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>>> GOOD AFTERNOON, EVERYBODY WELCOME TO THE WEDNESDAY AFTERNOON LECTURE ON I’M HERE TO INTRODUCE OUR SPEAKER TODAY, DR. MARIO APPLESEL HE IS A WORLD RENOWNED LEADER SHE IS PROFESSOR AND DIRECTOR AT THE DEPARTMENT OF BIOPHYSICS AND BIOPHYSICAL CHEMISTRY AT THE JOHN’S HOPKINS UNIVERSITY SCHOOL OF MEDICINE HER RESEARCH FOR UNDERSTANDING MECHANISMS OF ENZYMES INVOLVED IN REACTIONS AND PHOSPHORALATION TRANSFERS USING VARIOUS TECHNIQUES, INCLUDING X-RAY DEFRACTION, BIOCHEMICAL AND BIOTYPICAL METHODS HIS LAB ALSO DOES COMPUTATIONAL METHOD DR. APPLES SELL IS NOT ONLY A GREAT SCIENTIST BUT ALSO A GREAT TEACHER MENTOR — AMZEL HE WON THE GRADUATE STUDENT TEACHER OF THE YEAR AWARD AND UNIVERSITY TEACHING AWARD IN FACT, AFTER THE TALK, HE IS ON HIS WAY OUT TO RECEIVE A PRESTIGIOUS AWARDXñ/ç BY THE ARG TEENIAN MINISTER OF SCIENCE AND TECHNOLOGY THE GROUP DESCRIBED THE STRUCTURE OF P13 KINASE ALPHA ABOUT WHICH HE WILL BE TALKING TO US TODAY THANK YOU >> THANK YOU VERY MUCH FOR THE INTRODUCTION THANK YOU FOR THE INVITATION TO PRESENT MY DATA HERE I HAVE HAD NICE CONVERSATIONS WITH POSTDOCS THERE ARE PROJECTS IN THE LABORATORY AND THE ONES I CHOSE TO TALK TODAY INVOLVES THE STRUCTURE OF THE TERMINATION AND INTERPRETATION OF THE MECHANISM OF ACTIVATION OF P13K-ALPHA THIS IS AN ENZYME WHICH IS NOT MEMBRANE BOUND, IT’S MEMBRANE ASSOCIATED HERE IS THE LOCATION, AND IT IS RESPONSIBLE THROUGH A SERIES OF RECEPTORS OR THE SUBSTRATES AND IT’S FORMED WITH 2 SUBUNITS, ONE THE UNIT IS 10 KILOS WHICH CONTAINS THE CATALYTIC SITES AND 85 UNITS OF KILLADALTONS THAT IS ASSOCIATED WITH BRINGING THE PROTEIN TO THE MEMBRANE THE PROJECT IN THE LAB STARTED WHEN A GROUP AT HOPKINS3W)5f DIRECTED BY FOGELSTEIN, PUBLISHED IN 2004, THAT THE GENE THAT CAUSED 120 PROTEINS IN THIS KINASE WAS HIGHLY MUTATED IN SEVERAL CANCER MUTATIONS IN THIS PROTEIN — AND ASKED WHY ARE THESE MUTATIONS INVOLVED? SINCE THEN, MORE DATA HAS BEEN COLLECTED AND MORE CANCERS HAPPEN TO HAVE THESE MUTATIONS AND BY NOW, THEY ARE ABOUT 1,500 MUTATIONS THAT HAVE BEEN DESCRIBED IN THE GENE CODING FOR THE 120 KILLADALTON SUBUNIT OF THE PROTEIN SO, MOST OF THE MUTATIONS INCREASE THE ACTIVITY THE ONES WE DON’T KNOW ARE THE ONES — NOT TESTED SO THIS IS WHAT PEOPLE WANT TO CALL TYPICAL ON COJEAN YOU CREATE ACTIVITIES — ON COJEANS THE ENZYME, THAT ISP) KINASE WHICH HAS A VERY,á

SO, THERE ARE SOME QUESTIONS WE CAN ANSWER AND SOME QUESTIONS THAT WE CAN’T THAT SOME OTHER PEOPLE CAN AND THIS IS, WHY IS SO MANY MUTATIONS THAT HAVE INCREASED P13K ACTIVITY? THIS IS A VERY GOOD QUESTION AND WHAT SORT OF ANSWERED BY OTHER PEOPLE AND THEN, HOW DO THESE MUTATIONS INCREASE THE ACTIVITY BY PHYSICAL MECHANISTIC QUESTION? THAT IS THE GENERAL INTEREST WE HAVE IN THE SYSTEM SO, THE WHY IS VERY SIMPLE PI3K IS VERY SIMPLE THESE ARE RECEPTOR TYROSINE KINASES OR GROWTH FACTORoYj RECEPTORS WHEN THEY BECOME PHOSPHORYLATED, THE SUBUNIT P85 BINDS TO THEM THROUGH THE PHOSPHORYLATED SUBSTRATES AND THENG Ox ACTIVATED FROM THE MEMBRANE, THIS IS THE 110, AND REDUCE THIS PHOSPHATE AND THAT IS THROUGH PHOSPHORYLATION CONTROLS ALL THESE FUNCTIONS — [INAUDIBLE] MIGRATION, MOTILITY, THOSE ARE CHARACTERISTICS THE TUMOR CELL WILL TRY TO DEVELOP, WILL DEVELOP THROUGH THE NUMBER OF REPUTATIONS THAT ARE VERY, VERY LAST NUMBER BEFORE TUMORS BECOME DEDUCTIBLE JUST TO GIVE YOU MORE DETAIL, THERE ARE TWO CLASSES OF P110 CHANGE ALPHA, BETA AND DELTA BELONGS TO CLASS 1A AND THEY USE P85 AND THEN P110 GAMMA HAS DIFFERENT REGULATORY SUBUNITS AND ALSO IS NOT RESPONSIVE TO THE RECEPTIVE TYROSINE RECEPTORS BUT RESPONSIVE TO G PROTEIN COUPLE RECEPTORS SO RESPONSES TO CHEMOKINES I GET A LITTLE MORE DETAIL ABOUT THIS ONE THING THAT IS INTERESTING FOR PEOPLE WORKING IN5/>, MANY OF THESE THINGS, TO GET AN IDEA OF WHAT CONTROLS THIS PROCESS, IS THIS IS A COMPLICATED DIAGRAM NOT COMPLETE THAT WILL GIVE YOU AN IDEA THIS IS WHAT PI3K IS, IT BRUISES PIC3 AND RECRUITS TO THE MEMBRANE — PRODUCES — PLUS AKT AND PDK1 THAT CONTROLS THE PHOSPHORYLATION OF THIS PROTEIN SOME OF THE ACTIONS ARE MIG TORY AND SOME OF THE ACTIONS ARE ACTIVATING BUT MOST OF YOU RECOGNIZE MANY OF THESE PROTEINS THESE ARE THE PROTEINS THAT I SEE INVOLVED IN TRANSLATION THESE PROTEINS ARE INVOLVED IN APOPTOSIS SO, THROUGH THE ACTIVATION OF SOME OF THE PATHWAY, PI3K CONTROLS MANY OF THE ACTIVITY IN A LITTLE BIT MORE DIRECT, ALL MY TALK IS GOING TO BE CENTERED AROUND PI3K ALPHA AND THAT IS JUST FROM THE THINGS THAT I SHOW YOU, IT IS INVOLVING SEVERAL DISEASE PROCESSES, TUMORIGENESIS BECAUSE IT’S AN ALPHA AS A HUMAN COJEAN AND IN VIRAL INFECTION, SOME REQUIRE THE FUNCTION OF THE PI3K IN CARDIAC FUNCTION, THEY ARE INVOLVED IN SUPPRESSION OF CARDIAC HYPERTROPHY AND VENTRICULAR DILATION AND FIBROSIS IN SITUATIONS UNDER STRESS AND OF COURSE IT’S PART OF THE INITIAL STEP AFTER THE RECEPTOR IN THE INSULIN PATHWAY SO AS A GENERAL IDEA OF WHAT HAPPENS TO THE DIFFERENT CLASSES, ALPHA, BETA, AND DELTA ARE CLASS 1, GAMMA IS CLASS 1A AND GAMMA IS CLASS 1B AND THIS ARE THE FUNCTIONS THAT THEY HAVE THEY ARE VERY, VERY SIMPLIFIED I SHOW YOU A LITTLE BIT MORE DETAIL IN THE PREVIOUS SLIDE ANDWIv THESE ARE THE SPECIFICS THAT CAN BE TARGETED BY TARGETING THE PROBLEM PI3K ALPHA IS ACT SIGHTING MUTATIONS AND THROUGH THE — ACTIVATING — THROUGH THE RECEPTOR PI3K BETA AND INVOLVED IN ACTIVATION AND IS NOT MUTATED IN CANCERS THAT IS HOW THEY FUNCTION IN CANCERS WHICH ARE — TUMORS WHICH HAVE DEFICIENT PK FUNCTIONS AND THEN THESE TWO, THEY BELONG TO DIFFERENT CLASSES, THEY ARE RELATED WITH A NEW SYSTEM AND THEY ARE NOT INVOLVED IN RESPONSE TO IMMUNE IMPORTANT CYTOKINES THAT RESULT IN IMMUNE CELL MOTILITIY AND THEY ARE TARGET FOR INFLAMMATION [email protected]

SO, ONE WOULD LIKE TO CONTROL SOME HOW CHEMICAL IN THIS MOLECULE FOR BOTH BIOLOGICAL EXPLORATION FROM LABORATORY TESTS BUT ALSO FOR’ZxCHEMICAL INTERVENTION SO, WE ARE — THERE ARE SEVERAL ISOFORMS THAT CONTROL SEVERAL PROCESSES FOR EXAMPLE, IF ONE IS THINKING IN CANCER CHEMOTHERAPY, ONE WOULD LIKE TO INHIBIT ONLY PI3K ALPHA BECAUSE THAT’S THE ONLY DEMONSTRATED ONCOGENE FOR THIS PROTEIN SO, IN PRINCIPLE, THE IDEA SHOULD BE ISOFORM SPECIFIC.ñ?]d BUT EVEN IN THE INITIAL PI3K WILL HAVE SOME TO DETECT SO IN THE CASE OF CANCER CHEMOTHERAPY, ONE WOULD LIKE TO HAVE ISOFORM SPECIFIC BUT ALSO MUTANTS SPECIFIC SO THIS IS THE GENERAL IDEA OF WHERE THE DEVELOPMENT OF THE INDIVIDUALIZED MEDICINE AT THE LEVEL OF THE GENOTYPING OF THESE INDIVIDUALS OF THE PATIENT WILL GO WE ARE — WE CAN DO IT NOW BUT FOR NO USE THE ONLY USE HERE IS WHEN ALL THE CELL BIOLOGY AND ALL THE BIOPHYSICS IS DONE AND WE KNOW WHAT TO DO ONCE WE KNOW WHICH GENES ARE MEW TRADED SO THE DIRECTION TO TRY TO GET ALL THE INFORMATION NECESSARY TO MAKE GENOTYPING IN INDIVIDUAL PATIENTS TO BE CLINICAL REALITY SO, IN THE CASE OF THE ISOFORM-SPECIFIC MUTANTS-SPECIFIC INHIBITOR, ONE THING THAT IS CRUCIAL IS BIOPHYSICAL AND !gÑ FACTUAL INFORMATION SO ACTUALLY THE MUTATION SPECIFIC ARE VERY CLOSE ISOFORM-SPECIFIC INHIBITORS THIS IS A CARTOON, SHOULDN’T BLAME ME TOO MUCH FOR IT, IF YOU HAVE A WILDTYPE ENZYME, YOU HAVE AN INHIBITOR IF YOU HAVEd” REPRESENTED BY3;; ADDITIONAL — IT WILL BIND TO THE MUTANT ALSO HOWEVER, IF YOU ARE ABLE TO ASSIGN AN INHIBj THAT, IT WILL — [INAUDIBLE] TO THE WILDTYPE AND THIS LOOKS LIKE A CARTOON THAT DEPICTS THIS BUT IN REALITY, WE ASSUME THAT KIND OF INITIATION IN THE CASE OF ONE OF THE MOST STUDIED FOR INHIBITORS, HIV PROTEASE THIS IS THE WILDTYPE PROTEASE BINDING TO ONE OF THE INHIBITORS AND THEN IF YOU LOOK AT THE MUTANT, THIS AREA, IS COMPLETELY DIFFERENT IT’S VERY CLEAR THAT YOU COULD MAKE AN INHIBITOR TO BE MORE SPECIFIC FOR THAT MUTANT THAN IT IS FOR THE WILDTYPE SO AGAIN, THIS DIFFERENCE ISc VERY DIFFICULT TO REALIZE THEY WILL REQUIRE BIOPHYSICAL INFORMATION SO, NOW LET’S LOOK IN MORE DETAIL ON THIS STRUCTURE OF PI3K ALPHA 1,068 AMINO ACIDS AND HAS FIVE DOMAINS AND THE BINDING DOMAIN, — [INAUDIBLE] THE REGULATORY PROTEIN ALSO HAVE FIVE DOMAINS AND THIS IS: AND THE DOMAIN WAS MEANING INTERVENING SH2 THAT IS NOT AN SH2.hV]Bç FOR THE STUDIES WITH OVER 50 OR SO DIFFERENT CONSTRUCTS AND THE ONE THAT WORKED IN CELLS WAS ONE IN WHICH WE HAD THE COMPLETE P110 AND TWO DOMAINS OF THE P85, WHICH ARE THE SH2 AND YOU WILL REALIZE THAT THIS WAS NOT COMPLETELY RANDOM THIS IS PROBABLY THE MOST IMPORTANT DOMAIN TO HAVE AND WE REPORT IN 3 STRUCTURES, THE WILDTYPE WITH THE NI WITH THESE TWO WE REPORT IN ONE OF THE ONCOGENIC MUTANTS AND WE WILL DISCUSS THIS ONE AND THE SAME

ONCOGENIC MUTANTS IN THE INHIBITOR AND I’M GOING TO JUST TALK ABOUT THIS IN GENERAL IN VERY FEW CASES IT WAS REFERRED TO SOMETHING SPECIFIC ABOUT THESE STRUCTURES SO, THIS IS THE STRUCTURE OF THE PROTEIN AND HERE ARE THE DIFFERENT DOMAINS AND IT’S A VERY COMP CALLED STRUCTURE THE WAY THE — COMPLICATED — IT HAS NOTHING TO DO WITH THE LIN AR SEQUENCE LI SHOW YOU THIS THIS IS THE DOMAIN THAT INTERACTS WITH THIS ONE, THE HEEL CALL DOMAIN INTERACTS WITH THESE TWO AND THE KINASE DOMAINS HERE IT IS A QUITE COMPLICATED STRUCTURE — TO GET ANOTHER GENERAL VIEW IS THIS SUBUNIT THAT I SHOW YOU OF THE 120 STRUCTURE, THIS IS THE EXTENDED COIL AND THIS IS THE NSH2 AND I’M GOING TO TALKi : ABOUT THIS DOMAIN JUST IN THE SENSE, WHAT IS THEIR FUNCTIONAL IMPORTANCE SO, HERE WE COME BACK TO THE MUTATIONS AND YOU REALIZE THERE ARE TWO SPOTS ONE IN THE MEAL CALL DOMAIN, ONE — HELICAL DOMAIN AND ONE IN THE DOMAIN THAT HAS THE PHOSPHORYLATION ACTIVITY AND THERE ARE SOME MUTATIONS IN THE C2 DOMAIN AND OTHER MUTATIONS THAT I’M GOING TO TALK ABOUT ARE THESE THREEUHAAó MUTATIONS STARTING WITH THE PHENOTYPE AND THEN THE KINASE DOMAIN AND ONE THING THAT PROBABLY WOULD APPEAR IN ONE OF MY NEXT TALKS IS WE LOOK AT THE MUTATIONS NOT ONLY TO SEE WHAT THE MUTATIONS DO THEY ARE ACTIVATING THE ENZYME AND THAT IS THE FUNCTION TO ACTIVATE UP ON INTERACTION WITH THE RECEPTOR TYROSINE KINASES WE EXPECT THAT THE MUTATIONSR:V5 AN EASIER WAY TO LOOKING AT THESE ACTIVATION OF THE MOLECULES THIS IS THE QUESTION HOW DO THESE MUTATIONS INSIST IN ACTIVITY AND HOW DO THESE INCREASES INp%- ACTIVITY RELATE TO STRUCTURAL CHANGES? WHAT WE EXPECT IS WHAT I JUST SAID, INFORMATION WILL BE GIVING US INSIDE THE ACTIVATION ALSO SO, WE DISCUSSED MANY OF THESE MUTATIONS IN THE PUBLICATIONS AND THERE WOULD BE NO TIME TO WRITE THIS DOWN BUT THESE ARE PUBLICATIONS THAT PROBABLY DISCUSS MOST OF THE MUTATIONS THATgr WERE OBSERVED AND WE ARE GOING JUST TO DISCUSS A FEW OF THEM IN SOME DETAIL SO THE FIRST THING THAT HAPPENED HERE THE MUTATION THAT YOU SAW OCCUR HERE, HERE, THIS IS AN ACTIVE BINDING DOMAIN AND THIS IS THE C2 DOMAIN WITH ANOTHER SET OF MUTATIONS AND THIS IS THE KINASE DOMAIN WITH ANOTHER SET OF MUTATIONS HERE SO, BESIDES THE MUTATIONS IN THE KINASE DOMAIN, ALL OTHER MUTATIONS OCCUR AT DOMAINS SO, ONE THING WHICH !’i IS ALSO IMPORTANT, I HAVE TO TURN THIS 90 DEGREES TO ([b YOU WHERE THE BINDING SITE IS BECAUSE THE SITE IS HERE AND HERE AND ALL THE MUTATIONS HAVE A VERY FAR FROM THE SITE YET THEY ARE ALL ACTIVATED SO, HOW DO THEY DO IT? FOR SOME OF THE MUTATIONS, THE ANSWER LIES ON THIS SUBUNIT HERE WHICH IS THE NSH2 SUBUNIT OR DOMAIN OF THE REGULATORY SUBUNIT SO, IT WAS KNOWN FROM BIOCHEMICAL DATA THAT THIS DOMAIN HAS A PANEL IN — PARTIAL INHIBITORY AFFECT SO IF YOU LOOK AT THE FIGURE, NEW NEED THE REGULATORY DOMAIN FOR THE PROTEIN BUT WITH THE DOMAIN, THE PROTEIN IS LESS ACTIVE THAN WITHOUT IT SO AND IT WAS DETERMINED THAT THAT WAS USED TO THE NSH2 DOMAIN SO THIS IS SLIGHTLY INHIBITORY SO ONE WAY THAT SOME MUTATIONS MAY WORK IS BY RELIEVING THE INHIBITION BY THE NSH2 DOMAIN SO, NOW WE ARE LOOKING INTO THE

STRUCTURE STRAIGHT INTO THE NSH2 DOMAIN HERE IS THE MORE REALISTIC SHOW AT THE SURFACE OF THE NSH2 DOMAIN AND YOU REALIZE THERE ARE THREE DOMAINS OF THE LARGE CATALYTIC SUBUNIT THIS IS WHERE THE BINDING SITE IS SO HERE IS WHERE THE NSH2 DOMAIN EXPRESSES, FAR FROM THE BINDING SITE BUT IT SEEMS TO INTERACT WITH THREE OF THE SUBUNITS AT THE VERY, VERY CRUCIAL POINT WHERE THEY JUST [email protected]á POSITION OF EACH OTHER ONE OF THE STRONG INTERACTIONS OCCUR BETWEEN THE HELIX OF THE NSH2 DOMAIN AND THE POSITION OF THE HELICAL DOMAIN AND THE KINASE DOMAIN SO IN THIS GROUP, THIS HELIX IS THERE THAT’S ONE OF THE IMPORTANT INTERACTIONS AND WE ARE GOING TO LOOK AT THIS INTERACTION FROM TWO PERSPECTIVES ONE IS FROM THE PERSPECTIVE OF THE BINDING OF THE PEPTIDE WHICH WILL BE THE EQUIVALENT OF BINDING TYROSINE KINASES PHOSPHOTYROSINE SIGNALING< GROUPS AND THE THE OTHER IS THESE MUTATION THAT IS ARE FREQUENTLY MUTATED IN CANCER ARE THOSE RELATED TO THE ACTIVATION SO WE WERE NOT ABLE TO GET THE STRUCTURE OF THE PROTEINS WITH THE PEPTIDES, BUT OVER TEN YEARS AGO, THE STRUCTURAL OF THIS IDENTICAL DOMAIN, ISOLATED, IN THE PRESENCE OF PHOSPHOSEEN PEPTIDES WAS DETERMINED THAT -- THAT THIS IS THE ONE FROM THE GROUP -- [INAUDIBLE] AND THIS IS THE NSH2 OF THE SAME MOLECULE WHEN A PHOSPHOTYROSINE PEPTIDE BOUNCED HERE IF WE ALIGN THIS NSH2 WITH THE ONE FROM OUR STRUCTURE, THEY ARE IDENTICAL SO INés5XjT)T&E BY THIS, WE KNOW THE POSITION OF THE PHOSPHOTYROSINE PEPTIDE IN OUR STRUCTURE SO, THIS IS WHY IT WOULD BIND THIS IS THE NSH2 DOMAIN AND THIS IS WHY THE PHOSPHOPEPTIDE WILL BIND IF WE LOOK AT THE COMPLETE STRUCTURE, THAT SAME GROUP IS OCCUPIED BY A LOOK AND THAT LOOK CARRIES THE TWO HOTSPOT MUTATIONS OF THE HELICAL DOMAIN A GRADUATE MATE 542 AND GLUTAMATE 545 SO, IT ESSENTIALLY THE PHOSPHOTYROSINE SOMETHING SIMILAR IS BEING DONE BY THIS MUTATION THEY ARE NOT IN THE SAME PLACE SO, AFTER THE STRUCTURE WAS PUBLISHED, A GROUP OF COMPARISON HAD THE FOLLOWING DRAMATIC DATA ON THE ONE SIDE AND SOME OF THE MUTANTS AND HERE IS THE ACTIVITY -- [INAUDIBLE] THIS IS THE ACTIVITY OF THE WILDTYPE THIS IS THE ACTIVITY OF ONE OF THE HELICAL DOMAIN MUTANTS AND THIS IS THE ACTIVITY OF THE OTHER AND HERE ARE THE DATA THE ACTIVATION IS ALWAYS IN SIGNALING MOLECULES, THERE ARE TWO OR 3 VERY, VERY SMALL ACTIVATION BUT IT IS IMPORTANT FOR INITIATING SIGNALS IF WE NOW NORMALIZE THESE THREE ACTIVITIES TO 1 AND THOSE ARE THE -- [INDISCERNIBLE] THEN THIS IS THE ACTIVITY WITHOUT ANYTHING ON IT IF WE LOOK AT THIS BLACK BOX, IT IS HOW MUCH THE ACTIVITY INCREASED IF YOU ADD PHOSPHOTYROSINE PEPTIDES AND THE ONE SIDE GETS INCREASING ACTIVITY BY THE FACTOR OF 2, IT'S THE CORRECT ACTIVATI THE TWO MUTANTS THAT I DESCRIBED DO NOT INCREASE THE ACTIVITY THAT MEANS THEY USED UP THE SAME ACTIVATION MECHANISMS, REMOVING THE NSH2 FROM THå y INHIBITORY POSITION SO THIS IS MORE OR LESS WHAT HAPPENS AND THAT IS THE DOMAIN HAS AN INHIBITORY AFFECT ON THE KINASE DOMAIN AND THAT, INHñÑ4O BINDING PHOSPHOTYROSINE PEPTIDE WHICH WILL MOVE THIS SOMEWHERE

ELSE, NSH2 DOMAIN OR BY FOLLOWING THIS MUTATION IN THE SAME SURFACE THAT WEAKEN THE INTERACTION OF THE HELICAL DOMAIN WITH THE NSH2 SO THIS SEEMS TO LOOK MORE OR LESS LIKE THIS THIS IS THE MOLECULE AND WE ARE LOOKING ON MUTATIONS HERE AND THIS GROUP, THIS DOMAIN, IS IN HIBITORY OF THE ACTIVITY, SO HERE THE INHIBITION IS SHOWN BY THIS ARROW THE FACT THAT THE MUTATION HERE ALWAYS BINDING PEPTIDES, WE DISLODGE THE NSH2 SUBUNITS AND THAT RESULTS IN ACTIVATION SO THAT WOULD BE THE GENERAL SCHEME FOR THE MECHANISM OF ACTIVATION BY THE RECEPTOR TYROSINE KINASES OR BY MUTATIONS SO, THERE IS A CATCH TO ALL OF THIS IN THE MUTATION THEY ARE VERY SIMPLE BUT WHAT HAPPENS WHEN WE TRY TO LOOK AT HOW THIS ACTIVATION OCCURS? THIS HAS TO BE LOCATED THERE IS NOT ENOUGH BINDING FREE ENERGY CLOSE BY TO BIND THEM AND MOVE AND PUSH SOMEBODY AWAY THAT DOESN’T HAPPEN THIS SITE HAS TO BE AN&@#Jú OCCUPIED PART OF THE TIME SO, THAT MEANS MR. IS SOME SORT OF DYNAMIC EQUILIBRIUM BETWEEN THE HELICAL DOMAIN AND THE NSH2 BEING BOUND TO EACH OTHER AND SOME PARTS OF THE NSH2 DOMAIN — [INDISCERNIBLE] ONE HAS TO LOOK AT DYNAMIC AFFECT AND TO LOOK AT DYNAMIC EFFECT, WE WENT IN TWO DIFFERENT WAYS THE PROBLEM IS TOO LARGE AND WE DON’T KNOW ENOUGH SO WE TRY TO DO IT IN AS WE KNOW ONE WAY IS TO JUST DO COMPUTATIONALLY AND TRY TO SEE WHAT KIND OF CONFORMATIONS — [INDISCERNIBLE] BECAUSE THE MOLECULE IS VERY LARGE AND ONE WANTS TO HAVE INDICATIONS OF MOTION, WE WENT TO DO NORMAL ANALYSIS THIS IS ONE OF THE ANALYSIS AND I SHOW YOU MORE IT EXAGGERATED JUST FOR THIS SO WE DID NORMAL IN.MPY THELG TÑ WILDTYPE AND IN THE MUTANTS, DOUBLE MUTANTS, IN THE POSITIONS OF THEkm<% MUTANTS OF THE HELICAL DOMAIN, WHICH ARE ONES -- [INDISCERNIBLE] SO THE FIRST THING THAT HAPPENS IS THAT WHEN YOU TRY TO MODEL THAT MUTANT, THE7t [INDISCERNIBLE] JUST PUSH THE STRUCTURE AWAY AND WHEN YOU LOOK AS IT COMPARED WITH THE WHILE TYPE, THE DOMAIN IS ALREADY SLIGHTLY MOVED AWAY WHEN WE TRIED TO ANALYZE ITS WHAT THE EFFECT OF WEAKENING THIS INTERACTION, DOESN'T DISAPPEAR ON THÄ* GENERAL DYNAMIC MOTIONS OF THE MOLECULE SO, WHAT WE ARE SCRIBING AS THIS DISLODGING OF THE NSH2 BINDING TO THE LARGE DOMAIN, IT IS MORE THAN ANYTHING ELSE, IT IS WEAKENING THE BINDING AND THAT GIVES ACCESS TO DYNAMIC STATES WHICH ARE A WAY FROM THE POSITION OF NORMAL SO, ONE THING WE LOOKED AT IN THIS, IS DYNAMICS OF WHAT THE NSH2 DOMAIN DO AND HERE THE LAST NUMBER OF NORMAL MOUSE WHAT IT IS, IT REPRESENTS UNITS THE MOVEMENT OF THE INDIVIDUAL ATTRIBUTES THE NSH2 DOMAIN IN DIFFERENT NORMAL MODES AND HERE IN THE MOVIES THEY ARE BEING REPRESENTED THE ONE OF THE FIRST NORMAL MODES AND IF YOU LOOK AT THIS OR WHAT THIS SAYS IS THE WILDTYPE GENERIC -- IT'S A GRAY LINE AND IT HAS A VERY, VERY SMALL AMPLITUDE OF MOTIONS FOR THE NSH2 ONCE YOU WEAKEN THE INTERACTION OF THE NSH2 BY THE MUTATIONS YOU GET LARGE FLUCTUATIONS THAT ARE POSSIBLE AT LOW ENERGY SO HERE IS THE NSH2 OF THE WILDTYPE AND HERE IS THE NSH2 OF THE MUTANT AND THEY ARE MUCH LARGER MOTIONS IN THE MUTANT THAN THEY FOR WILDTYPE AND THOSE MOTIONS CHANGING THE INTERACTION OF THIS REGION WITH RESPECT TO THE KINASE DOMAIN

SO WE LOOK ALSO A VERY, VERY GENERAL IDEA — [INDISCERNIBLE] I SEE TWO GROUPS OF NORMAL MODES IN ORANGE IS THE WILDTYPE AND THESE ARE GENERALLASMITUDES OF MOTIONS AS A FUNCTION OF AMINO ACID NUMBER IN GREEN IS ONE OF THE MUTANTS AND IT IS CLEAR FOR THE NSH2 DOMAIN, THE AMPLITUDE IN EVERY RESPECT OF MOTION BECOMES MUCH, MUCH LARGER THAN THAT OF THE WILDTYPE THAT IS WHERE THE PEPTIDE IS GOING TO GO IN THIS CASE, THIS LARGE MOVEMENTS ARE THE ONES THAT DIRECTLY ACTIVATE THE ENZYME THIS LARGE MOVEMENTS IN THE WILDTYPE IN THE ORANGE ARE THE ONES THAT WE WILL ALLOW TYROSINE KINASE TO BIND THE ISH2, THE LONG HELICAL DOMAIN, FOR EXAMPLE, IT DOESN’TT-÷ EXPERIENCE THE SAME KIND OF MOTION THEY ARE MOTIONS BUTINATE AND I HAVE MUTANTS ARE EXTREMELY SIMILAR YOU CAN NOT RECOGNIZE HERE WHICH ONE IS GREEN, WHICH IS ORANGE AND WHICH IS GRAY THE SAME IS TRUE, THERE ARE TWO LOOPS IN THE MOLECULE WHICH FOR THE MOMENT I SAID WITH THE INHIBITION AND WITHIN ACTIVITY BUT THERE SHOULD BE A MECHANISM OF ACTIVITY AND THAT PART OF COURSE IS WE DON’T KNOW YET THAT IS WHAT WE ARE TRYING TO DO BUT ONE THING THAT IS CLEAR IS THERE ARE TWO LOOPS CALLED THE ACTIVATION LOOP AND0 THE — [INDISCERNIBLE] THAT IS VERY, VERY VALUABLEÓ CONFIRMATION WHEN YOU LOOK AT THE LARGE NUMBER OF KINASES HERE, WE BUILD MODEL OF THE ACTIVATION LOOP AND THEN WE LOOK AT THE SITUATION IT’S THE SAME COLOR THE WILDTYPE IS ORANGE AND THE MUTANT IS IN GRAY AND THERE ARE MANY MOVEMENTS THAT ARE LARGE AMPLITUDES FOR THE MUTANT WHICH ARE NOT LARGE AMPLITUDES FOR THE WILDTYPE WE EXPECT, WHEN WE ANALYZE THEM, THAT SOME OF THIS MOVEMENT WILL SHOW SOME POSITIONING OF RESIDUE THAT INCREASE CATALYTIC ACTIVITY WE ARE NOT THERE YET HERE, YOU CAN SEE WE ARE REPRESENTING AGAIN THE ONE WITH THE RED DOT AND THERE ARE SOME LARGE MOVEMENTS IN THE WILDTYPE BUT HERE, IT IS VERY SMALL AND IF YOU LOOK HERE IN THE ACTIVATION LOOP, IT IS VERY LARGE SO AGAIN, THE DYNAMIC EFFECTS WILL MAKE CONFIRMATION ACCESSIBLE, PARTS OF THE TIME CONFIRMATIONS MORE ACTIVE CATALYTIC AFFECTS THERE ARE NOT CONFIRMATIONS, JUST BECOME ACCESSIBLE THERE ARE MANY STRUCTURES THAT HAVE BEEN DONE BY US AND BY OTHERS AND IT DOESN’T SEEM TO BE A HEMOGLOBIN TYPE STRUCTURE WHERE YOU HAVE ONE INSTRUCTOR HERE AND ANOTHER STRUCTURE HERE — [INDISCERNIBLE] CHANGES IN THE DYNAMIC LIKE THIS ONE WHO SEEMS TO CONTROL THE CATALYTIC ACTIVITY REMEMBER THIS ACTIVITY IS ONLY A FACTOR AT THE MOST OF 4 SO THEY ARE VERY, VERY SUBTLE CHANGES IN ACTIVITY SO, YOU HAVE TO GIVE AN IDEA, WHAT IS SAID HERE IS THE MUTATIONS WEAKEN THE INTERACTIONS AND CHANGING THE CHARACTERISTICS OF THE — [INDISCERNIBLE] AND THEN PHOSPHOTYROSINE PEPTIDES ON THE MUTANTS HAVE SIMILAR EFFECT ONE THING WE ARE DOING IS TO TRY TO SEE IF WE CAN GET ANY INFORMATION ABOUT BINDING OF PEPTIDES TO THE MOLECULE THE MOLECULE PROBABLY BECAUSE NSH2 IS DISLODGED AND IF KEPT MOVING, IS RESISTING THE PEPTIDES, SO WE WENT TO A TECHNIQUE CALLED — [INDISCERNIBLE] SO THE KIND OF INFORMATION YOU GET IS THEN FROM THE MOLECULE THE ENVELOPE HERE IS SHOWN AT THIS EGG CRATE IT IS USED TO CALCULATE THAT ENVELOPE AND HERE IN THIS ENVELOPE IS THE FITTING OF THE MOLECULE THE FITTING LOOKS MUCH SMALLER THAN THE ENVELOPE ITSELF BUT THIS ENVELOPE INCLUDE WATER WHICH IS IMMOBILIZER AND TRACKING THE PROTEIN SO REFLECTS WATER AS ASSOCIATED WITH THE MOLECULE WHEN WE HAD PHOSPHOTYROSINE PEPTIDE FROM THE INSULIN RECEPTOR 1 THAT WE KNOW BINDS TO

THEE PORTION OF THE ENVELOPE CAN JUST DRAMATICALLY — THERE ARE OTHER PLACES WHERE IT CHANGE [INDISCERNIBLE] I CONCENTRATE IN THIS PORTION THIS IS WHERE THE NSH2 IS THIS IS THE NEW PORTION OFuWmlñ THE ENVELOPE SO, WHAT IS HAPPENING IS THAT THIS NSH2 NOW CAN SAMPLE ALL THE CONFIRMATIONS BETWEEN THESE TWO POINTS AND NOW THE ENVELOPE BECOMES BIGGER HERE SO NSH2 SAMPLES FROM HERE TO HERE THAT IS CONSISTENT AND SIMILAR TO THE OBSERVATIONS THAT WE HAVE FROM THE NORMAL THE DATA, EXPERIMENTAL DATA IS HERE YOU REALIZE INFORMATION CONTENT OF THE DATE DATA ISqH # VERY SMALL BUT IT’S GOT ENOUGH INFORMATION THIS ENVELOPE AS I’M COMPLETELY CONVINCED DO NOT REFLECT ANYTHING OF THIS STRUCTURE THEY ARE JUST BASED ON THE EXPERIMENT OF DATA AND THEN WE DID IT WITH ANOTHER PEPTIDE JUST TO MAKE SURE IT WAS NOT A FLUKE LIKE WE DID HERE ALL THE PEPTIDES, THESE ARE ALL EXPERIMENTAL HERE IS THE ENVELOPE WITH ANOTHER PEPTIDE FROM ANOTHER AND RECEPTOR TYROSINE KINASE ASSOCIATED PROTEIN AND AGAIN THIS REGION HAS GROWN WITH RESPECT TO THE WILDTYPE EACH OF THESE ENVELOPE CALCULATED ENZYMES WITH TEN DIFFERENT DATASETS SO THERE ARE THINGS WE WILLD+ñ SIGNAL THERE SO THAT’S MORE OR LESS WHERE WE ARE WE ARE NOW TRYING TO LOOK WITH MOREe0/Z SOPHISTICATED WAYS OF MATCHING THE EXPERIMENTAL DATA THAT TAKES A BIGGER ADVANTAGE OF THE POSSIBLE COORDINANTS OF THE STRUCTURE OR KNOWING THE DYNAMICS HAVING ALL THE?k CONFIRMATIONS THAT ARE BEING SAMPLE IN SO, JUST TO SUMMARIZE THIS TALK, THE BINDING OF THIS PHOSPHOTYROSINE PEPTIDE RELEASES THE INHIBITION BY WEAKENING THE INTERACTION WITH THE NSH2 DISLODGING THE NSH2 FROM THE CENTER POSITION CHANGES THE STATES OF THE DYNAMICALLY ACCESSIBLE IT’S NOT A NEW CONFIRMATION IT’S JUST CONFIRMATION THAT SOMETHING BECOMES JUST — SPENDS MORE TIME AWAY THIS CHANGES, ONCE THIS HAPPENS, THEY ARE CHANGES THAT IS IN THE CONFIRMATIONS ACCESSIBLE TO THE CATALYTIC GROUP OF THE KINASE DOMAIN, THAT PROBABLY WILL BE ABLE, SOMEBODY WILL BE ABLE TO FIND THEY WILL HAVE CHANGES IN THE CATALYTIC ACTIVITY WHICH IS SOME OF THIS CONFIRMATION THAT HAS THE LIMITATION OF CATALYTIC RESIDUES AND THE PHOSPHOTYROSINE BINDING SEEMS TO BE THE SAME AS THE ONCOGENIC MUTATIONS IN THE HELICAL DOMAIN SPOT AND ANOTHER GROUP OF MUTATIONS OCCUR IN THE INTERACTION OF THE DOMAIN WITHIN THE HELICAL THIS IS THE PRESENTATION OF THAT BIG GLOB OR COIL, AND I DON’T HAVE TIME TO DISCUSS IT BUT THERE ARE A SERIES OF SPECIFIC> INTERACTIONS WHICH INVOLVEK]Ñ CHARGES AND HYDROGEN BOMBS BUT THERE IS A LARGE SURFACE OF INTERACTION THERE IS ONE MUTATIONqhD WHICH IS QUITE PREVALENT IN THE C2 DOMAIN AND THIS IS A — [INDISCERNIBLE][p6 MAKES HYDROGEN BOMBS OF THE COILED COIL OF THE IL2 DOMAIN — [INDISCERNIBLE] 5 SCOUR AND 560 SO WE REASON THAT CHANGING THIS RESIDUE WILL CHANGE THIS TO A — AND WILL HAVE SOME AFFECT FROM THE ACTIVITY OF THE — THAT SEEMS TO BE THERE WAS ANOTHER PREDICTION ONE COULD MAKE WHY DID THESE MUTATIONS — [INDISCERNIBLE] THEY WILL HAVE THE SAME AFFECT AND THE REASON IS THAT IT DIDN’T LOOK FIRST SO ABOUT TWO YEARS AFTER THE STRUCTURE, A LARGE NUMBER OF GLOWO BLAST OHMS CAME OUT WHEN THEY DID THAT FROM THEIR PAPER, THEY FOUND THE RESIDUE THAT WAS PRODUCED THAT WILL HAVE THE SAME EFFECT AS MUTATED FREQUENTLY IN GLIOBLASTOMA SO IT’S VERY CLEAR THAT WE ARE

COMING OR GETTING IT HANDLED ON WHAT THESE MUTATIONS DO NOW THESE LARGE — THE GENERAL IDEA IS THIS IS INTERACTION FOR SOME MUTATIONS HERE AND THIS IS A COILED COIL SO,qKQm ATTENDING THE POSITION OF THE MUTATION HERE OF THIS ROD WILL ALSO HAVE AN AFFECT ON HOW STRONG IS THE INTERACTION OF THE NSH2 WITH THE HELICAL AND KINASE DOMAIN SO MUTATIONS HERE WILL HAVE THE SAME AFFECT WITH WEAKENING INTERACTION OF THE NSH2 SO, TO DISCUSS THE LAST KIND OF MECHANISMS, I’M GOING TO TALK A LITTLE BIT ABOUT MEMBRANE INTERACTIONS THIS IS A LIPID KINASE SO SUBSTRATES AND MEMBRANE COMPONENTS THEY ARE NEVER IN SOLUTION THEY ARE A MEMBRANE COMPONENT SOCIETY PROTEIN HAS TO SOMEHOW INTERACT WITH THE MEMBRANE SOME OF THE THINGS IN HERE — IF YOU LOOK AT THE SURFACE OF THE MOLECULE, IT HAS THE PERCENTAGE THAN USUAL OF BLUE IS POSITIVELY CHARGED, POSITIVELY CHARGED HERE IT REMAINS BUT THERE IS A LARGE NUMBER OFwu LYSINES ANDAL JANINES SO WE ARE NOT PRO PEASING THIS SULPHATE INTERACTS WITH THE MEN BRAIN Tha ISH2 DOMAIN THAT IN REALITY HAVE NOT BEEN PREDICTED THIS DATA USING ANTIBODIES THIS IS THE ADAPTER BINDING DOMAIN THAT ALSO THE C2 DOMAIN INTERACTS SONGLY WITH THE ISH2 AND THE C2 DOMAIN IN MEMBRANE INTERACTION DOMAIN SO EVERYTHING MAKES GENERAL AND THE5÷5çñ CATALYTIC SIDE IS SOMEWHAT HERE AND I SHOW IT TO YOU IN MORE DETAIL, ACCESS TO SUBSTRATE THIS IS AN OTHER REPRESENTATION OF THE SAME AND HERE IS WHERE THE CATALYTIC SITE IS I CANNOT SEE IT FROM HERE BUT MAYBE IN THE SCREENING RIGHT THERE THERE IS AN ATP BUILT IN THE SITE OF THE CATALYTIC SITE AND HERE IS A DIRECT CONNECTION TO A MEMBRANE WHERE THIS COULD HAVE BEEN A LITTLE BIT MORE IN THE MEMBRANE AND THEN THE ACCESSIBLE DIRECT PHOSPHORYLATION ONE THING THAT IS INTERESTING, THESE ARE ONE SET OF INTERACTIONS WHICH ARE NOT SPECIFIC INTERACTIONS WITH THE MEMBRANE BY DOMAINS OF THEóBñ PROTEIN HOWEVER, THERE IS STRUCTURE OF THE SAME PROCESS OF ANOTHER ISOFORM WITH RAS SO RAS BINDS TO THE BINDING DOMAIN WE TRANSFER THAT BINDING TO+fÑ MODEL AND THEN THIS IS THE DERMINOUS AND THIS IS MORE OR LESS WHAT THE LENGTH OF THE PATHWAY IS NOT PRESENT IN THE STRUCTURE BECAUSE — [INDISCERNIBLE] AND THAT’S WHERE THIS ONCOR FOR6ISU(j IS IT’S POSSIBLE THAT SOME CONDITIONS, THE FUNCTION OF RAS IS TO AID THE PROTEIN IN THE MEMBRANE AND THERE ARE SOME STORIES YOU CAN SEE RELATED TO THAT SO AGAIN, THIS IS WHERE THE BINDING SITE IS AND THIS>=Ñ IS WHY THE LIPID SUBSTRATES WILL BE SO, WHAT HAPPENS WHEN THIS MUTATION AND WHY AM I MENTIONING THE MEMBRANE WHEN I TALK ABOUT THAT MUTATION? THIS IS ONE OF THE MOST PER VALIANT MUTATIONS AND IT IS CORRELATING QUITE STRONGLY WITH AGGRESSIVENESS OF THE TUMOR IT IS LOCATED CLOSE TO THE END OF THE MOLECULE THE MOLECULE HAS 1,068 AMINO ACIDS MOST OF THE TIME IT IS AN ARGININE IN THE MUTANT LOOK AT THE STRUCTURE OF THE MUTANT AND THERE ARE SOME SMALL DIFFERENCES I SHOW YOU IT IS ALMOST IDENTICAL TO THE ONE OF THE WILDTYPE.N IN THE RIGHT PLACE

SO, WHAT IS DIFFERENT? SO IF WE LOOK AT THE PATH RESIDUE 478, HERE IS WHAT 1047 IS IN THE WILDTYPE THE WILDTYPE IS A HIS TEEN AND THEN THE CELL CHAIN IS COMPLETELY DISORDERED AFTER ONE AMINO ACID, 1047, NEVER SAW ANYMORE OF THEM WHEN WE LOOK AT THE MUTANT, MANY THINGS HAPPEN THIS CONFORMATION CHANGES BECAUSE THE ARGININE NOW FUNCTIONS IN THAT DIRECTION AND NOW THIS WHOLE LOOP UP TO AMINE ON ACID 1062 BECOMES ORDER FOR THESE LOOP TO BE ORDER, THIS OTHER LOOP HAS TO CHANGE CONFORMATION”í>Q÷ AND ESE TWO LOOPS NOW FORM A SULPHATE HERE YOU LOOK AT OUR SUGGESTION, THE MEMBRANE PORTION IS, WE CHANGE COMPLETELY ONE OF THE REASONS WHY THIS INTERACTS WITH THE MEMBRANE AND HERE ARE THE TWO SITUATIONS THIS IS THE WILDTYPE AND THIS IS THE MUTANT WITH THE TWO LOOPS INTERACTING WITHxxv THE MEMBRANE WE NEED SOMEñ&NK CALCULATIONS AND THE CALCULATIONS INDICATE NOT ONLY THAT BUT THERE IS CHANGING ORIENTATION AND THE SMALL CHANGING IN ORIENTATION WITH RESPECT TO THE MEMBRANE, THIS ONE WILL MOVE IN THAT DIRECTION TO MAKE A COMPOUND AND MORE OR LESS, IT SAYS THAT WEHvb [email protected] INCR EASING THE INTERACTION ENERGY WITH RESPECT TO THE FACTOR OFg 6-8 SO, THIS MUTANT INTERACTS A LITTLE BIT BETTER WITH THE MEMBRANE IF THAT IS THE CASE, THAT MEANS THAT IT MAY BE CHANGING THE ACTIVITY BY JUST HAVING MORE ACCESS TO — [INDISCERNIBLE] IF THAT IS THE CASE, THIS MUTANT SHOULD BE ACTIVATABLE BY THE ACTIVATOR, PHOSPHOTYROSINE PEPTIDE AND [INDISCERNIBLE] THE SAME SLIDE I SHOW YOU BEFORE THE WILDTYPE HAS NORMAL ACTIVITIES THESE ARE THE ONES I DISCUSSED BEFORE AND THIS IS THE MORE OR LESS THE SAME IF WE NORMALIZE, THOSE ARE THE WHITE BARS, TO ONE, AND THEN WE TAKE THEM IN THE PRESENCE OF ACTIVATING PEPTIDES, THEI it WILDTYPE INCREASES, AS IT SHOULD, THESE TWO DON’T BECAUSE THEY USE THE SAME MECHANISM BUT THIS ONE IS VERY ACTIVATED BY PEPTIDES WHICH MEANS WHATEVER MAKES THIS ACTIVATION IS NOT RELEASED IN THE ACTIVATION — INHIBITION SO CLEARLY THEY ARE TWO MECHANISMS AT PLAY ONE IS RELEASE OF NSH2 ACTIVATION AND THE OTHER IS IN CREASE OF THE — [INDISCERNIBLE] WE ARE NOT GREAT MEMBRANE CHEMISTS SO WE HAVE TO DEVISE AN EXPERIMENT THAT EVEN WE COULD DO AND WHAT WE DID IS, THIS IS OPERATIONAL DIFFERENTIAL ACCESS TO THE MEMBRANE WE SHOULD DETECT IT SOMEHOW SO, WE DID THE ASSAY IN THE PRESENCE OF — THAT CONTAINED DIFFERENT PHOS TO LITTICS — [INDISCERNIBLE] LIVER AND BRAIN LIP KIDS AND LIPIDSjx THEY WERE USED AS THEY WERE AVAILABLE NOT BECAUSE WE WANTED TO PUT ANY EMPHASIS ON THE FACT THAT WE ARE TUMOR CELLS WE CHECKED FOR THE INCORPORATIONlómD OF P CELL FORMATION INTO PIP3 AND WHEN YOU DO THAT, IFu LOOK AT THE WILDTYPE AND THE MUTANT, HERE IS THE NORMALIZED TO ONE FOR BASAL ACTIVITY, IF WE CHECK THE DIFFERENT LIPIDS, THEY RESPOND TO THE CHANGE IN LIPIDS IN VERY DIFFERENT WAYS SO, I HAVE EVERYTHING PROVEN WHAT IS PROVEN IS THAT IT IS DONE WITH THESE CHANGED LOOKS AND HAVE DIFFERENT WAY OF INTERACTING WITH THE MEMBRANE THAN THE WILDTYPE ONE THING JUST BEFORE I FINISH AND I HAVE TO SKIPâ SOME SLIDES WHAT DO WE KNOW ABOUT BINDING OF

INHIBITORS? SO THE ONE THAT WE TESTED WAS — [INDISCERNIBLE] IT’S A KINASE OF THIS TYPE INHIBITOR AND WHAT IT DOES IS MAKE COVALENT BOND IT IS THE BINDING OF THIS TYPE THAT HAS A COVALENT BOND TO A LYSINE RESIDENT THIS IS THE STRUCTURE AND THAT IS THE WAY WE KNOW WHERE THE ATP SITE IS — [INDISCERNIBLE] THIS IS YOU SEE HOW THE ACTIVE MEMBRANE TO THE CATALYTICp SO IF WE LOOK NOW AT TWO ISOFORMS AND LET’S LOOK FOR ISOFORM SPECIFIC INHIBITOR THIS IS THE ISOFORM GAMMA THIS IS PET FAR IN 55 — AS FAR AS CAN GET IF YOU LOOK AT THE BINDING, THIS STRUCTURE WAS DONEKóz BY THE GROUP OF — [INDISCERNIBLE] ALL THE RESIDUES — [INDISCERNIBLE] ALL RESIDUES INVOLVING ARE THE SAME SO IT IS CLEAR THAT — [INDISCERNIBLE]ö ISOFORM SPECIFIC INHIBITOR IT’S VERY GENERAL INHIBITOR HOWEVER, WHEN WE LOOKED AT THE STRUCTURE IN MORE DETAIL, WE SAW THAT THIS LOOK IN THE ALPHA EXPERIENCED BETWEEN UNBOUND AND BOUND, A LARGE CHANGE WHY THIS LOOKS HERE DOESN’T IN THE CASE OF GAMMA, IT’S THE OTHER WAY AROUND THIS LOOK DOESN’T EXPERIENCE ANY CHANGE THIS ONE EXPERIENCE A VERY LARGE CHANGE SO, EVEN THE STRUCTURES ARE EXTREMELY SIMILAR IN THE BINDING SITE, IT WOULD BE POSSIBLE TO DO ISOFORM-SPECIFIC INHIBITORS BY LOOKING AT THE CONFORMATIONAL AFFECT OF INHIBITOR BINDING I HAVE MORE THINGS LIKE THAT BUT I THINK I’M GOING TO SKIP THEM AND TO GIVE YOU AN IDEA WHAT OTHER THOUGHTS OCCUR WHEN WE LOOK AT THE BINDING SITES THIS IS THE A TP BINDING SITE IT’S LOCATED HERE AND I SHOW YOU WITH THE MEMBRANE THIS IS A VERY WELL STUDIED SITE AND ALL THE INHIBITORS TO DATE ARE GOING TO THE BINDING SITE BECAUSE THEY COME FROM THE BACKGROUND CHEMISTRY OF KINASES AND THAT IS WHAT THEY FOUND IN COMMON, NOT THE PHOSPHATE BUT THE ATP IT IS VERY DIFFICULT TO FIND BINDING TO THIS PROTEIN, IT’S VERY DIFFICULT TO SEE STRUCTURE.oz SO, WE LOOK FOR SOME EXPERIENCE LOOKING FOR PROTEIN SITES AND STRUCTURES AND WE CANNOT WITH THIS SITE, IN THE CORRECT POSITION TO BE PHOSPHORYLATED THIS IS THE POSITION TO BE PHOSPHORYLATED THE RELATION WITH THE MEMBRANE IS PERFECT SO, WE ARE NOW EXPLORING THIS SIDE AS IT IS POSSIBLE THAT WILL GIVE THE IDEA TO OPEN A NEW SITE FOR THE DEVELOPING OF INHIBITORS AND ALSO FOR SUBSTRATE88hj INHIBITORS, INHIBITOR THAT IS COVER THE ATP AND THE — [INDISCERNIBLE] THAT IS SOMETHING WE ARE EXPLORING JUST FROM THE BIOPHYSICAL POINT OF VIEW SO, JUST TO GET AN IDEA OF WHAT I TRIED TO TELL YOU IN 50 MINUTES, THERE SEEMS TO BE TWO GENERAL MECHANISM OF ACTIVATION ONE IS WHICH WE NEED NSH2 CLEARLY RELATED TO RECEPTOR TYROSINE KINASE BINDING AND SOME ONCOGENIC MUTANTS AND THEN CHANGING OF THE ENZYME WITH THE CELL, MEMBRANE AND BOTH OF THEM ARE PRESENT IN MUTATIONS BUT BOTH OF THEM ARE PROVIDING IMPORTANT — [INDISCERNIBLE] SO, HOW IS THE RELEASE OF ACTIVATION? THIS DOMAIN COMPACTS THREE DOMAINS AND POSSIBLY MODIFIES THE COMMUNICATION BETWEEN THOSE THREE(R ACTIVATING PHOSPHOTYROSINE PEPTIDES RELIEVES INHIBITION BYW 9Uz THE NSH2 DOMAIN WHICH IS THE DOMAIN THAT IS MODIFYING INTERACTIONS HOWEVER, ACCESS TO THIS SITE BY THE RECEPTOR TYROSINE KINASE REQUIRES THAT THIS DOMAIN IS AT

LEAST PART OF THE TIME REMOVED FROM THE POSITIONS SO THERE ARE THREE IN THE NEED FOR DYNAMIC EFFECTS FOR THE MUTATIONS, MANY OF THE MUTATIONS OCCUR AT INTERFACES BETWEEN DOMAINS AND SOME OF THESE MUTATIONS USE THE SAME MECANISM AND ACTIVATION AND THE MUTATIONS CAN OCCUR DIRECTLY IN THE INTERFACE AND WHEN THEY OCCUR VERY FAR, THEY ARE COMMUNICATED BY THIS COIL, WHICH IS A DROP OF THE MOLECULE THE AFFECTS OF WEAKENING THIS INTERACTION IS A CHANGING THE FACE OF DYNAMICALLY UPSET THERE ARE PROBABLY NOT VERY WELL DEFINED CONFORMATIONAL CHANGES IN THE CASE OF INHIBITOR BINDING, IT IS POSSIBLE THAT ISOFORM-SPECIFIC REAGENTS CAN BE PAID BY LOOKING AT THE CONFORMATIONAL CHANGE THAT IS OCCUR AFTER BINDING THE KNOWN CD4 AND THEN THE OTHER MECHANISMS INVOLVED INTERACTIONS WITH THE CELL MEMBRANE AND INTERACTIONS WITH THE CELL MEMBRANE SENSITIVITY OF THE INTERACTION WITH THE CELL MEMBRANE CORRELATE WELL WITH THE STRUCTURAL AND THE ACTIVATION BY THIS PARTICULAR ENZYME SO, I WANT TO THANK THE PEOPLE PEOPLE — [INDISCERNIBLE] HERE FROM THE BEGINNING AND DIRECTED MOST OF THE DAY-TO-DAY OPERATIONS OF THIS WORK THE STUDENT THAT DID THE STRUCTURE OF THE WILDTYPE [INDISCERNIBLE] [READING] A GRADUATE STUDENT GRADUATING IN A FEW WEEKS AND SHE LEAD ALL THE CALCULATIONS I SHOWED YOU SHE DETECTED FOR SOME TO THE LABORATORY [READING NAMES] AND THE OTHER COLLABORATORS FROM THE LABORATORIES AND WE DID MANY OF THESE EXPERIMENTS IN THE NATIONAL LABORATORY, FUNDING OF THEE’ç NCI AND THE NIH AND WE STARTED THIS PROJECT ON AN UNPHOSPHOR LATED TRANSFER PROJECT AND IT EVOLVED INTO THIS PROJECT AND WE HAD FUNDING FROM NICMH AND THESE ARE PEOPLE IN MY LAB THIS IS NOT A SLIDE PHOTOGRAPH BUT HERE ARE SOME OF THE PEOPLE I MENTIONED [INDISCERNIBLE] THANK YOU VERY MUCH FOR YOUR ATTENTION [APPLAUSE] I WOULD BE FLOOD TO ANSWER QUESTIONS I’M SURE NOT EVERYBODY BELIEVED EVERYTHING I SAID >> THERE IS A RECEPTION AT THE LIBRARY AFTER THIS >> YES? >> [OFF MIC] >> WHO HAS A MIC? >> THE MUTANTS WHO ALSO DIFFERENT DOMAINS SUPPOSED TO WORK ON THE SAME MECHANISMS, THE ACTIVATIONS OF THE KINASE IS SYNERGISTICS OR — YOU GET THE SAME EFFECT? >> IN THAT CASES, IT WAS — WHEN TUMORS ARE SEQUENCE, USUALLY YOU DON’T KNOW IF THE MUTATIONS OCCURRED MORE THAN ONE IN THE SAME SET TOOTS CONGLOMERATE OF CELL SO, WE DID NOT TRY MUTANT, DOUBLE MUTANT THAT SEPARATED IN SEQUENCE WE TRY THE HOTSPOT OR THIS HOTSPOT AND AS FAR AS I CAN

TELL, DOUBLE MUTATIONS OF DISTANT MUTANTS CHECK TO SEE — [INDISCERNIBLE] BUT WE WALK WITH THE MUTANT SPECIFIC WHO WOULD LIKE TO HAVE MUTANT THAT WALKS ON DIFFERENT DOMAIN BUT WALKS LIKE THE SAME MECHANISM NOT TO BE ADDED >> THAT IS CORRECT YES ONLY NONACTIVITY WAS SHOWN WITH RESPECT TO PHOSPHOTYROSINE BINDING BUTDbE NOT TO THE — NOT TOO MANY OF THEM CORRECT THAT’S A GOOD EXPERIMENT, >> SO YOU TALKED ABOUT THE DYNAMICS WHAT DO YOU THINK WHICH TIME SCALE DOES THE PROTEIN BEHAVE DYNAMIC? DO YOU THINK IT’S FAST ORN’?eñ SLOW? >> YOU REALIZE THAT SOME OF THE CALCULATIONS WERE NOT — [INDISCERNIBLE] ONE WAS DYNAMIC NETWORKS AND THEN — [INDISCERNIBLE] SO ALL THE SEQUENCE THAT IS WE ARE LOOKING AT ARE Aá FREQUENCY THE TIME SCALE IS DIFFICULT TO BELIEVE THIS WILL BE THE FREQUENCY AS O SUGGESTED — [INDISCERNIBLE] SO THEN 10 TO THE 7s OR SOMETHING LIKE THAT, WHICH IS I THINK DIFFICULT TO BELIEVE SO THE TIME SCALE IS DIFFICULT TO IT’S POSSIBLE — [INDISCERNIBLE] IT DOES REFLECT THE MOMENT FOR THOSE TO OCCUR, A STABLE STRUCTURE, IT HAS TO BE EASY TO MOVE IN BOTH DIRECTIONS THAT IS WHAT THIS CALCULATIONS REFLECT IF YOU’RE A STABLE STRUCTURE, THOSE DIRECTIONS THAT WE ARE SEEING ARE THE ONES WHERE IT’S EASIER TO MOVE ONCE YOU MOVE FROM THE STABLE STRUCTURE, HOW FAR OR HOW FAST IS MUCH MORE DIFFICULT TO DECIDE BUT IN A SYSTEMS OF THIS SIZE UNLESS NEW TECHNIQUES BECOME ACCESSIBLE, IT’S VERY DIFFICULT TO GET TIME SCALE >> JUST ONE — ACTIVITY NOT THAT MUCH HIGHER IN THE MUTATED — [INDISCERNIBLE] >> SAY IT AGAIN? >> THE ACTIVITY NOT THAT MUCH HIGHER, SO IT WAS NOT SOMETHING LIKE — [INDISCERNIBLE] >> NO, NO THAT WAS THE BIGGEST SURPRISE TO ME WHEN I GOT INTO THIS I WAS INTO — I COME FROM OTHER ENZYMES AND WE DO A MUTATION, AND WE MAKE DECISIONS WITH A FACTOR OF THREE AND THAT HAS NOTHING TO DO WITH THE ACTIVITY AND THE MECHANISM OF THE ENZYME THAT HAS BEEN MY STANDARD RESPONSE THEN I GET INTO SIMILAR AND VERY SMALL CHANGES WITH VERY LARGE AFFECTS I’M NOT COMPLETELY SURE HOW THAT IS ACCOMPLISHED IN THE SENSE THAT IF YOU DO HAVE THAT ACTIVITY, — [INDISCERNIBLE] YOU COULD SAY YOU HAD NO ACTIVITY BUT P10, THE PHOSPHATASE, IT IS ALWAYS JUST TAKING IT AWAY BUT SOMETHING$% [INDISCERNIBLE] WHY WOULD YOU PHOSPHORYLATE SOM% TO KEEP IT IN CONTROL? YOU CAN’T DO IT — [INDISCERNIBLE] SO I’M AS BAFFLED AS YOU ARE I SHOULDN’T BE BUT I AM, YES >> ANYMORE QUESTIONS? IF NOT, LET’S THANK THE SPEAKER AND PLEASE JOIN US AT THE RECEPTION

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