEX OF LIGAND DETECTED HERE,
AND THIS SLIDES ARE POTENTIALLY
THE LIGAND OF BYPASSING SITES
AND OF MARKETING MAGENTA HERE ON
THE GBCR MEMBRANE STRUCTURE BUT
WE WERE VERY HAPPY AND AT THAT
TIME, WE REALIZED THIS STILL
CANNOT HELP US TO CRACK THE
POSITION FOR CI INTO LIGAND FOR
THE BINDING POCKET OF THIS PC R
BECAUSE OF PHOTO CROSS LINKING
IS NOT A RESIDUE SELECTIVE SO WE
CANNOT KNOW THE DISTANCE
CONSTRAINTS BETWEEN THE PEPTIDE
LIGAND IS THE GPCR RECEPTOR.
AND HERE GEX PROVIDED THE
SOLUTION, SINCE WE ALL KNOW, GEX
IS THE RESIDUE AND THE DISTANCE
SPECIFIC, WE COULD USE THE 2
PINPOINT THE EXACT CONTACT
POINTS BETWEEN THE CRF AND THE
RECEPTOR.
SO WHAT WE DID IS INTRODUCE THIS
EFFECT ON END TERMINAL ACID ON
DIFFERENT POSITIONS OF THIS CIF
1 R, AND ASSISTING IN THE
PEPTIDE LIGAND.
WE THROW IN THIS PEPTIDE LIGAND
INTO THE LIVE CELLS, AND THEN
ANALYZE, IF THE RECEPTOR
CAPTURED A LIGAND USING WESTERN
BLOT ANALYSIS OF THE CELL
LIAISON SAIT.
AND THIS RESULT TELLS US WHICH
RESIDUE IN THE RECEPTOR IS IN
CONTACT WITH THE SPECIFIC
RESIDUE IN THE PEPTIDE LIGAND AS
SHOWN BY THE MATCHING COLOR IN
THIS DIAGRAM HERE.
SO BASED ON THIS CONSTRAINT
INFORMATION AS WELL AS THE
STRUCTURE OF INDIVIDUAL GBCR
SEPARATELY, NOWY WERE ABLE TO
BUILD A MODEL FOR THE CRF LIGAND
BINDING TO THE FULL LENGTH TPC
R, PROVIDING THIS INSIGHT INTO
RECEPTOR ACTIVATION AND
INHIBITION.
AS YOU CAN SEE HERE, ALL THE
INFORMATION WE HAVE IS ON THE
CELL SURFACE WHICH CAN
COMPLEMENT WHAT PEOPLE CAN
PORTEND LATER USING THIS OR
CRYOEM STRUCTURES.
TAKING A BOLDER STEP BEYOND THE
CELL SURFACE, WE NEXT APPLY GEX
INSIDE LIVE CELLS TO KACCT
TININE PATHWAY IRB
PROTEIN-PROTEIN INTERACTIONS SO
THE STRATEGY IS TO INCORPORATE
THE LATENT END TERMINAL INTO OUR
TARGET PROTEIN WHICH IS
EXPRESSED WITHIN LIVE CELLS.
WHENEVER AN UNKNOWN PROTEIN
ACTIVATES PROTEIN OF INTEREST,
THEN THE BIO'S ACTIVE TERMINAL
ACID REACT WITH FLRL RESIDUE ON
TO THIS UNKNOWN PROTEIN
IRREVERSIBLY CAPTURE IT AND
ALLOW US TO DO SUBSEQUENT
PROCESSING AND IDENTIFY THIS
UNKNOWN PROTEIN USING MASS SPEC.
AS CAN YOU SEE, YOU KNOW IN
ADDITION TO WORKING IN LIVE
CELLS, OUR GEX MECHANISM HAS A
MAJOR ADVANTAGE AS SPONTANEOUS
REACTION AND LONG REACTION
WINDOW WHICH ALLOW IT TO ENRICH,
WEAK AND INTRENSENT PROTEIN ISHT
ACTIONS INIMPROVING SENSITIVITY
FOR IDENTICTION.
THIOREDOXIN IS AN ENZYME THAT
CAN REDUCE THE DISULPHIDE BOUNDS
OF THE PROTEINS.
TO IDENTIFY THIS IN THE PROTEIN
IN OH COAL I WE INCORPORATE THIS
UAA BPRY INTO NEAR THE ACTIVE
SITE AND PROTEIN, WE EXPRESS
THIS CONTINUOUS REDOXON IN
E.COLI, AND WESTERN BLOT AND
ACID OF THE CELL LIAISON SAIT TO
SHOW THE MULTIPLE BENS WITH
HIGHER MOLECULAR WEIGHT AND
REDOXON AND THIS BEN WILL PULL
DOWN AND IDENTIFY MASS
SPECTROMETRY.
THIS SCHEMA HAS 205 PROTEINS
THAT DIRECTLY BINDS TO THE
THIOREDOXIN AND THE RESULTING
PROTEIN ISHT ACTION NETWORK
PROVIDES NEW SIZE INTO THE
REDUCTION ROW IN REGULATING
METABOLISM, DNA DAMAGE REPAIR
AND OTHER PROCESSES.
OF COURSE, THE SYSTEM IS NOT
ALWAYS AVAILABLE FOR TARGETING.
AND THAT WHY WE DEVELOP THE FSY
AND FSK ON THESE 2 END TERMINAL
ASIT TO TARGET THE MORE ABUNDANT
TYROSEEN AND EFTHIMIOSINE
RESIDUE, WE SHOULD HAVE A BROAD
RANGE OF PROTEINS SO WE CAN
CAPTURE.
ADDITIONALLY, WE THINK FSY, AND
FSK CAN BE PLACED INTO THE
PERIPHERY OF THE PROTEIN INSTEAD
OF THE BINDING IRPT FACE OR THE
ACTIVE SITE TO MINIMIZE
INTERFERENCE.
SO TO TEST THIS IDEA, WE
INCORPORATE FSY AND FSK AGAIN
INTO THIOREDOXIN BUT THIS TIME
FURTHER AWAY FROM THE ACTIVE
SITE.
WOE THEN EXPRESS THIS MUTANT
PROTEINS IN E.COLI AND NOW YOU
CAN SEE ON WESTERN BLOT, WOO WE
CAN ADENTIFY MORE SUBSTRATE
PROTEINS OF THIOREDOXIN COMPARED
TO USING THE BPRY OUT OF THE
ACTIVE SITE.
IN ADDITION TO COVALENT, THE
PROTEINS WE RECENTLY ENABLED
GENETIC ENCODED CHEMICAL CROSS
LINKING OF RNA, AND OTHER
DIFFERENT CLASS OF BIOMOLECULES.
PROTEIN INTERACTIONS REGULATE
NEAR ALL ASPECTS OF RNA
INCLUDING PRERNA SPICING, RNA
MODIFICATION TRANSLATION AND
DEGRADATION.
RECENT STUDIES HAVE ALSO
REVEALED THAT ON THE BINDING
PROTEINS, CAN BIND RNA THROUGH
THE INTRINSIC DISORDER REGIONS
SO TO FULLY UNDERSTAND THIS
COMPLEX REGULATORY MECHANISM,
AND UNCOVER NEW INSIGHTS, IT IS
CRUCIAL TO IESHES DENTIFY
PROTEIN INTERACTIONS IN VIVO,
OOH DIALLY WITH SINGLE
NUCLEOTIDE AND A SINGLE IMMUNO
RESOLUTION.
THE MOST WIDELY USED METHOD
CURRENTLY IS THE NUCLEICIDE
BASED CROSS LINKING.
WHEN EXPOSED TO UV LIGHT, THE
BASEOT NUCLEOICIDE CAN GENERATE
RADICALS, WHICH CAN CROSS LINK
AMINO ACID RESIDUES COMBINED
WITH THE IMMUNOPRECIPITATION AND
HIGH THROUGH PUT SEQUENCING WE
CAN IDENTIFY THE TARGETS OF THE
SPECIFIC RNA BYPASSING PROTEINS.
UNFORTUNATELY, CROSS LINKING AS
BIAS PREFERENCE FOR THE
[INDISCERNIBLE] MAKING IT
DIFFICULT TO STUDY REGIONS OF
[INDISCERNIBLE].
AS I MENTIONED EARLIER IT IS
NONRESIDUE SELECTIVE AND
THEREFORE MAKING IT A CHALLENGE
TO ACHIEVE AMINO ACID RESOLUTION
EMPLOY AND, BECAUSE THE REACTION
IS NONSPECIFIC, THEREFORE, IT'S
INFEASIBLE TO ENGINEER PRECISE
COVALENT LINKAGES BETWEEN
PROTEINS AND RNA.
AND THIS UAA, WILL REACTIVE WITH
NUCLEOTIDEOT RNA WHEN THE RNA
BINDING PROTEIN IRPT ACT WITH
THE RNA.
AS CAN YOU SEE, NOW THE
REACTIVITY COMES FROM THE
PROTEIN INSTEAD OF COMING FROM
THE RNA.
IF WE ARE ABLE TO TALK AT A 2
PRIME HYDROXYL GROUP ON THE
[INDISCERNIBLE], THEN WE SHOULD
BE ABLE TO ELIMINATE THE
NUCLEOTIDE BIAS, AND SINCE
APPROXIMATELY ENABLED
REACTIVITY, DOESN'T REQUIRE AN
EXTERNAL TRIGGER, SO WE THINK
THIS IS--SHOULD HAVE WORKED BOTH
IN CELLS AND IN VIVO.
IN ADIG THE REACTION OCCURS
EXCLUSIVELY BETWEEN THE UNEND
TERMINAL AND THE BIONUCLEOTIDE
AND THEREFORE SHOULD PROVIDE A
SINGLE AMINO ACID RESOLUTION AND
SINGLE NUCLEOTIDE RESOLUTION.
WE USE THE [INDISCERNIBLE]
MATURE RNA, TO STUDY MATURE RNA.
ON THE CRYSTAL STRUCTURE, NEAR
THE CLEAVAGE SIDE POSITIONS IN
THE SITE TO [INDISCERNIBLE] THE
BOWPPED RNA.
AFTER INCUBATING THIS MUTANT
CAS13 B, WE FOWBD IT WAS ABLE TO
CAPTURE THE PRECURSOR RNA
REGARDLESS OF NUCLEOTIDE
IDENTITY OUT OF THE CLEAVAGE
SITE.
THIS DEMONSTRATES THAT GECX-RNA
CAN TARGET THE RTHIS,A WITHOUT
THE NUCLEOTIDE BIAS.
TO DEMON TRAIT THE UTILITY OF
GECX- RNA, WE DEVELOP THE
TECHNOLOGY TO MAP THE
MODIFICATION ACROSS THE
TRANSCRIPT ORDER OF MICRONS.
IT REGULATES MRNA FUNCTION.
WHILE SEVERAL DETECTION METHODS
ALREADY EXIST THEY EITHER LACK
SINGLE NUCLEOTIDE RESOLUTION OR
LACK OF PROTEIN SPECIFICITY OR
THEY RELY ON THE ANTIBODY FOR
INVITRO RECOGNITION.
OUR GOAL IS TRYING TO DETECT THE
M6A DIRECT IN SALES WITH BOTH
SINGLE NUCLEOTIDE RESOLUTION AND
PROTEIN SPECIFICITY.
OUR STRATEGY INVOLVES EXPRESSING
THE M6A READER PROTEIN IN LIVE
CELLS TO RECOGNIZE M6A.
AND LATENT BIOREACTIVE END
TERMINAL ACID IS INCORPORATED
INTO THE READER PROTEIN NEAR THE
M6 A BINDING SITE.
ONCE THE READER PROTEIN
RECOGNIZE THE M6 A THROUGH THE
GECX-RNA MECHANISM, THEN WE
CROSS LINK THIS NUCLEI O TIDEOT
RNA OF THE AND THEN THIS COMPLEX
IS PULLED DOWN AND THE PROTEIN
WAS DIGESTED TO RELEASE THE
CAPTURED RNA.
THIS CAPTURED RNA, IS THEN
REVERSE TRANSCRIBED WITH THE
PROCESS TERMINATED AT THE CROSS
LINKING SITE.
DUE TO THE PRESENCE OF THE END
TERMINAL ACID, AND THIS RNA
TRANSCRIPTS WILL BE PC R
AMPLIFIED AND THEN SEQUENCED IN
HIGH THROUGH PUT AND THIS WILL
REVEAL ALL THE CROSS LINKING
SITES, THEN WOANS WE KNOW THE
CROSS LINKING SITES WE THEN
EXPECT THE M6A SITE SHOULD BE
ADJACENT TO THE IDENTIFY THE
CROSS LINK IN SIZE, SO FOR THIS
EXPERIMENT, WE USED THE WIDE
URGE OF THE MEN OF THE M6A
READER PROTEIN, AS M6 A READER
PROTEIN.
AND WE INCORPORATE END TERMINAL
ACID FSY INTO THE M6A BINDING
SITE OF THIS YTH DEMAND AND
PUTTING IT CLOSE TO THE M6A
SHOWN IN BLUE HERE.
WE TESTED THAT TESTED THIS
[INDISCERNIBLE] TECHNOLOGY IN
THE CELLS.
HERE IT SHOWS THE PEAKS OF 1
EXAMPLE AS YOU CAN SEE THE PEAKS
TERMINATED ALREADY, SOME OF THEM
AT THE M6A SITE, WE THEN
ANALYZED ALL THE NUCLEOTIDES
SURROUNDING THIS TERMINATION
PEAKS AND FOUND THEY ARE
ENRICHED WITH THIS DARK MOTIF
WHICH IS CONSISTENT WITH THE
CONSENT MOTIVE OF M6A, AND IN
ADDITION WE FOUND THAT CROSS
LINKING SITE, PRIMARILY AT THE
MINUS STARTER POSITION RELATIVE
TO THE M6A MODEL
CITIZENIFICATION INDICATING THIS
APPROACH GAVE US A SINGLE
NUCLEOTIDE RESOLUTION EMPLOY SO
USING THIS APPROACH WE PREDICTED
A TOTAL OF ALMOST 14,000 M6 A
SIDE FOR DEMAND.
AND 6000 ISA A KNOWN M6A SITE
APPROACH AND THE REST ALMOST
8000 NEWLY IDENTIFIED.
IN SUMMARY AS YOU CAN SEE, OUR
METHOD CAN RECOGNIZE M6A
DIRECTING LIVE CELLS HAS
PROTEIN--READER PROTEIN
SPECIFICITY, AND THEREFORE WE
ANTICIPATE THIS CAN BE SIMILARLY
BE USED TO MAP ALL THE OTHER
MODISHS IN CELLS AS LONG AS YOU
HAVE A PROTEIN TO RECOGNIZE THE
RNA MODIFICATION.
NEXT I WOULD LIKE TO DISCUSS
GENETIC ENCODED CHEMICAL CROSS
LINKING OF CARBON HYDRATES AND
METHOD WE REFER TO AS
GECX SUGAR.
PROTEIN GLYCAN INTERACTIONS PLAY
A CRUCIAL ROLE IN VARIOUS
BIOLOGICAL PROCESSES, RAINCHLING
FROM INTERSPECIES COMMUNICATION
TO INTERSPECIES RECOGNITION
EMPLOY FOR INITANCE, THE BINDING
OF C-GLYCOPROTEIN TO THE FLI
COLSIS CAN AND REACH THE
SELF-RECOGNITION IN OUR IMMUNE
SYSTEM.
AND IN FACT, VIRAL PATHOIENS CAN
EXPLOIT THE [INDISCERNIBLE] TO
INHIBITOR C-GLAC ON THE CELLS
THEREFORE TO EVADE IMMUNE
DETECTION.
SIMILARLY TUMOR CELLS CAN
UPREGULATE THE SALIC GLUE
MARIOUS GAND ON THE SURFACE AND
USE THIS TO ENGAGE THE SIGLEC ON
THE IMMUNE CELLS'DING TO IMMUNE
ESCAPE AND CANCER PROGRESSION
AND METASTASIS.
>> DESPITE THE ESSENTIAL ROLE IN
MOLECULAR RECOGNITION, THE
PROTEIN GLYCAN INTERACTIONS HAVE
BEEN VERY DIFFICULT TO STUDY,
MANY OTHER DIFFICULTIES IN MAJOR
CHALLENGE OF 1 HIGHLIGHTER HERE
IS THE LO AFFINITY BETWEEN A
SINGLE GLYCAN, A SINGLE SUGAR
PROTEIN RECOGNITION WITH KD,
DISASSOCIATION CONITANT OFTEN IN
THE HIGH MICROMOLAR AND IN THE
MINIMOLER RANGE.
SINCE THE BINDING AFFINITY IS SO
LOW, SO WE THOUGHT CAN WE ON
COVALENTLY STABILIZE PROTEIN FLI
COLSIS KAN--KANA INTERACTION
THEREFORE TO BETTER STUDY IT?
HOWEVER, WE FOUND THERE WAS NO
REACTION THAT CAN REACT WITH
GLYCAN UNDER VERY MILD
CONDITIONS, SO WE HAVE TO TART
TO IDENTIFY A FUVENGZAL GROUP
THAT IT CAN REACTIVE WITH FLI
COLSIS KAN--KANA UNDER
BIOCOMPATIBLE CONDITIONS.
TO DO THIS, WE DEVELOP A PLAN
AND A PLAN, AND A CROSS LINK
[INDISCERNIBLE].
WE FIRST INCUBATE THE
C-GLYCOPROTEIN THE LIGAND OF
GD3, WHEN THEY BIND WITH EACH
OTHER, THEN WE THROW IN A BUNCH
OF CROSS LISTEN INCHINGERS.
ONE END REACTS RAPIDLY WITH THE
PROTEIN, PRIOR TO THE CROSS
LINKING OF THE PROTEIN AND CAST
AT THE OTHER END TOWARD THE
BOUND TBLI KAN--KANA, IF THIS
END REACTIVE WITH OUR GLYCAN,
THEN COVALENT COMPLEX OF A
GLYCAN PROTEIN WILL BE FORMED
AND,A LOW DETECTION EMPLOY SO WE
DECIDED AND THE SLIDE TO SEVERAL
CROSS LINKERS AND DORPHED THAT
THIS FUNCTIONAL GROUP,
[INDISCERNIBLE] FLUORIDE WAS
ABLE TO REACT WITH TBLI
KAN--KANAS WITH MILD CONDITIONS
AND IT'S THE ONLY POSITIVE SPOT
WE DETECTED.
NEXT WE THEN DETERMINED THIS FSY
TO CONTEND TO IDENTIFY THE
FLUORIDE FUMPLEG ALGORITHMS UPON
GROUP AND TO IPT GREATER
CORPORATE THIS END TERMINAL ACID
INTO THE BYPASSING PROTEIN.
AND WE FOUND ONCE THIS END
TERMINAL IS INCORPORATED INTO
THIS SPECIFIC SIZE, IT ENABLED
TO BYPASSED THE SUITE
COVALENTLY.
NEXT WE THEN INCUBATED THE
CANCER CELL LINE THAT HAS HIGH
LEVEL OF THE TBLI CAN WITH THE
[INDISCERNIBLE] OR COVALENT 7
FSY.
AS YOU CAN SEE THE COVALENT
VERSION OF SIGLEC 7 HAS DRAMATIC
INCREASE CELL BINDING COMPARED
TO THE WILD-TYPE SIGLEC 7, AS
SHOWN IN THIS GRAPH HERE,
FURTHER MODEL CITIZEN IF WE
PRETREAT THE CELLS WITH
[INDISCERNIBLE] TO REMOVE THE
SIALOGLYCAN, NOW YOU CAN SEE
IT'S REDUCED TO 1 TENTH OF THE
ORIGINAL LEVEL.
THAT SUPPORTS THE SIGLEC 7 V,
SFY, FOR THIS CANCER CELL
SURFACE.
MANY TUMORS UPREGULATE CELL
SURFACE GLYCAN AND THIS DPLI
KAN--KANA CAN ENGAGE WITH THE
SIGLEC RECEPTOR 7 ON THE HUMAN
CELLS.
THIS IS A INHIBITOR SIGNAL TO
TAXONOMY DOWN THE ABILITY TO
KILL THE CANCER CELLS.
WE ENVISIONED IF WE THROW IN AN
IRREVERSIBLE BINDER THIS SIGLEC
7 V, SFY, WHEN IT BIEBDS TO THE
CANCER CELL SURFACE CELL,
GLYCAN, THIS WILL RELEASE THE
CELL FROM THE INHIBITORY
SIGNALING ACCESS AND THEREFORE
ENHANCED NK CELLS ABILITY TO
KILL THE CANCER CELL.
TO TEST THIS HYPOTHESIS, WE ARE
TREATED 3 DIFFERENT CANCER CELL
LINES, THAT HAS HIGH LEVEL
[INDISCERNIBLE] WITH EITHER
WILD-TYPE SIGLEC 7 OR ANOTHER
VERSION OF SIGLEC7 SFY, AND WE
THEN WASH THE CELLS AND ADD THEM
BACK IN AND MEASURE THE CANCER
CELL DEATH.
AS YOU CAN SEE HERE IN THIS
GRAPH, SHOWN IN RED IS OUR
COVALENTERTION OF THE SIERK GLEC
7 V, AND THEY ENHANCE THE
ABILITY TO KILL CANCER CELLS
COMPARED TO THE WILD-TYPE IN
BLACK HERE.
AND THIS FINDING SUGGESTS THAT
COVALENT TARGETED THE GLYCANOT
CANCER CELL SURFACE, MIGHT IT BE
A NEW APPROACH FOR CANCER
IMMUNOTHERAPY.
IN THE FINAL PART OF MY TALK, I
WILL DISCUSS THAT THE CO
VALENTINEDENT PROTEIN DRUGS
USING PRACTICALS MALLY ENABLED
THERAPEUTICS OR PERX FOR SHORT.
COVALENT SMALL MOLECULES CAN
FORM A COVALENT BOND WITH THEIR
TARGET AFTER THE BINDING, AND
THIS LINK CANCER CENTER OFFERS
SEVERAL ADVANTAGE OVER THE
NONCOVALENT DRUGS SUCH AS
INCREASED POTENCY, PROLONGED
DURATION OF ACTION, LESS
PREQUENT AND DOSING AND THE ABLE
TO TARGET THE PREVIOUSLY
UNDRUGGABLE PROTEINS.
ALTHOUGH THE PHARMACEUTICAL
INDUSTRY, IS TOTALLY AVOIDED
THEM DUE TO CONCERN OF OFF
TARGETED REACTION, COVALENT
SMALL MOLECULE DRUGS HAVE
RESURGED WITH GREAT SUCCESS IN
THE RECENT YEARS.
IN CONTRAST THE POTENTIAL OF
COVALENT PROTEIN DRUGS REMAINS
LARGELY UNEXPLORED DUE TO
PROTEINS TYPICALLY LACK THE
ABILITY TO FORM COVALENT BOUNDS
WITH THEIR TARGETS.
TO ENABLE COVALENT PROTEIN DRUGS
WE DEVELOP THE TECHNOLOGY
RECORDED APPROXIMATELILY ENABLED
THERAPEUTICS, IN THIS APPROACH,
SIMILARLY, WE INCORPORATE LATE
IN THE BIOTERM NASAA.COMITY,
INTO THE PROTEIN DRUG NEAR THE
BINDING INTERFACE.
ONCE THE PROTEIN DRUG BINDS WITH
THE TARGET, THEN THE END
TERMINAL REACT WITH THE RESIDUE
IRREVERSIBLY CROSS LINK THE
PROTEIN DRUG TO ITS TARGET
ESSENTIALLY SERVING AS COVALENT
PROTEIN DRUG.
DURING THE COVID PANDEMIC, WE
DEVELOPED A COVALENT NANO BODIES
TRYING TO INHIBIT CROSS
INFECTION USING THIS PERX
MECHANISM.
AS WE ALL KNOW NOW, THE VIRUS
ENTERS THE CELLS WITH THE SPIKE
PROTEIN WITH THE HUMAN AEC2
RECEPTOR, THE CONVENTIONAL NANO
BODIES AND ANTIBODIES CAN BIND
TO THE SITE TO INHIBIT THIS
PROCESS BUT SUCH BIND SUGGEST
NONCOVALENT AND THEREFORE IT'S
REVERSIBLE AND THIS MAKES
COMPLETE INHIBITION QUITE
CHALLENGING.
WE THOUGHT IF WE USE THE
COVALENT NANO BODY TO
IRREVERSIBLY BIND IT TO THE
SPIKE PROTEIN OF THE VIRUS, THIS
MAY REDUCE THE LIKELIHOOD OF A
VIRAL ESCAPE AND SIGNIFICANTLY
IMPROVE TO ACHIEVE A MORE POTENT
INHIBITION.
WE THEN WORK WITH NANO BODY MMB6
TO FIND OUT A SITE THAT IT CAN
DO THE CROSS LINKING.
WE ACTUALLY KREENED ALL SITES IN
THE REGIONS OF THIS NANO BODY
USING FSY.
AS YOU CAN SEE IN THIS CROSS
LINKING ANALYSIS WE FOUND A
MULTIPLE SIZE ENABLING CROSS
LINKING OF THE VIRAL SPEAK OF
THE PROTEIN IN CDR1 REGION HERE
AND IN CDR3 REGION WE FOUND
MULTIPLE SITES THAT ENABLE CROSS
LINKING.
BECAUSE THIS VIRUS INFECTION IS
A VERY ACUTE PROCESS.
QUEE WOULD LIKE TO ACCELERATE
THE REACTION, TO INTRODUCE THIS
WE INTRODUCED FLUORINE GROUP ON
INTO THE FSY CREATING NEW END
TERMINAL TO THE FFY, AND THIS
INCREASED THE REACTION RATE BY
2.4.
IN BEST CASES WHEN WE
INCORPORATE FFY OF THIS SITE OF
THE MMB6, IT CAN CROSS LINK THE
SPIKE PROTEIN INVITRO WITHIN 10
MINUTES.
TO EVALUATE THE NEUTRALIZATION
EFFICIENCY, WE TREATED THIS
PSEUDOVIRUS OF A SARS-COV-2 WITH
EITHER WILD-TYPE NANO BODY OR
COVALENT NANO BODY AND THENEE
WIEWS THIS MEX TOUR TO EFFECT
THE CELLS, AND THE EFFECTOR
CELLS WERE MEASURED AND PLOTTED
AGAINST THE CONCENTRATION OF THE
NANO BODIED ADDED.
AS YOU CAN SEE IN THIS PLOT, IN
GRAY IS THE WILD-TYPE NANO BODY
WHICH HAS 36 NANO MOLAR.
IN CONTRASTURE COVALENT NANO
BODY WITH THE 1 NANO MOLAR,
SUGGESTING 36 FOLD IMPROVEMENT,
WE FURTHER TESTED THESE NANO
BODIES AGAINST THE AUTHENTIC
SARS-COV-2 VIRUS, AGAIN AS CAN
YOU SEE HERE, THE CO VALENTINED
END NANO BODY ACHIEVED A 40 FOLD
IMPROVEMENT, COMPARED TO THE
WILD-TYPE NANO BODY.
REPRESENTING, SO THIS RESULT
INDICATED THAT A SIGNIFICANT
INCREASE IN POTENCY WHEN YOU
HAVE A COVALENT BOUNDING ABILITY
IN NEUTRALIZING THIS WILD-TYPE
SARS-COV-2.
IN ADDITION TO THE WILD-TYPE
VIRUS, MMB6 WAS ALSO ABLE TO
BIND DIFFERENT VALID AND
RELIABLE YABTS INCLUDING ALPHA,
DELTA, EPISILLSON, AND LAMDA,
AND OF COURSE WE TESTED THE
CROSS LINKING WE FOUND OUR
COVALENT NANO BODY WAS ABLE TO
CROSS LINK THIS CO VALENCE AS
WELL.
IN ADDITION THEY SHOW THAT
DRASTIC INCREASE IN THE POTENCY
IN NEUTRALIZING THIS SARS-COV-2
VARIANTS.
BUT THE MMB6 NANO BODY WAS
UNABLE TO BIND THE LATEST
OMICRON VERYIANT SO WE THEN
ENGINEERED A NANO BODY CALLED
MD70 WHICH WAS ABLE TO BIND TO
THE OMINOCRON VARIANT AND IT
CROSS LINKED THE VARIANT IN HIGH
EFFICIENCY, THIS ACTUALLY CAN
ACHIEVE A SIGNIFICANT CROSS
LINKING WITHIN 5 MINUTES.
AND AS YOU CAN SEE HERE, IT NANO
BODY SHOWED 8-10 FOLD INCREASE
IMPROVEMENT IN THE WILD-TYPE
NANO BODY IN NEUTRALIZING THIS
OMICRON VARIANT.
SO IN SUMMARY WE FOUND THE CO
VALENTINED EPT NANO BODY ARE
SIGNIFICANTLY MORE POTENT THAN
THE WILD-TYPE, IN NEUTRALIZING
WILD-TYPE SARS-COV-2 AND ITS
VARIANTS.
IN A SECOND EXAMPLE, I WOULD
LIKE TO DEMONSTRATE HOW PERX CAN
ADDRESS A LONG STANDING
CHALLENGE IN THE TARGETED RADIO
AND NUCLEI, TRT, TRT BASICALLY
AIMS TO THEREFORE RADIO ACTIVITY
INTO TUMORS VIA MINIMIZING THE
EXPOSURE TO NORMAL TISSUE.
AND THE RADIATION HAS A VERY
UNIQUE ABILITY TO KILL CELLS
REGARDLESS OF THE DEC MECHANISM
AND THEREFORE IS CURRENTLY
EXPERIENCE CLINICAL RESURGENCE,
HOWEVER A MAJOR CHALLENGE IN THE
FIELD HAS BEEN HOW TO BALANCE
EFFICACY WITH SAFETY.
AS CAN YOU SEE HERE, YOU CAN USE
CONVENTIONAL ANTIBODY TO
THEREFORE THE RADIO ISOTOPE, CAN
YOU ACHIEVE A VERY EFFECTIVE
TUMOR ACCUMULATION BUT BECAUSE
ANTIBODIES CAN AT A IN THE BLOOD
STREAM FOR A LONG TIME, THIS
INCREASE THE DAMAGE TO NORMAL
TISSUES.
IN CONTRAST, YOU CAN USE LO
MOLECULAR RATE FOR THE RADIO
ISOTOPE, THIS CAN BE CLEARED
FROM OUR BLOOD VERY QUICKLY,
PROVIDING A SAFER PROFILE, BUT
IN THE MEAN TIME REDUCE TUMOR
UPTAKE AND REDUCE EFFICACY.
AND IDEAL RADIO PHARMACEUTICAL
WOULD BE COMBINING THIS, TOO,
WHAT IF WE HAVE A FAST PLOOT OF
CLEARANCE AND HIGHER TUMOR
UPTAKE, THAT WOULD BE PERFECT.
SO WE THOUGHT THAT A SMALL
COVALENT PROTEIN WILL BE--COULD
SERVE AS AN IDEAL VEHICLE FOR
RADIO PHARMACEUTICAL, BECAUSE
IT'S LOW, WE ALLOW IT TO RAPIDLY
BE CLEARED FROM THE BLOOD
STREAM, BUT IN THE MEAN TIME,
ONCE IT'S IRREVERSIBLE TO THE
BINDING OF THE CANCER ANDROGEN
GEN, YOU SAY AT A THERE FOR A
LONGER TIME TO ACHIEVE A MORE
THERAPEUTIC.
SO TO TEST THIS CONCEPT WE BEGAN
WITH COVALENT NANO BODY SPECIFIC
TO THE HER2 RECEPTOR.
SIMILARLY WE INCORPORATED FSY
INTO THE NANO BODY OF THIS P54
SITE TO TARGET THE HER2 BY
TARGETING LYSINE 150 SITE AND WE
SHOWED THIS COVID AND NANO BODY
FSY COULD CROSS LINK THE HER2
EXTRA CELLULAR DOMAIN VERY
EASILY AND IT COULD BE DETECTED
WITHIN 10 MINUTES.
WE THEN SHOWED THAT THIS NANO
BODY WAS ALSO ABLE TO CROSS LINK
AT THE ENDOGENOUS HER2 RECEPTOR
ON CELL SURFACE ON DIFFERENT
CANCER CELL LINES.
IN THIS 2 CELL LINES THAT LACK
HER2 EXPRESSION AND THEREFORE WE
DIDN'T DETECT CROSS LINKING
SUGGEST THANKSGIVING CROSS LINK
XG HIGHLY SPECIFIC.
WE FURTHER SHOWED THAT THIS MB
NANO BODY FSY WAS ABLE TO CROSS
LISTENING THE HEERROR 2 RECEPTOR
TO THE TUMOR DIRECTLY IN MICE AS
WELL.
WE NEXT LABELED THIS NANO BODIES
WITH RDON 124 AND INYECT THEM
INTO MICE XENO GRAPHED WITH THE
HER2 EXPRESSING TUMORS FOLLOWED
BY THE PET IMAGING.
TO YOUR RIGHT SIDE FROM THE 3210
POST INYEKS, AS YOU CAN SEE
THERE WAS NO SIGNIFICANT
DIFFERENCE BETWEEN THE WILD-TYPE
NANO BODY, THE TOP HERE, AND THE
COVALENT NANO BODY IN TERMS OF A
TOMB'RE TUMOR SIGNATURES NOX
FAMILY ACTIVATOR.
HOWEVER A DRAMATIC REDUCTION
THETIC DEFENSE CAN BE FOUND
AROUND 24 HOURS TO 72 HOURS.
WILD THE NANO BODY, THE
WILD-TYPE NANO BODY WAS NO
LONGER DETECTABLE, THE
COLVALENTINEDDENT NANO BODY
REMAINS CLEARLY SEEN.
FURTHER MORE IF WE DO 3D IMAGING
AS YOU SEE HERE FROM 2472 HOURS,
THE TUMOR SIGNAL WAS DISTINCTLY
VISIBLE USING THE NANO BODY BUT
NOT VISIBLE IN THE WILD-TYPE
NANO BODY.
AND THE--ANOTHER EXCEPTIONAL
OBSERVATION IS THAT WE SEE VERY
CLEAR BACKGROUND AUTONATIONAL
LIBRARY OF MEDICINAL TISSUES
EXCEPT THE TIARAS ROADWAY
BECAUSE IT IS THE NATURAL SIDE
FOR R-DINE METABOLISM.
AND THE TOTAL RADIATION
ACCUMULATED IN THE TUMOR SHOW ON
LEFT HERE IS 81% MORE WITH THE
COVALENT NANO BODY, SO THIS
RESULTS TELL US THAT THE
COVALENT NANO BODY SIGNIFICANTLY
PROLONGED TUMOR RETENTION AS
WELL AS INCREASE THE TUMOR
ACCUMULATION OF THE RADIO
ISOTOPE.
WELL, WOULD THIS BE SUFFICIENT
TO IMPACT ANTITUMOR EFFECT.
SO TO TEST THIS WE THEN LABELED
THE NANO BODY WITH THE 225,
POTENT [INDISCERNIBLE].
AND THEN INJECT THIS NANO BODY
INTO THE MICE XENOGRAFTICALLY TO
HER2 TO EXPRESS IN TUMORS.
AS CAN YOU SEE IN THIS GRAPH
HERE, COMPARED TO THE SALINE IN
PLAQUE, IT SHOWED NOW DIFFERENCE
IN TUMOR INHIBITION, IT
CONTRAST, IN ORANGE HERE THE
COVALENT ANTIBODY LABELED
[INDISCERNIBLE] 25, SHOWS A
SIGNIFICANT TUMOR INHIBITION.
AND END POINT ANALYSIS OF THE
TUMOR WEIGHT ALSO INDICATE THE
COVALENT NANO BODY REDUCED TUMOR
WEIGHT SIGNIFICANTLY BUT NOT
WITH THE WILD-TYPE NANO BODY,
AND THE BODY WEIGHT OF THE MICE
DID NOT CHANGE SIGNIFICANTLY
ACROSS ALL THRESHOLD GROUPS,
SUGGESTING THERE'S NO SYSTEMATIC
TOXICITY.
WE FURTHER DID A TISSUE ANALYSIS
OF LIVER, THE KIDNEY THE HEART
AND THE BONE HERE, AND WE DIDN'T
FIND ANY ANOMALIES SUGGESTING
THERE'S NO TOXICITIES EITHER,
HERE.
SO IN SUMMARY THE RADIO
PHARMACEUTICAL USING THIS COVID
NANO BODY OF THE DELIVERY
VEHICLE SHOWS POTENTIAL FOR
EFFECTING TUMOR INHIBITION
WITHOUT A COURSE IN TISSUE
TOXICITY.
TO CONCLUDE MY TALK, BY
DEVELOPING LATENT BIOREACTIVE
UNNATURAL TERMINAL AMINO ARK
SIDS THROUGH THE APPROXIMATE
ENABLED BIOREACTIVE MECHANISM,
WE CAN NOW GENETICALLY INTRODUCE
COVID LINKAGES BETWEEN PROTEINS
AS WELL AS TEEN PROTEINS AND
OTHER BIOMOLECULES INCLUDING
RNA, AND THE CARBOHYDRATES.
THIS COVALENT CONNECKS ARE
SELECTIVELY STABLE AND
IRREVERSIBLE AND HOPING UP NEW
POSSIBILITIES FOR CREATING NOVEL
PROTEIN PROPERTIES WHICH
I--DON'T COVER TODAY.
AND WE CAN USELESS USE THESE
COVALENT LINKAGES TO STUDY CELLS
IN VIVO.
ADDITIONAL WE DEVELOPED THIS
PLATFORM TECHNOLOGY PERX FOR
COVALENT PROTEIN DRUGS WHICH HAS
SHOWN PROMISE IN CANCER'MUNE O
THERAPY, SARS-COV-2
NEUTRALIZATION AND A TARGETED
RADIO NUCLEIDE THERAPY.
WE ANTICIPATE THAT SHIFTING THE
PARADIGM FROM NONCOVALENT TO
COVALENT INTERACTIONS IN PROTEIN
MOLECULAR ADVANCE AMS CAN HELP
BASIC BIOLOGICAL RESEARCH AND I
BIOTHERAPEUTICS.
FINALLY, I WOULD LIKE TO THANK
MY GROP MEMBERS BOTH PAST AND
PRESENT FOR MAKE THANKSGIVING
POSSIBLE AND I'M ALSO DEEPLY
GRATEFUL FOR THE GENEROUS
SUPPORT OF MANY WONDERFUL
COLLABORATORS THAT ARE LISTED
HERE AND VARIOUS FOUNDING
ORGANIZATIONS WITH A SPECIAL
THANKS TO NIH FOR YOUR
CONTINUOUS SUPPORT AND THANK YOU
VERY MUCH FOR YOUR ATTENTION.
I WILL BE HAPPY TO TAKE
QUESTIONS.
[ APPLAUSE ]
SO THERE ARE MICs ON EITHER
SIDE, NO QUESTIONS YET COMING ON
LINE.
>> HI, LEI, GREAT TALK.
SO I HAVE SORT OF A CONCEPTUAL
LEVEL QUESTION.
SO YOU HAVE DIFFERENT LATENT
BIOREACTIVE AMINO ACIDS AND THEY
HAVE SORT OF DIFFERENT LEVELS OF
REACTIVITY, DO YOU THINK YOU
PUSHED REACAS FAR AS CAN YOU
WITHOUT ENCOUNTERING SOME SORT
OF AUTOREACTIVITY OR DO YOU
THINK IT WILL BE POSSIBLE TO
MAKE AMINO ACIDS THAT HAVE
HIGHER RATES OF REACTIVITY IN
THE CONTEXT OF FULL SIZE
PROTEINS?
WOO HAVE A WIDE RANGE OF OF
REACTIVITY.
WE STUDIED THIS SYSTEM WITH
DIFFERENT BYPASSING AFFINITYS,
ET CETERA WE FOUND THAT THE
REACTION DEPENDS ON MULTIPLE
FACTORS.
THE INTRINSIC REACTIVITY OF THE
WAR HEAD IS 1 ASPECT OF IT, THE
AFFINITY IS ANOTHER PART AND THE
ORIENTATION IN THE DISTANCE,
EVERYTHING ENCLOUDED CONTRIBUTES
TO THE FINAL REACTIVITY, TO GIVE
YOU EXAMPLE, SOME OF THE
REACTIONS MAY TAKE MULTIPLE
HOURS, 6 HOURS, 8 HOURS, TO
ACHIEVE 80% AND SOME ASSISTANCE
WE FOUND THAT THIS CROSS LINKING
CAN OCCUR WITHIN 5 MINUTE ANDS
IN SOME SYSTEMS WE FOUND THE
CROSS LINKING CAN REACH 99%
COMPLETION WHEREAS MANY OTHER
SYSTEMS, AS YOU SUGGESTED WE DO
SEE INTRA MOLECULAR CROSS LINK.
AND GENERALLY AS YOU SUGGESTED
THIS LINK IS CORRELATED WITH THE
REACTIVITY OF THE WAR HEAD, SO
I'M HOPING THAT WITH THE
ADVANCEMENT OF NEW CHEMISTRY, WE
MAY BE ABLE TO FIND AN EVEN
BETTER [INDISCERNIBLE] THAN WHAT
WE CURRENTLY HAVE TO MINIMIZE
CROSS LINKING WHILE
SIGNIFICANTLY EXPAND, ACCELERATE
THE FINAL REACTION FOR EXAMPLE,
BETWEEN 2 PROTEINS INSTEAD OF
HAVING BY INTERNAL MOLECULAR
REACTIONS.
GREAT, THANK YOU!
>> HI THIS, IS AN AMAZING,
CREATIVE AND THOUGHT PROVOKING
PRESENTATION EMPLOY
CONGRATULATIONS.
I'M JUST WONDERING ABOUT THE
PATH TO COMMERCIALIZATION, AND
WE KNOW HOW DIFFICULT IT IS HERE
TO PUT OUR SMALL MOLECULES ON TO
A TRANSLATIONAL PATH.
I IMAGINE THAT IF YOU PROPOSE TO
ACCOMPANY THAT HAVE YOU SOME
COVALENT WAR HEAD ON YOUR
PROTEIN OR ON YOUR SMALL
MOLECULE, THE PATH WOULD BE EVEN
HARDER.
WHAT CAN YOU SAY ABOUT THE
DERISKING OR POTENTIAL TOXICITY
OF THE WAR HEAD.
YOU MEKSED IN 1 CASE YOU DIDN'T
HAVE ANY REACTIVITY WITH GLUTEA
THE IPAD OWN, BUT WHAT EVERYONE
DO YOU HAVE THAT THERE'S NOT
OTHER TOXICITY FOR INSTANCE THE
TYROSEEN DERIVATIVES WITH THE
DISCIPLINARY SULFER
NEOFLUORIDES.
>> GREAT QUESTION.
THANK YOU VERY MUCH FOR POINTING
THAT OUT.
O MY THINKING IS COVALENT SMALL
MOLECULE DRUG VS BEEN SO
SUCCESSFUL AND INITIALLY PEOPLE
ALSO WERE AFRAID OF MANY OFF
TARGET AND TOXICITIES THE REASON
WE STARTED THIS PROGRAM ALMOST
10 YEARS AGO IS WE THINK
PROTEINS COMPARED TO SMALL
MOLECULES, I THINK CO VERSION, I
IT SHOULD HAVE ADVANTAGES OVER
SMALL MOLECULES BECAUSE 1,
PROTEINS HAVE LARGER INTERACTION
AND INTERPHASE WHICH CAN ACHIEVE
A BETTER SPECIFICITY, AND
PROTEINS ALSO HAVE A LOT OF
LOCAL MICROVIERM, IDEALLY YOU
CAN MANIPULATE BUT MANY TIMES
YOU COULD HAVE A CHANCE TO FOR
EXAMPLE, BLOCK AMINO REACTION TO
TRIGGER THE REACTIVITY AND
ANOTHER THING I WANT TO POINT
OUT IN OUR CASE, WHEN THE
COVALENT--IN ORDER FOR THE
COVALENT BOND TO FOR THE
PURPOSE, THERE ARE 2 STEPS
INVOLVED, 1 IS PROTEIN A, NEEDS
TO BIND WITH PROTEIN B,
REQUIREMENT 1.
THIS REQUIRES SOME SPECIFICITY,
AND THE REQUIREMENT OF B, IS
THIS END TERM NOWS EXCITAIEN IS
CORRECT IN THE ORIENTATION IS
DISTANCE TOWARDS ITS TARGET OF
NATURAL RESIDUE, THEREFORE THESE
2 GAUGE, THE SPECIFICITY I THINK
IS VERY HIGH.
IN ADDITION WE DID SOME
OFF-TARGETED REACTIONS BOTH IN
MICE AND IN HUMAN SERUM, AND
USING WESTERN BLOT WE HAVEN'T
DETECTED ANY OFFTARGET CROSS
LINKING YET.
OF COURSE I UNDERSTAND THIS IS
NOT SUFFICIENT AND FOR EXAMPLE
IN OUR RADIO TRT EXPERIMENT, WE
ALSO MONITORED THE SECRETION OF
THE COVALENT NANO BODY VERSUS
WILD-TYPE NANO BODY RADIO
LABELED AND WE DIDN'T SEE ANY
SIGNIFICANCE EITHER, SUGGESTING
THERE'S NO SIGNIFICANT MUTATION
OR OFF TARGET REACTIVITY,
EITHER.
BUT YOU HAVE A IMRIT POINT.
IT'S REALLY IMPORTANT TO ADDRESS
THIS AND I THINK AS THIS
COVALENT PROTEIN DRUG, AT EARLY
STAGE, THERE'S A LOT OF WORK TO
BE DONE AND WE THINK THIS' A
GREAT POTENTIAL BUT WE NEED TO
ARE VERY CAREFUL TO EVALUATE ALL
THIS ASPECTS AS YOU SUGGESTED.
THANK YOU.
NTHANK YOU.
>> HI, LEI, THAT WAS BEAUTIFUL.
I HAVE 2 QUESTIONS.
THE FIRST QUESTION ACTUALLY SORT
OF RELATES TO THE RATE
ENHANCEMENT OF THE SULFINATE
FLUORIDE REACTION.
SO HAVE YOU DONE ANY SORT OF
INVITRO MEASUREMENTS THAT GIVE
YOU AN IDEA OF WHAT YOU HAVE A
PROTEIN-PROTEIN BYPASSING
FOLLOWED BY THEN THE REACTION
WITH THE HYDROXYL WITH THE
SULFINE OLDER PEOPLE FLUORIDE,
WHAT ENHANCEMENT ARE YOU
ACHIEVING COMPARED TO LET'S SAY
JUST IN SOLUTION REACTION, AND
COUPLED TO THAT I HAVE ANOTHER
QUESTION WHICH IS THAT WHEN YOU
THINK ABOUT JUST THE FEN
ILLEGALS SULFIN FLUORIDE AND
THEN THAT ON A PROTEIN, ARE YOU
ACTUALLY RETARDING ITS REACTION
BY ESSENTIALLY PUTTING ON A
BULKY SUBSTITUTE WHICH IS THE
PROTEIN?
SO IN OTHER WORDS IS THE
REACTION ON SUCH ON A NONENGAGED
SURFACE RETARDED BECAUSE IT HAD
STATIC INHIBITION BY HAVING
BULKY PROTEIN, CAN YOU COMMENT
ON THAT?
>> REALLY INTERESTING COMMENT.
THE FIRST 1 IS HOW MANY PHOTO
IMPROVEMENT WE HAVE WITH THIS
EFFECT.
SO WHEN THE COMEMMISTRY WAS
DEVELOPED BY BARRY SHARPLESS ON
THE SMALL MOLECULE LEVEL, THEY
NEED CATALYST TO ALLOW THE
REACTION TO OCCUR, BUT LATER
FURTHER DEVELOPMENT SHOWS CAN
YOU ALSO HAVE SOME
[INDISCERNIBLE] IN THE CATALYST.
IN OUR CASE, BARRY AND JEFF
[INDISCERNIBLE] AT SCRIPTS THEY
DID BEAUTIFUL SPRMS TO TEST THE
REACTION OF THIS ON THE SMALL
MOLECULE LEVEL, YOU CAN
BASICALLY BOIL UPON FLOWERRAL
SULFONATE WITH [INDISCERNIBLE]
AND THEN BOIL, MANY, MANY HOUR
ANDS THERE'S NO REACTION, IF WE
LET THIS REACTION TO OCCUR AS
YOU SUGGIESTED WITHOUT THIS
PROXIMITY EFFECT, THEY ACTUALLY
DON'T REACT.
>> I SEE.
NTHAT'S WHAT--THEY HAVE MULTIPLE
PAPERS TO DEMONSTRATE THAT.
SO I ACTUALLY CANNOT MEASURE
ENHANCEMENT BUT WHAT WE DID IS
WE MEASURED THE KINETETTIC ON
THE PROTEIN LEVEL WHERE WE HAVE
2 PROTEINS BINDED TOGETHER, WE
MEASURED THE K22nd WAY OF THIS
REACTION AND OUR RESULT WAS IN
THE RANGE OF 5-50 PER MOLE PER
SECOND.
SO THIS IS NOT GREAT BECAUSE
QUICK CHEMISTRY ARE IN THE RANGE
OF 1-210 TO THE FIFTHS.
IN THE RAW IT HAS GREATER
REACTION, SHE SHOWED ME, REACH
TO THE HIGHEST LEVEL OF
[INDISCERNIBLE] TO 10 TO THE
FIFTH EMPLOY FOR US THE
[INDISCERNIBLE] IS 5 TO 50, BUT
WE RECENTLY FOUND R-GENINE, AND
AND SLOWER THAN THE CLICK
CHEMISTRY LEVEL, AND THIS IS
MANY REACTIONS AND THAT'S
QUESTION 1 QUESTION 2 IS VERY
INTERESTING SO YOU'RE THINKING
THIS REACTION IS TABLE IN OUR
PROTEIN COMPLEX BECAUSE PROTEINS
ARE DIFFICULT TO--AS IN 2 LIVING
GREEP IS DIFFICULT TO LEAVE
BECAUSE HAVE YOU THIS BINDING
AFFINITY.
>> SO ESSENTIALLY LIKE IF YOU
HAVE A BULKY PROTEIN, DOES THAT
DISCOURAGE REACTION THE SULFA
NEAL UNLESS HAVE YOU A
PROTEIN-PROTEIN BINDING BECAUSE
THE PROTEIN ACTS AS ESSENTIALLY
SORT OF--DOESN'T SORT OF
ENCOURAGE,--THAT'S--LOSE WORD.
>> THIS IS GREAT QUESTION.
SO, THAT'S I THINK THAT'S 1
ADVANTAGE OF USING PROTEINS AS
COVALENT.
YOU CAN PUT A WAR HEAD INTO THE
PROTEINS AND PROTEINS HAVE THIS
BULKY EFFECT TO PREVENT OR AT
LEAST MINIMIZE REACTIONS TO
[INDISCERNIBLE].
>> SO I HAVE ANOTHER QUESTION IF
THAT'S OKAY, FOR YOUR 2 NANO
BODIES AGAINST THE SPIKE
PROTEIN, THERE ARE I THINK A
NUMBER OF NANO BODIES, DID
YOU--HOW DID YOU SELECT THE NANO
BODY?
DID YOU CHOOSE THE MOST TIGHT OR
THE 1 AVAILABLE AT THE TIME?
>> WE ACTUALLY DIDN'T CHOOSE
THAT TIME IS DURING THE PANDEMIC
IT WAS ALL IN A MESS.
SO WE USE WHATEVER IN THE
LITERATURE.
THE MMB6 WAS DEVELOPED BY PETER
WRIGHT GROUP AT UCSF, AND MD7
WAS DEVELOPED BY SOMEONE AT
[INDISCERNIBLE] UNIVERSITY.
SO WE DIDN'T PICK WHICH NANO
BODY TO USE, OUR STRATEGIES,
THAT SPEAKS SOMETHING OF OUR
STRATEGY THAT WORKS INTO--SO
FAR, IN OUR HANDS IT WORKS ON
ANY NANO BODY WE USE IT ALL.
>> I SEE.
>> AND MANY OTHER PROTEINS
BECAUSE THE TYROSEEN, LYSINE AND
HISTODEANS IS JUST EVERYWHERE,
SO WE NEVER FAILED SO FAR TO
CROSS LINK THE TARGETED PROTEIN
INTEREST IN THE EFFECTS
APPROXIMATELILY ENABLED
REACTIVITY.
AND THESE 2 HAVE 10 FOLD
DIFFERENCE, 1 NANO BODY HAS 1
NANO MOLE OAR BIENING AFFINITY,
THE OTHER HAN .1 NANO MOLAR.
MD70 AS .1.
THEREFORE YOU SEE THE
IMPROVEMENT IS 8 TO 10.
THE OTHER HAS 1 NANO MOLAR IS
LIKE 44 EMPLOY NTHANK YOU.
>> OKAY, WE'RE AT THE TOP OF THE
HOUR THERE'S A RECEPTION RIGHT
OUTSIDE.
PLEASE AT A, 1 MORE TIME, THANK
THE SPEAKER.
[ APPLAUSE ]
>> THANK YOU.