TRACO 2019 – TGF beta and Small molecules
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OUR FIRST SPEAKER IS IN THE CANCER TRAINING BRANCH OF THE NCI. SHE DID IS A Ph.D. AT RUTGERS UNIVERSITY AND THEN WENT TO FRANCE TO DO A POST-DOCTORAL FELLOWSHIP AND SHE CAME BACK TO THE STATES AND STARTED WORKING WITH A FAMOUS CANCER PREVENTION PERSON, MICHAEL SPORNE AND HAS BEEN AT NCI EVER SINCE. SHE’LL TALK BEEN TRANSFORMING FACTOR BETA. SONYA. >>THANK YOU, TERRY. TODAY I’LL BE TALKING ABOUT TRANSFORMING TRANS GROWTH FACTOR BETA IN LUNG CANCER GENESIS. YOU MAY ASK WHY STUDY THIS TOPIC. LUNG CANCER HAS BECOME THE FIRST COMMON CAUSE OF CANCER DEATH IN BOTH MEN AND WOMEN IN THE U.S. IT HAS SURPASSED EVEN LUNG CANCER IN WOMEN. LAST YEAR THERE WERE OVER 234,000 NEWLY DIAGNOSED CASES OF LUNG CANCER IN THIS COUNTRY. AND OVER 154,000 DEATHS DUE TO THE THIS DISEASE. IT’S A REALLY IMPORTANT DISEASE. NOW, WITH THE ADVENT OF ANTI-SMOKING CAMPAIGNS AND TOBACCO CAMPAIGNS IN THIS COUNTRY, MOST CASES OF LUNG CANCER NOW OCCUR IN FORMER SMOKERS AND THE AVERAGE AGE OF ONSET OF DISEASE IS ABOUT 70 YEARS OF AGE. IT’S A LONG-TERM DISEASE. ESPECIALLY IF YOU START SMOKING AS A TEENAGER. IN ADDITION TO THIS WHILE THE CURRENT HAS BEEN GOING DOWN IN THIS COUNTRY AND EUROPE AND ASIA, THE INCIDENT OF SMOKING AND TOBACCO USE HAS IMPROVED. SO NO MATTER WHAT WE DO, LUNG CANCER WILL BE A DISEASE WITH US FOR MANY YEARS TO COME. NOW, THE FIVE-YEAR SURVIVAL RATE IS STILL LESS THAN 150%. 50%. WHY STUDY TRANS GROWTH FACTOR BETA OR TGF BETA FOR SHORT. IT’S A MULTIFUNCTIONAL REGULATOR OF CELL GROWTH. IT’S BEEN SHOWN TO BE A POTENT INHIBITOR OF THE PROLIFERATION OF MOST NORMAL EPITHELIAL CELLS. IT SHOWS EXPRESSION IN MAMMALS AND PLAYS A PIVOTAL ROLL IN HOMEOSTASIS AND HAS BEEN ASSOCIATED WITH VARIOUS FORMS OF CANCER INCLUDING LUNG CANCER AND A STIMULATION OF CELL PROLIFERATION IN THE NEOPLASTIC TRANSFORMATION. IT’S IMPORTANT IN NORMAL CELLS AS WELL AS IN TUMOR CELLS. SO IT COULD BE AN ATTRACTIVE CANDIDATE SO THAT’S WHY WE WANT TO KNOW MORE ABOUT THIS GROWTH FACTOR. TO UNDERSTAND GROWTH FACTOR BETA WE HAVE TO GO BACK TO THE BEGINNING. IT HAS A GROWTH FACTOR. IT’S A POLY PEPTIDE AND FIBROBLASTS CAN STIMULATE AND FORM COLONIES AND THIS WAS THE TRANSFORMATION EFFECT USED TO IDENTIFY THIS POLY PEPTIDE IN THE ’70s. THERE’S TWO TYPES. ONE CLASS WAS SHOWN TO COMPETE WITH EPIDERMAL GROWTH FACTOR FOR BINDING AND THIS WAS ONE AND THE SECOND CLASS WAS CALLED TGF BETA AND TGF ALPHA EQUALS THE GROWTH FACTOR. THIS IS SHOWN IN THE EARLY ’80s. SO FOLLOWING THE IMPLICATION OF THE TGF BETA THERE WERE THREE TYPES IDENTIFIED BY EARLY INVESTIGATORS. SO TYPICALLY IT’S A TYPE THAT WAS AFFECTED AND SENT TO REMOVE THE DEBRIS AND IT WAS ALLOWED OVERNIGHT IN THE COLD WITH A FORM OF ETHANOL. THE NEXT MORNING IT WAS REDISSOLVED IN ACIDIC ACID AND ONE LITER WAS COLLECTED AND LOOKED AT FOR IDENTIFICATION. TYPICAL YIELD WAS 6 MICROGRAMS PER TON. TO GIVE YOU AN IDEA THIS WAS SHOWN BY THE PINK ARROW IS THE 55 GALLON DRUM. THESE WERE FOR BIG PURIFICATION SYSTEM. THE SYSTEM EMPLOYED NORMAL KIDNEY CELLS AND SO TECHNICALLY A MIXTURE OF SERUM AND CELLS AND OTHERS WERE COMBINED IN THE PLATE. IT WAS THEN ALLOWED TO GROW FOR ONE WEEK AT THAT STAGE AND THE COLONIES WERE COUNTED AND THIS IS SHOWN AT THE BOTTOM. IF TGF BETA WAS CONSISTENT COLONIES WOULD GROW AND THEY WERE COUNTED FOR SIZE ON THE RIGHT. IF NO TGF BETA IS PRESENT WE WOULDN’T EXPECT COLONY TO GROW AS SHOWN ON THE LEFT OF THE SLIDE. AS SHOWN ON THIS SLIDE IS THE FINAL PURIFICATION OF A SYSTEM AND SHOWN BY THE ARROW IS THE EN TIGHTY THAT WAS IDENTIFIED TO BE TGF BETA. AT THAT TIME WE DIDN’T KNOW THERE WERE MORE THAN ONE CALLED TGF BETA. SO IT’S CREDITED WITH IDENTIFYING AND PURIFYING TGF BETA AT THE NCI. UNFORTUNATELY, WE LOST ANITA ROBERTS IN 2006. WE STILL REMEMBER HER BECAUSE SHE HAS A LECTURE EVERY YEAR NAMED AFTER HER. NOW, FOLLOWING THE PURIFICATION OF TGF BETA THE SEQUENCE OF BETA 1 WAS DONE. IT WAS SHOWN TO BE EXIST AS AN ENTITY OF 391 AMINO ACIDS CONTAINING A SIGNAL PEPTIDE AT THE AMINO TERMINAL FOLLOWED BY THE LATENCY FOUND IN THE LAB AND THE TGF BETA POLY PEPTIDE OF 112 AMINO ACIDS. I SHOW AT THE PICTURE IN THE TOP IN THE CARTOON AT THE TOP IS THE 112 AMINO ACIDS OF TGF BETA 1. AND THERE REALLY HAVE BEEN SIM ASIMILE. FOLLOWING THE STRUCTURE AND IDENTIFICATION OF TGF BETA THE CRYSTAL STRUCTURES OF TGF BETA WERE IDENTIFIED IN THE EARLY ’90s AND IT’S A REPRESENTATION OF TGF BETA 2 BECAUSE TGF BETA 2 WAS EASIER TO CRYSTALIZE THAN TGF BETA 1. SO TGF BETA EXISTS FROM THE ENVIRONMENT OF TWO IDENTICAL MON MONOMERS HELD TOGETHER AND THE FAMILY IS NOT OATH TGF BETA WHICH EXISTS WITH ISO FORM OF TGF BETA, 2 AND 3. TGF4 AND 5 HAS BEEN IDENTIFIED IN CHICKENS AND TGF BETA 4 AND 5 ARE FROM THE MAMMALIAN TGF BETA 1. AND IT ALSO CONTAINS THE MORPHOGENIC PROTEINS. SO THIS IS WHAT WE CALL THE TGF BETA SUPER FAMILY. SO TO SUMMARIZE, THERE’S HOMOLOGOUS FAMILIES EXIST. AND NO CELLS EXPRESS TCG PAY TA AND I’LL GO INTO RECEPTORS SHORTLY. IT’S USUALLY SECRETED IN WHAT WE CALL INACTIVE FORM WHICH HAS TO BE ACTIVATED IN ORDER FOR TGF BETA TO DO ITS THING. AND INCLUDES THE BETA AND THE ACTIVE INHIBITORS BMPs AND GDFs. AND THEY’VE HELD ON TO BEING A CONTROL MODULE FOR MANY PROCESSES. INCLUDING THE PROCESSES OF DEVELOPMENT, IMMUNE SYSTEM FUNCTION, REPRODUCTION AND ANGIGENESIS, AGING, TISSUE REPAIR AND RESPONSE, METABOLIC REGULATION AND PROLIFIC HOMEOSTASIS. SO THERE’S A LOT OF KEY BIOLOGICAL PROCESSES THAT ARE CONTROLLED BY THIS SUPER FAMILY. OTHER PROCESSES ARE PROLIFERATION IN SOME INSTANCES AS WELL AS TO REGULATE APOPTOSIS AND DIFFERENTIATION IN THE IMMUNE CELL FUNCTION DEPENDING ON WHICH YOU’RE LOOKING AT. SO IT’S ALSO ABLE TO STIMULATE THE ACCUMULATION OF RED CELL MATRIX AND HEMOTAXIS. AND IF YOU FOLLOW AT THE TOP LEFT, SO THE BLUE REPRESENTS THE LEFT BLUE REPRESENTS TGF BETA LIGAND AND THAT COULD BE ANY MOLECULE IN THE SUPER FAMILY. IT BINDS TO THE TYPE 2 RECEPTORS SHOWN IN PINK. THIS COMBINED AND THEN THE RECEPTOR SHOWN IN BRIGHT PINK IS THEN RECRUITED AND TRANSPHORYL CORRELATED AND IT’S ABLE TO HOOK UP WITH ANOTHER FAMILY OF PROTEINS. THEY’RE CALLED SMADS. THEY’RE THE COMMON PROTEIN FOR BINDING TO TGF BETA. IT’S ACTIVE. ONCE THEY BIND 2 AND 3 WITH THE TGF BETA THEY’RE PHOSPHORYLATED AND THEY’RE ABLE TO GO TO WHAT WE CALL 4 AND WHEN THEY COMBINE WITH THE 4 PROTEIN, WE’RE ABLE TO TRANSLOCATE TO THE NUCLEUS FOR TRANSCRIPTION. THE WHOLE THING CAN BE CIRCUMVENTED BY WHAT WE CALL INHIBITORS. AND THESE CAN TURN OFFER THE WHOLE SYSTEM IF — TURN OFF THE WHOLE SYSTEM IF THEY’RE ALLOWED TO DO SO. NOW, CLINICALLY, TGF BETA HAS ALSO BEEN SHOWN TO ACT IN A CLINIC. IT’S SHOWN TO BE ABLE TO ACT AS A TUMOR AND THERE’S THREE INSTANCES OF GERM LINE MUTATIONS IN CERTAIN PARTS OF THE TGF BETA PATHWAY AND SHOWN AS A PREDISPOSITION TO CANCER. 4 HAS SHOWN TO BE A MUTATED AND JUVENILE SYSTEM. LIKEWISE, TGF BETA COMPONENTS HAVE BEEN SHOWN TO EITHER BE SOMATICALLY MUTATED IN SOME CANCERS. THIS HAS BEEN SHOWN FOR THE TYPE 2 RECEPTOR IN COLORECTAL CANCER AND IN PANCREATIC CANCER. AND LASTLY, REDUCING FRICTION OF TGF BETA OR OVER EXPRESSION OF PATHWAY INHIBITERS HAVE BEEN SHOWN TO BE ASSOCIATED WITH PROGRESSION OF DISEASE. AND THIS ENCOMPASSES THE TYPE 2 RECEPTORS. THERE’S AN INTERESTING PROTEIN. NOW, TGF BETA IN THE CLINIC HAS ALSO BEEN SHOWN TO BE A PROVIDER. TGF BETA 1 IN THIS RESPECT HAS BEEN SHOWN TO BE ELEVATED IN MANY TYPES OF ADVANCED HUMAN CANCERS AND TO BE CORRELATED WITH BOTH POOR PROGNOSIS AND IT’S BEEN SHOWN IN A LARGE VARIETY OF CANCERS INCLUDING LUNG CANCER. NOW, THIS IS A CARCINOMA OF THE PROSTATE STAINED WITH THE TGF BETA 1 ANTIBODY. YOU CAN SEE THE STAINING IS AT THE INTERFACE BETWEEN THE TUMOR AND MICROENVIRONMENT. SO SIT A VILLAIN OR HERO. IT CAN BE BOTH. IT CAN BE A MALIGNANT PHENOTYPE AND ALSO A TUMOR SUPPRESSER AND A METSTATIC FACTOR. IT DEPENDS ON A LOT OF CONTEXT. AND TGF BETA HAS BEEN SHOWN TO SWITCH TO A PROMOTER. SO WHEN WE HAVE A NORMAL EPITHEL EPITHELIUM THE SUPPRESSER DOMINATES BUT AS YOU HAVE CHANGES MADE DURING GENETICS AND EPIGENETICS YOU START TO LOSE THE RESPONSE TO TGF BETA AND INCREASE ITS EXPRESSION AND/OR ACTIVATION AND THEN THE PRO OXYGENIC ACTIVITIES SEEMS TO DOMINATE OVER THE IMMUNOSUPPRESSOR AND YOU HAVE INVASIVE CANCER AND METASTASES. I’VE BEEN TALKING MAINLY IN THE FIRST PART ABOUT THE ONES INVOLVED. IN ADDITION, TGF BETA HAS INDEPENDENT PATHWAYS BY WHICH IT CAN OPERATE. THIS INCLUDES BOTH THE TACK PATHWAY AND INCORPORATES AS WELL AS THE RISK PATHWAY AND THE TGF PATHWAY. BECAUSE WE’RE INTERESTED IN LUNG CANCER WE’RE INTERESTED. THE ONCOGENE HAS SHOWN TO HAVE AN EXTRA MUTATION IN AT LEAST 25% TO 50% OF HUMAN LUNG CARCINOMAS. IT’S BEEN SHOWN TO HAVE AMPLE PATHWA PATHWAYS AS WELL AS SIGNALLING PATHWAY. ACTIVATION OF THE PATHWAY HAS SHOWN TO MODULATE TGF BETA. AND LASTLY, IN VITRO STUDIES PARTICULARLY IN CELL CULTURE HAS SHOWN TGF BETA CAN DOMINATE AND THE ACTIVATED GRAPH CAN OVERRIDE THE TGF BETA. SO WE’RE PRETTY CONFIDENT THE K-RAS PATHWAY IS INVOLVED. HOW CAN THE TUMOR EXPRESSION, TUMOR PROMOTER ACTIVITY WORK? WELL, SHOWN ON THE LEFT SIDE OF THE SLIDE IS THE NORMAL TUMOR SUPPRESSION PATHWAY WHEREAS TGF BETA CAN HAVE OTHER PATHWAYS. HOW CAN THIS BE TURNED OFF? ONE WAY IS BY DECREASED PRODUCTION OR ACTIVITY OF THE BETA 1 OR BETA RECEPTOR. THIS IS DECREASED AND COULD RESULT IN TUMOR PRODUCTION. REMEMBER, THE SWITCH BETWEEN TUMOR EXPRESSION AND TUMOR PROMOTION. ANOTHER IS THE HYPERACTIVE ACTIVITY OF THE PATHWAY. IT CAN LEAD TO TUMOR EXPRESSION. THE OTHER WAY IS BY DECREASED LEVELS OR ACTIVITY OF THE SMAD PROTEINS. THEY’RE VERY ACTIVE IN THE DEFENDING PATHWAYS AND IF YOU THINK OF A FOURTH PATHWAY YOU CAN SEE A COMPROMISED FUNCTION IN THE TUMOR EXPRESSER ACTIVITIES OF THE TGF BETA. OKAY. SO WE’RE KEYING INTO THE HYPERACTIVE PATHWAY HERE. OUR GOAL WAS TO LOOK AT THE ROLE IN EPITHELIAL CELLS. THIS WORK WAS DONE IN THE EPITHELIAL SECTION OF THE LUNG CANCER BIOLOGY BRANCH IN SEATTLE. WE FIRST DETERMINED THE EFFECT OF TGF BETA 1 DELETION AND THE MUTATION IN THE LUNG AND THEN IN COMBINATION A LUNG TUMOR INCIDENT AND PATHOLOGY. THIS IS GOING TO DETERMINE THE EARLY EVENTS IN THE LESION AND HOW THEY PROGRESSED ON TO TUMORS AND THIRDLY TO LOOK AT POTENTIAL SIGNALLING TRANSCRIPTION PATHWAY CHANGES THAT WERE OCCURRING WITH TUMOR GENESIS. NOW, THERE WERE THREE MODELS IN OUR STUDIES. FIRST WAS THE AGING SYSTEM. THE TGF BETA 1 PROVIDE THE SYSTEM AND THE COMBINATION THAT WAS TGF BETA K-RAS. SO WE ASKED TWO QUESTIONS. DOES LUNG TUMOR GENESIS REFLECT THE TGF SIGNALLING PATHWAY AND THE TYPICAL QUESTION IS THE SIGNALLING PATHWAY AFFECT THE LUNG TUMOR. WHY DID WE USE THE MATH MODEL SYSTEM? WELL, IT’S SHOWN TO BE VERY REFLECTING CHEMICALLY IN THE LUNG TUMORS. AND THE LUNG TUMORS THAT DEVELOP IT’S SIMILAR TO ABOUT WHAT WE SEE IN THE HUMAN CONDITIONS GOING THROUGH HYPERPLASIA AND ON TO CARCINOMA. THE CARCINOMAS THAT APPEAR HAVE SHOWN TO BE SIMILAR TO HUMAN LUNG ADENOCARCINOMAS AND THE SAME MUTATIONS HAVE BEEN SHOWN IN BOTH HUMAN AND OTHER TOUMORS WITH LOW EXPRESSION OF K-RAS EXPRESSION. IT WORKS THROUGH TWO DIFFERENT PATHWAYS. A DETOX PATHWAY. IT FUNCTIONS DOWN TO FORM HARMLESS ETHANOL, CO2 AND THERE’S ALSO AN ACTIVATION PATHWAY WHICH. AND THIS INVOLVES AN INTERMEDIATE WHICH RESULTS IN POSSIBLE BINDING TO AN ENTITY THAT MAY BIND TO MACRO MOLECULES LIKE DNA. SO TO DO THE STUDIES IN THE MODEL SYSTEM, WE TOOK TWO MONTH-OLD AGEING MICE AND INJECTED THEM AND WAITED FOR A PERIOD OF MONTHS AND LOOKED AT TGF BETA IN TYPE 1 AND 2 RECEPTORS. HERE IS TUMORS AT TWO MONTHS, FOUR MONTHS AND EIGHT MONTHS AND STAINED FOR TGF BETA 1 AND THE VENTER AND TYPE 2TGF BETA RECEPTOR. SHOWN IN THE LEFT AND MIDDLE PANEL, YOU’LL SEE RATHER INTENSE STAINING FOR TGF BETA 1 LIGAND AND IF YOU LOOK AT THE PANEL ON THE RIGHT FOR THE TYPE 2 RECEPTOR YOU’LL SEE COMPARATIVELY LIGHT STAINING IN THE TUMORS AND VIRTUALLY NO STAINING IN THE OLDER TUMORS. THIS INDICATES THE RECEPTOR PROTEIN IN THE TUMORS. AND THIS IS SHOWN BETTER IN THIS SLIDE. ON THE LEFT THESE ARE INDUCED LUNG CANCERS STAINED FOR THE TYPE 1 AND TYPE 2 RECEPTORS. WHILE THE TYPE 1 RECEPTOR SHOWS RATHER INTENSE BROWN STAINING IN THE TUMOR WITH THE PINK ARROW BUT ALSO THE NORMAL BRONCHIOLE TISSUE SHOWN BY THE RED ARROW. WHEN YOU LOOK AT THE STAINING BY THE TYPE 2 RECEPTOR ON THE RIGHT, YOU SEE VIRTUALLY NO STAINING IN THE TUMOR SHOWN BY THE PINK ARROW AND RATHER THE SAME AMOUNT OF STAINING IN THE NORMAL EPITHELIUM. WE’VE SHOWN DECREASED PRODUCTION OF TYPE 2 RECEPTOR PROTEIN IN TESE TUMORS. NOW, WE ALSO LOOKED AT THE RNA PATTERN FOR THE TYPE 1 AND TYPE 2 RECEPTOR AND CELL LINES SHOWING THE TUMOR AND I’LL DRAW YOUR ATTENTION TO THE LM1 AND PCC4 CELL LINE WHICH IS AN INDUCED MASS CELL LINE. HERE WE SEE REDUCED EXPRESSION OF THE TYPE 2 RECEPTOR MRNA IN THE TWO CELL LINES COMPARED TO THE OTHER CELL LINES. SO WE’RE ABLE TO SHOW IN MASS TISS TISS TISSUE AND CELL LINES THE REDUCED EXPRESSION OF THE TYPE 2 RECEPTOR PROTEINS IN THE MRNA IN THE CELL LINE. WE ALSO LOOKED AT EXPRESSION OF THESE PROTEINS AND THE MRNA FOR THE PROTEINS AND TUMORS. AND SHOWN ON THE TOP OF THE SLIDE, AGAIN, FOCUS YOUR ATTENTION ON THE ARROWS, WE SEE INTENSE BROWN STAINING WITH TGF BETA LIGAND AND RECEPTOR IN COMPARISON STAINING OF THE TUMORS. IF YOU FOCUS ON THE MIDDLE PANEL, THIS IS HYBRIDIZATION FOR THE MRNA FOR THE PROTEINS AND WHILE YOU SEE RATHER INTENSE HYBRIDIZATION OF THE TGF BETA 1 LIGAND IN THE TYPE 2 RECEPTOR EXPRESSION OF THE METHOD FOR TYPE 2 RECEPTOR IS VERY LOW IN COMPARISON. SHOWN ON THE BOTTOM OF THE SLIDE ARE STAINING PATTERNS FOR THE PROTEINS FOR AND THEY’RE SHOWING WHERE THEY’RE ALMOST IDENTICAL AMOUNT OF STAINING. SO WE’VE BEEN ABLE TO SHOW IN THE INDUCED LUNG TUMORS THERE’S ALSO A DECREASE MRNA AND PROTEIN IN A TUMOR FOR THE TYPE 2 RECEPTOR. SO OUR MODEL REFLECTS DECREASED PRODUCTION OF AN EXPRESSION OF TYPE 2 RECEPTORS CAN LEAD TO TUMOR PROMOTION IN THE MODEL SYSTEM. NOW, OUR NEXT QUESTION WAS, DOES THE DELETION OF TGF BETA 1 REDUCE GENESIS. WE LOOKED AT THE BETA 1 MOUSE. SO IF TGF BETA IN A KNOCKOUT MOUSE WAS BORN IN THE ’90s AND WHILE THE MICE ARE BORN THEY TEND TO START TO DIE WITHIN A WEEK AND AFTER ABOUT 21 DAYS THEY GET A SYNDROME. THIS KNOCKOUT MOUSE IS NOT VERY GOOD VEHICLE FOR STUDYING CARCINOG CARCINOGENESIS BUT THIS TYPE OF MOUSE SEEMS TO THRIVE AND GO ON TO BE A GOOD CANDIDATE FOR STUDYING THE EFFECTS OF TGF BETA 1. SO IN CORRELATION WITH NCI WHO IS INTERESTED MORE THANE LIVER CARCINOGENESIS SHE HAD MICE WITH A LIVER CARCINOGEN AND GOT PRODUCTION OF THE LIVERS OF THE MICE AND FOUND SHE GOT EVEN MORE LUNG TUMORS IN THESE MICE. THE TOLD ME IT MAY INVOLVE THE TGF BETA IN LUNG CARCINOGENESIS AND SO THEY’RE NOT REALLY A GOOD VEHICLE FOR STUDYING THE TUMORIGENESIS AND WE LOOKED AT THE HETEROZYGOUS MOUSE WITH THE AGING MICE WHICH WERE WILD TYPE FOR TGF BETA 1 TO DERIVE THE F1 GENERATION WHICH CONTAINED BOTH TGF BETA 1 AS WELL AS WILD TYPE PHENOTYPES. AND WE CAN LOOK AT THE LUNG TUMOR MAY DEVELOP. WE HAVE HERE THE STAINING FOR THE BETA 1 LIGAND. WE WANTED TO ESTABLISH IF THE MICE WERE BEHAVING AS WE EXPECTED THEM WITH THE TGF BETA. WE SAW ONE COMMON STAINING FOR TGF BETA 1 IN THE WILD TYPE IN THE UPPER LEFT PANEL AND THE HYBRIDIZATION SHOWING THE MRNA. WHEN YOU LOOK AT THE HETEROZYGOUS MOUSE IN THE MIDDLE PANEL, THIS WAS FOR THE PROTEIN AND IF YOU COMBINE THE ANTIBODY WITH THE ANTIGEN YOU LOSE THE STAINING PATTERN AND THE HYBRIDIZATION. COMPARED TO THE BOTTOM LEFT OF THE SCREEN YOU’LL SEE DECREASED EXPRESSION OF THE TGF BETA 1 MESSAGE IN THE HETEROZYGOUS MICE COMPARED TO THE WILD TYPE. IF YOU DO A COMPARISON OF YOU’LL ALSO SEE REDUCED COMPETITION IN THE HETEROZYGOUS COMPARED TO THE WILD TYPE. THE MODEL IS PERFORMING THE WAY WE THOUGHT IT WOULD. ONCE AGAIN WE TOOK 2-MONTH-OLD MICE. THIS TIME WE HAD TWO PHENOTYPES. TWO GROUPS, ONE TGF BETA 1 HETRO HETEROZYGOUS AND WILD TYPE AND WE INJECTED THEM AND SACRIFICED THEM IN MONTHLY INTERVALS AND LOOKED AT THE TUMORS THAT RESULT. WE SAW INCREASING AMOUNTS OF HYPERPLASIA AND AZEN — ADENOMAS IN THE HETEROZYGOUS MICE COMPARED TO THE WILD TYPE IN GREEN. I’D LIKE TO FOCUS YOUR ATTENTION TO THE CARCINOMA. WHEREAS TUMORS AND CARCINOMAS STARTED FORMING FOUR MONTHS IN THE HETEROZYGOUS MICE IT TOOK 12 MONTHS OR LONGER BEFORE WE SAW CARCINOMAS IN THESE MICE. WE SHOWED INCREASED TUMOR INCIDENT AS WELL AS MULTI PLISI PLISITY COMPARED TO THE WILD TYPE. AGAIN, WE WENT BACK AND STAINED FOR THE TYPE 2 RECEPTOR PROTEINS IN THESE TWO PHENOTYPES AND IF YOU LOOK AT THE LOOK AT THE HYPERPLASIA AND ADENOMA YOU SEE SIMILAR STAINING IN THE TYPE 2 RECEPTOR PROTEIN. BUT WHEN YOU LOOK AT THE CARCINOMA AND COMPARE THE HETEROZYGOUS ON THE LOWER RIGHT COMPARED TO THE WILD TYPE, YOU SEE DECREASED EXPRESSION OF THE TYPE 2 RECEPTOR PROTEINS IN THESE TUMORS OF THE HETEROZYGOUS MICE COMPARED TO THE WILD TYPES. WE LOOKED AT THE RELATIVE LEVELS OF TYPE 2 RECEPTOR MRNA LESION FROM THE HETEROZYGOUS MICE TREATED WITH THE CARBONATE OVER A PERIOD OF MONTHS. WHEN YOU LOOK AT HYPERPLASIA VERSE THIS CARCINOMAS YOU SEE HOW DECREASING AND RNA SHOWED INCREASING REGENESIS. AND AGAIN IN THE HETEROZYGOUS MICE. THE THIRD QUESTION WE ASKED DOES DELETION OF TGF BETA 1 AND MUTATION OF KRAS TOGETHER EFFECT LONG-TERM RE-GENESIS? FOR THIS WE EMPLOYED WHAT WE CALL THE TGF BETA 1 HETEROZYGOUS. TO STUDY THE INTERPLAY OF THE TGF BETA 157BD KRAS WE LOOKED TO ACTIVATE THEM IN THE MICE. WE USED THE KRAS. WE WERE ABLE TO DERIVE FOUR PHENOTYPES USING THE DOUBLE MUTANT AND SINGLE MUTANT AND THE SINGLE TGF BETA 1 AND THE WILD TYPE GLUTAMATE. AND FROM HERE IS A TYPICAL MOUSE LUNG FROM THE FOUR PHENOTYPES. ARE PROMINENT CARCINOMAS ON THE LUNGS OF THE PANEL A IN A SINGLE IN PANEL B. PANEL B AND C CONTAINED THE SINGLE HETEROZYGOUS MOUSE AND GLUTAMATE SHOWED LONG-TERM RISK. AN OCCASIONAL SPONTANEOUS TUMOR. SO WE NEXT LOOKED AT THE EFFECT O TGF BETA 1 CELL MUTATION FOR SURVIVAL COMPARED TO THE DOUBLE MUTANTS SHOWN IN PANEL A. THE SINGLE MUTANT SHOWN HAS A LONGER LIFE TIME COMPARED TO BOTH THE TGF BETA 1 HETEROZYGOUS AND THE WILD TYPE SHOWN IN C AND D. SO WE CONCENTRATED ON OUR DOUBLE MUTANTS AND SINGLE KRAS. THEY BOTH SHOWED DECREASED LIFE SPANS COMPARED TO TGF BETA 1 HETEROZYGOUS MOUSE OR THE WILD TYPE GUTE — GLUTAMATE. WE LOOKED AT THE PATHOLOGY OF LUNG LESIONS AND SHOWED INCREASED HYPERPLASIA AND CARCINOMA SHOWED IN GREEN COMPARED TO THE ONE IN THE RED. I’LL FOCUS YOUR ATTENTION ON THE ADENO CARCINOMA AND HERE MORE IFFY BUT WE SHOWED THE DOUBLE MUTANTS SEEMS TO HAVE GREATER AMOUNTS OF OUR CARCINOMA. AGAIN, WE LOOKED AT STAINING FOR TGF BETA 1 IN THE RECEPTORS AND SHOWED DECREASED EXPRESSION OF THE TGF BETA LIGAND AND IN THE DOUBLE MUTANT AUTHENTICATE THE PINK ARROWS. SINCE IT’S IMPORTANT IN THE TGF BETA PATHWAY WE LOOKED AT THE TYPE 2 RECEPTOR IN LUNG TUMOR GENESIS. I’LL BRING YOUR ATTENTION TO THE TWO PANELS IF YOU COMPARE THE EXPRESSION AND THE KRAS WITH THE DOUBLE MUTANT SHOWN ON THE RIGHT YOU SEE THE DOUBLE EXPRESSION COMPARED TO THE SINGLE MUTANT. THE WILD TYPE AND HETEROZYGOUS TGF BETA 1 SHOWS VIRTUALLY NO CHANGE. SO IN THE DOUBLE MUTANT WE’RE SEEING THE REDUCTION OF THE TYPE 2 RECEPTOR AND PROJECTION. WE ALSO LOOKED AT THE PROTEINS FOR THESE AND ESSENTIALLY WE SAW REDUCED EXPRESSION OF THE FORM OF SMAD 7 WITH RECEPTORS IN SMAD 3. WE LOOKED AT THE KRAS INTERACTING PATHWAYS OF PROTEINS FOR PROJECTION OF BOTH OF THESE PROTEINS AS WELL. THE THIRD TGF BETA HAS SHOWN TO BE IMPORTANT FOR APOPTOSIS IN THE SINGLE ACTIVATABLE MOUSE WITH THE DOUBLE MOUSE. AND IF YOU LOOK AT THE RED COMPARED TO THE GREEN, IT SHOWS APOPTOSIS IN THE DOUBLE MUTANT COMPARED TO THE ACTIVATABLE MOUSE TUMORS. SO, THE SYSTEM WE’VE BEEN MODELLING SHOWS THE ABILITY FOR THE TYPE 2 RECEPTORS TO BE EQUAL AS WELL AS BEING REACTIVATED. AND APOPTOSIS TO BE COMPROMISED WHICH CAN LEAD TO ADVANCING LUNG TUMOR PRODUCTION IN THE LUNG MODEL SYSTEMS. NOW WE’VE COME TO UNDERSTAND THE TGF BETA CAREFULLY, ARE WE ABLE TO USE TGF BETA FOR CLINICAL TRIALS? IT’S COMING ALONG SLOWLY BUT THERE’S FIVE TYPES OF TGF BETA COMPOUNDS INCLUDING ANTIBODIES, AND RECEPTOR INHIBITORS AND BEING ACTIVELY PURSUED BY VARIOUS PHARMACEUTICAL COMPANIES. IN DISSATISFACTION — ADDITION TO THAT I’VE FOCUS THE TALK ON THE TGF BETAS AND SUPER FAMILY BUT THERE’S ALSO QUITE ACTIVE INVESTIGATION GOING ON INTO THE BNP COMPOUND INHIBITERS IN CLINICAL TRIALS. THAT’S MOVING THIS WAY ALONG. THEY’RE NOT SIMPLE TRIAL OR MAKING THE COMPOUNDS TO INHIBITERS ARE NOT THAT SPAM BECAUSE TGF BETA IS SO GLOBALLY ACTI ACTIVE EVERYWHERE IN THE SYSTEM, IT’S KIF TO TRY TO — DIFFICULT TO TRY TO TARGET THESE AND MORE USEFUL IN PREVENTING DISEASE. THAT’S THE NEXT LEVEL OF COMPLEXITY TO MAKE THEM SPECIFIC. THESE WERE THE PEOPLE WHO WORKED ON THIS PROJECT. I’LL ENTERTAIN ANY QUESTIONS YOU MIGHT HAVE. [APPLAUSE] [OFF MIC]>>WELL, I’LL ANSWER THE SECOND QUESTION FIRST, OKAY BECAUSE MY BRAIN WORKS THAT WAY. I THINK WITH THE QUESTION, WERE THERE TUMORS EXPRESSIVE VERSUS TUMOR PROMOTER. A LOT DEPENDS ON THE TYPE OF THE CELL AND THE CELL TYPE AND PHASE THE CELL IS IN. IS IT A NORMAL CELL, A CLOSE TO NORMAL CELL? SIT STARTED TO BECOME A METASTATIC CELL? THAT HAS A LOT TO DO WHAT ROLE IT PLAYS AND IT SWITCHED FROM BEING A TUMOR EXPRESSER TO A TUMOR PROMOTER. NOW, YOUR FIRST QUESTION WAS THE ISO FORMS. CERTAIN TISSUES AND CERTAIN TUMORS HAVE MORE TGF BETA 1, 2 AND 3 THAN OTHERS. WE’RE CONCENTRATING ON TGF BETA 1 BECAUSE IT’S BEEN THE MAJOR PLAYER HERE AND YOU CAN GET A LOT OF IT. THE BETA 2 AND 3 ARE A LITTLE MORE TRICKY. THEY’RE LESS INVOLVED. BUT IN THE PROJECT IT SEEMS TO HAVE MORE TGF BETA 2 OR 3 THAN 1. IT HAS TO DO WITH WHERE IT IS AN WHAT ITS ROLE IS. I SHOWED YOU A SLIDE THAT WAS INVOLVED IN DEVELOPMENT OF IMMUNE SUPPRESSION. I TOOK A GLOBAL APPROACH JUST TO TALK ABOUT IT BUT TGF BETA 2 MAY BE MORE IMPORTANT IN THE IMMUNE FUNCTION THAN TGF3.>>YES. [OFF MIC]>>IT’S SECRETED IN AN INACTIVE FORM AND THE FORM HAS TO BE ESSENTIALLY CUT TO AN ACTIVE FORM WITH THE ENZYMES IN THE SPECIFIC ENVIRONMENTAL CONDITIONS THAT ENABLE ITS ACTIVATION. THAT’S IMPORTANT THERE. WHAT WAS THE OTHER PART OF YOUR QUESTION? [OFF MIC]>>IT’S DONE EXTRA CELLULARLY AND ALLOWS IT TO BE ACTIVE INSTEAD OF BINDING TO THE RECEPTOR. YES. [OFF MIC]>>NO. WE’RE TRYING TO MIMIC AS CLOSELY TO THE HUMAN CONDITION AS WE CAN. THERE’S REALLY NO PURPOSE MODEL SYSTEM THAT CAN GO ON THERE. YOU’RE RIGHT IN THE CONTEXT OF WHAT YOU’RE CALLING IT.>>YES. EXACTLY. YES. [OFF MIC]>>WELL, WE PLAN TO GET ENOUGH FOR STATISTICAL ANALYSIS. AND WHEN YOU’RE LOOKING AT ONE MONTH, TWO MONTHS, THEY’RE NOT ALL HAVING TUMORS. MOST OF THEM DON’T HAVE TUMORS. I’LL TURN YOUR ATTENTION TO THE SLIDE, A, B AND C. THE ADENOMA, CARCINOMA AND DEPENDING ON THE PHENOTYPE VERSUS WILD TYPE, YOU SEE THE INCREASE IN THE TUMORS VERSUS NOTHING. RIGHT. IS THAT CLEAR? I’M SORRY. MAYBE WE CAN TALK BRIEFLY LATER. I’LL POINT YOU TO THE SLIDE. YES. [OFF MIC]>>THAT’S HARD TO SAY. I COULDN’T ANSWER THAT. IT’S POSSIBLE. WHATEVER MOUSE YOU’RE INJECTING AND I WASN’T AWARE PEOPLE WERE BEING [INDISCERNIBLE] BUT IT’S POSSIBLE I SUPPOSE. THANK YOU. ANTON GOT HIS Ph.D. AT THE UNIVERSITY OF SOUTHERN CALIFORNIA. I’M A NATIVE CALIFORNIAN SO I KNOW ALL ABOUT THAT AND DID HIS POST-DOC IN CALIFORNIA AND WORKED AT THE BOARD OF LIFE SCIENCES FOR SEVERAL YEARS AND THEN CAME IN 2004, 2011 HE WAS THERE AND HIS TITLE EARLY THERAPEUTIC DISCOVERY AND OPPORTUNITIES.>>THANK YOU. IT’S GREAT TO BE HERE AGAIN. MY JOURNEY WITH NIH STARTED FOUR FLOORS UP IN THIS BUILDING. WE WERE PART OF THE GENOME STUDENT-ATHLETE BEFORE WE BECAME NCATS BACK IN 2004. SO IT’S BEEN AN INTERESTING JOURNEY OF ACTUALLY HELPING CREATE WHAT IS A START-UP ORGANIZATION WITHIN NIH. I WAS THE SIXTH PERSON HIRED TO BUILD THE SCREENING CENTER AND WE EXPANDED OUR OPERATIONS TO LATER STAGE PRE-CLINICAL DEVELOPMENT. WHAT WE DO AT NCATS IS DEAL WITH THE NEBULOUS SPACE BETWEEN THE SCIENCE AND NATURE PAPER AND THE ACTUAL THERAPEUTIC INTERVENTION THAT HELPS PATIENTS. ON THE NCATS ADVISORY COUNCIL THERE’S A CHILD WITH A RARE GENETIC DISORDER WHO SAID SEVERAL TIMES PUBLICLY, IT’S GREAT THAT YOU GUYS PUBLISH SCIENCE AND NATURE PAPERS BUT I CANNOT FEED MY CHILD A SCIENCE PAPER TO IMPROVE HER HEALTH SO WE NEED TANGIBLE INTERVENTIONS. SO HOW DO YOU THINK ABOUT THAT PROCESS? THERE’S ACTUALLY A VERY SORT OF NAIVE WAY OF THINKING ABOUT IT WHICH IS TO THINK ABOUT THE DISEASE MECHANISM AND HOW TO INTERVENE AND WHAT TO INTERVENE ON AND IT MAY BE BY INDIVIDUAL UNIQUE END DESIGN, KINASE IN CANCER OR A PATHWAY AND THEN CREATE A TEST SYSTEM TO DISCOVER THERAPEUTIC CANDIDATES. YOU TEST MANY CHEMICALS IN THAT SYSTEM. THAT’S AN EXAMPLE WITH SMALL MOLECULE DRUG DISCOVERY TO IMAGINE HOW IT WOULD WORK WITH THERAPEUTIC ANTIBODIES AND OTHER TECHNIQUES. SO YOU RUN THE SCREEN AND TEST MULTIPLE CANDIDATES. THEN YOU HAVE TO MODIFY THEM THROUGH EXTENSIVE CHEMISTRY TO IMPROVE THEIR PROPERTIES, PREPARE THEM FOR IN VIVO USE AND TEST IN ANIMALS AND EVENTUALLY TEST THE CANDIDATE MOLECULE IN HUMANS. SO IT SEEMS VERY LINEAR. IT SEEMS AUTOMATED AND SEEMS LIKE SOMEONE ELSE’S JOB. THEY’LL DO IT FOREVER AND PROBLEMS WILL BE SOLVED. SO YOU LOOK AT THE REALITY THOUGH EXPRESSED IN THE BOTTOM TWO GRAPHS ON THE LEFT SIDE WE SEE THE GREAT NEWS, THE SCIENCE PAPERS, THE EXPLOSION OF KNOWLEDGE IN TERMS OF ACTUAL MOLECULAR MECHANISMS ASSOCIATED WITH MORE AND MORE DISEASES. THAT IS WE BELIEVE WHEN THERE’S A BASIS FOR ACTUALLY WELL OVER 6,000 DISEASES RIGHT NOW AND THE NUMBER IS GROWING THAT ONLY ABOUT 500 COME WITH THERAPY. AND AT THE PACE OF CURRENT DRUG APPROVES WORLDWIDE IT MAY TAKE ABOUT 2,000 YEARS TO ADDRESS ALL DISEASES. SO THAT’S A PRETTY LONG TIME. THEN ON THE RIGHT SIDE YOU LOOK AT THE FINANCIAL ASPECT OF THE OPERATION THE PRODUCTIVITY AS THE INVESTMENT HAS BEEN GOING DOWN AND THIS IS INDICATING IT’S PROBABLY THE OPPOSITE TREND OF COMPUTER CHIP WITH TIME. SO HOW DO YOU ACTUALLY REVERSE THE TRENDS AND IMPROVE THE PACE OF THERAPEUTIC DEVELOPMENT? THAT’S WHAT WE TRY TO ADDRESS AT NCATS IN A DISEASE AGNOSTIC MANNER. WE CANNOT AFFORD TO WORK ON ONE DISEASE AT A TIME AND NOT EQUIPPED TO DO THAT. SO WE’RE FOCUSSING ON THE OVERALL PROBLEMS THAT ARE DISEASE INDEPENDENT AS MUCH AS PROBLEMS IN PREDICTING THE SAFETY AND EFFICACY OF NEW DRUG CANDIDATES. THEY WORK GREAT IN CELL-BASED ASSAYS AND IN ISOLATED ASSAYS. MAYBE MOUSE MODEL AND THEN FAIL IN HUMANS. FOR BOTH EFFICACY AND TOXICITY REASONS. HOW COME WE COULDN’T PREDICT THAT EARLIER AND SAVE $500,000 IN UNNECESSARY PRE-CLINICAL DEVELOPMENT. HOW CAN WE SCALE APPROACHES TO FIND TREATMENTS FOR MORE THAN ONE DISEASE AT A TIME? CAN WE PROBE THE PROTEOME IN A SCALEABLE MANNER BY MOLECULAR BASIS. OTHERS ARE PRE-CLINICAL. SOME PROBLEMS ARE SCIENTIFIC IN NATURE. ROGER — OTHERS ARE ORGANIZATIONAL IN NATURE. HOW DO YOU PRIORITIZE THE SPACE WHERE YOU HAVE MANY INDIVIDUALS. WHAT IS THE COURTESY THAT OPERATES IN THE SPACE? IT CANNOT BE P.I.-DRIVEN RESEARCH. HOW ABOUT IT’S ORGANIZATIONAL, NOT OTHER WAYS AND I’LL TALK PRIMARILY ABOUT THE FIRST BULLET. DEALING WITH THE PRE-CLINICAL EARLY DISCOVERY AND GIVING YOU EXAMPLES OF HOW WE TRY TO TACKLE INEFFICIENCIES IN THIS SPACE AND HOW IT MAY APPLY TO ACTUAL PROJECTS WITH SPECIFIC EXAMPLES FROM ONCOLOGY AND ELSEWHERE. SO THE JOURNEY TYPICALLY STARTS WITH SCREENS YOU DESIGN TEST SYMPTOM LOOK FOR THE DRUG CANDIDATE OUT OF SAY A MILLION TEST MOLECULES. YOU CAN SET UP LARGE ROBOTIC SCREENING SYSTEMS AND RUN THE SCREENS AND OVER THE PAST 20 YEARS OR SO, HIGH THROUGHPUT SCREENING HAS BECOME PRETTY STANDARD STEP IN THE DRUG DISCOVERY PROCESS AND FOR THOSE WHO PLAN ON VISITING OUR SHOP WILL SEE THE SYSTEM IN OPERATION. IT’S BEEN RUNNING FOR 13 YEARS. FROM DAY ONE WE ACTUALLY TRIED TO IMPROVE THE UNDERLYING PROCESS OF HIGH THROUGHPUT SCREENING. ONE GLARING DEFICIENCY OF THE PROCESS IS TYPICALLY A PHARMACEUTICAL COMPANY AND ELSE WHERE, PEOPLE HAVE BEEN TESTING THE DRUG CANDIDATES IN A SINGLE DOSE. IT MAY BE DUPLICATE IT MAY DEPEND ON THE BUDGET AND ASSAY COMPLEXITY. WE KNOW WELL FROM ECOLOGY MEANS THE EFFECT DEFENDS ON THE DOSE. THERE’S THE EQUATION AND AN ASSOCIATION WITH TYPICAL DRUG EFFECTS. WHY NOT DO THE OPS. IT’S SHOWN ON THE LEFT. THE DRUG CANDIDATES IN MULTIPLE DOSES. WE DESIGNED A WORK THROW TO DO JUST THAT. WE USED TOP OF THE LINE MINIATURIZATION TECHNIQUES TO SHRINK THE ASSAY VOLUMES TO 8 MICROMETERS PER TEST AND DEVELOPED NEW INFORMATICS TIME LINES TO LOOK AT THE DOSE RESPONSE CURVES IN THE TIME SPAN OF 20 MINUTES WITH MINIMAL MANUAL INTERVENTION. THE PRISM WILL NOT ALLOW YOU. HVE TO LABOR EXTENSIVELY OVER A DOZEN CURVES. MA WHAT DO WE GET — WHAT DO WE GET IN THE END? WE GET FAPHARMACO LOGICAL
PROFILES SO RELIABILITY WENT WAY UP AND FALSE NEGATIVES AND FALSE POSITIVES WERE REDUCED DRAMATICALLY AND WERE ABLE TO RANK MOLECULES WHETHER THEY’RE INACTIVE A RESPONSE OR ADDING MORE UNIQUE PHARMACOLOGY AND WEAK POTENCY,EST SET RA, FROM THAT — ETCETERA FROM THAT ROBOTIC SCREEN IT. HELPS YOU SAVE MONEY DOWN STREAM ON NOT HAVING TO FOLLOW UP ON THE FALSE POSITIVES. WE’RE PART OF NIH. PART OF THE MANDATE IS TO MAKE DATA PUBLIC WITH PRETTY MUCH EVERY SINGLE DATA POINT OF MILLIONS OF DATA POINTS FROM SCREENS. THEY’RE AVAILABLE FOR OTHERS TO ANALYZE AND BUILD PREDICTIVE MODELS AND ACTUALLY IMPROVE THE PROCESS OF PREDICTING WHAT CHEMICALS WILL BE ACTIVE AGAINST NEW TARGETS. AGAIN, THIS IS DISEASE AGNOSTIC IN ALL THE DETAILS HAVE BEEN MADE AVAILABLE FOR OTHERS TO REPLICATE THE PROCESS. PROBE TARGET DIFFERENT PATHWAYS AND ENZYME. THEY’RE NOT DISCOVERED DIRECTLY FROM SCREENS. YOU NEED TO PROOF THEM TO CHEMISTRY. WE HAVE ADDITIONAL CHEMISTRY OPERATION. CHEMISTS WORK NOT ONLY TO IMPROVE POE TEN SIF THE MOL — POTENCY OF THE MOLECULE AGAINST THE TARGET. THIS IS THE FIRST IN CLASS INHIBITOR SPECIFIC PROTEASE USB1. SO CHEMISTRY YIELDED QUALITY IMPROVEMENT IN THE INITIAL SCREENING KIT AND THE OPERATION ALLOWS FOR MULTI-PARAMETER AUTHORIZATION AND OTHER PHYSICAL CHEMICAL PROPERTIES REQUIRED FOR DOWN STREAM ANIMAL EXPERIMENT AND EVENTUALLY HUMAN CLINICAL TRIAL. WE HAVE PROBE AND THE CONCEPTS. THE DARK MATTER OF THE GENOME. EVERYBODY CONTINUES TO STUDY THE SAME KINASE. WHAT ABOUT THE OTHER TARGET CLASSES? THAT’S HOW WE POSITIONED OVER THE YEARS. AS A RESULT WE OPENED UP ENTIRE SEGMENTS IN TERMS OF NOVEL TARGET CLASSES AND THERAPEUTIC HYPOTHESES AND SOME OF THESE PROBES AND WE HAD A SHOT TO RUN HIGH THROUGHPUT SCREENING TO HAVE ACTIVATORS FOR THE KINASE AROUND THE TYPE WHEN THE REGULATION IN CANCER WAS COMING TO THE FORE SO IT WAS 10 YEARS AGO WITH VERY LITTLE KNOWLEDGE IN THE SPACE TO HIGH THROUGHPUT SCREENING AND CHEMISTRY SEVERAL DIFFERENT PHENOTYPES WERE DISCOVERED AND SHARED WITH ACTUALLY SEVERAL HUNDRED LABS AROUND THE WORLD. THIS IS AN EXAMPLE OF AN EARLY PAPER WITH THE INITIAL PROFILING OF THE MOLECULES. AS WE LOOK AT THE OTHER EXAMPLE IN THE LOWER RIGHT, THIS IS VERY RECENT ACTIVATION WAS ASSOCIATED WITH CONTROL OF PANCREATIC BETA CELLS INDICATING THESE MOLECULES MAY HAVE USE IN DIABETES. PKM2 DID NOT HAVE ACTIVATORS FOR IT SO THE HYPOTHESIS ON THE LOWER RIGHT WAS NOT AVAILABLE TO GUIDE US IN TERMS OF ULTIMATE UTILIZATION OF THE ACTIVATORS. THE PROSPECTIVE DEVELOPMENT OF SUCH ACTIVATORS ALLOWED THE BIOLOGY TO BE STUDIED IN THESE NEW THERAPEUTIC APPLICATIONS TO BE DISCOVERED. ANOTHER EXAMPLE FROM MORE RECENT WORK IS SOMETHING A TRADITIONAL MEDICINAL CHEMIST WOULD NOT LIKE TO SEE WHICH IS DISCOVERY AND DEVELOPMENT OF THE INHIBITOR OF AN ENZYME. ONE THINKS OF THESE AS BEING HIGHLY PROMISS — PROMISCUOUS OF THE DEVELOPMENT. IN THIS CASE THE REDUCTASE WAS AN UNDER STUDIED SUBJECT IN ONCOLOGY LOOKING AT CLEARING OF SPECIES BUT THERE WERE NO INHIBITORS FOR THE ENZYMES SELECTED FOR IT. IMPORTANTLY SHOWN IN THE SECOND BULLET POINT HERE WHEN WE RAN THE ROBOTIC SCREEN AND FOUND THE NEW INHIBITOR HIGHLIGHTED HERE IT WAS HIGHLY SELECTED AND DID NOT HAVE A GOOD AMOUNT ON THE REDUCT PHASE WHICH IS AN ENZYME THAT STARTED CONTROLLING THE REDUX WHICH IS ESSENTIAL FOR ALL THE CELLS IN THE BODY. ESSENTIALLY THE THERAPEUTIC HYPOTHESIS WAS TO GET THE REDUCTASE COULD NOT HAVE BEEN SELECTED WITHOUT THE SELECTIVE INHIBITOR SO WHEN THE MOLECULE WAS TEST AND PROFILED HOW WOULD IT BE NORMALLY IF SOMETHING IS COVALENT IT WILL HIT THE OTHER RELATED TARGETS. IT TURNS OUT THERE WAS A REDUCTASE RESPONSIBLE AND THE COMPOUND WAS ACTING WITH THE RESIDUE. THE COMPOUND ITSELF WAS NOT REACTIVE AGAINST THE ENZYMES. SO ON THE RIGHT YOU CAN ACTUALLY SEE SOME IN VIVO RESULTS IN THE COMPOUND B TESTED WITH SEVERAL MODELS IN VIVO. THIS WAS PUBLISHED LAST YEAR AND ACTUALLY IT IS TREATED AS A DRUG CANDIDATE THROUGH A SPIN-OFF COMPANY OUT OF SWEDEN WHEN WE COLLABORATED ON THIS. SOMETHING THAT WAS PUBLISHED JUST A FEW WEEKS AGO, YOU GO AFTER THE SINGLE TARGET AND DEVELOP A GREAT MOLECULE. IT GOES IN EQUAL AND SHOWS GOOD RESULTS ESSENTIALLY AND THEN THE RESISTANCE DEVELOPS. THIS IS THE CASE WITH KINASE INHIBITORS WITH AML. THE TEAM HERE PERFORMED IN DEPTH PROFILING TO THE CELL’S RESPONSE TO THIS INHIBITION AND DISCOVERED THE CELLED — CELLS WERE BASICALLY ACTIVATING IN THE IMMUNE PATHWAYS AND THAT WAS ESSENTIALLY CREATING A MECHANISM FOR THE INHIBITION. IF YOU TACKLE THE PROBLEM YOU DON’T WANT TO BE HAVING A SITUATION WHERE YOU TREAT THE PATIENT WITH THE INHIBITOR AND WAIT FOR RESISTANCE TO DEVELOP AND THEN TRY TO TREAT WITH THE INHIBITOR. THE GROWTH DEVELOPED A DUAL ACTING MOLECULE TO EXTENSIVE WORK TO TARGET BOTH KINASE SIMULTANEOUSLY. YOU CAN READ THE PAPER. THE MOLECULE IS SHOWN ON THE LOWER RIGHT. IT ACTUALLY PERFORMED BETTER THAN THE INHIBITOR AND ESSENTIALLY EXPRESSING THE ACTIVATION OF THE PATHWAY FROM THE GET GO. IT REMAINS TO BE SEEN WHAT HAPPENS WITH THE PROLONGED TREATMENT. IT HASN’T BEEN TESTED IN THE TREATMENT. I HAD TO GO TO PRE-CLINIC DEVELOPMENT AND THE INITIAL RESULTS ARE ENCOURAGING. WHAT HAPPENS WHEN YOU DON’T HAVE A MOLECULAR TARGET? THIS IS THE SO-CALLED PHENOTYPIC SCREENS OR ASSAYS WE DISCOVERED A NUCLEAR ORGANELLE AND ITS PREVALENCE IS CORRELATED WITH CANCER SPREADING WITH METASTASIS. HOW DO YOU GO ABOUT LOOKING AT THE MODULATORS TO TEST THE METASTASIS WHEN YOU DON’T KNOW WHAT THE PNC ORGANELLE IS COMPOSED OF. YOU DON’T HAVE TARGETS OR I PATHWAY AND NO OBVIOUS TARGET AND AN APPROACH IN THIS CASE IS TO SCREEN FOR THIS FEATURE FOR COMPOUNDS THAT MAKE IT GO AWAY BUT YOU CAN SEE HOW IT WOULD WORK. WE AASSEMBLED A TEAM OF EXPERTS WE’RE VERY GOOD AT SCREENING. ACTUALLY INCLUDING SOMEONE FROM NCI YOU MAY RECOGNIZE WE SCORED THE CELLS FOR WHETHER THEY RETAINED THE COMPARTMENT AFTER TREATMENT OR DON’T. THE COMPOUND WAS DISCOVERED AND THE FURTHER STUDY SHOWED THE COMPOUNDS HAD EFFECTS AND THE MOLECULE THE GROUP SHOWED IT IN ME TAS IS A TA SIS. — METASISIS AND SHRINKS TUMORS OR EFFECTED ME TAS — YOU CAN SEE FOR EXAMPLE ON THE LEFT SIDE THE SEPARATION BETWEEN THIS AND THE PREVALENCE AND CANCER SPREADING. SO WHERE IS THE PROGRAM? IT’S STILL IN-HOUSE. THIS IS SUCH A RISKY THERAPEUTIC HYPOTHESIS YOU CANNOT JUST SAY I’LL TACKLE IT AND LICENSE IT AND PFIZER WILL JUST BUY IT OVERNIGHT AND WORK ON THE DEVELOPMENT. COMPANIES SEE THIS PAPER AS TOO RISKY OF AN ASSAY. TOO UNPROVEN. COMPANIES WOULDN’T TOUCH THIS ASSAY. IT’S STILL TOO RISKY. WE’RE SUPPORTING THE MOLECULE AND COMING UP WITH BETTER THIS SYNTHETIC STRATEGY AND ENABLING THE TOX STUDIES IN SEVERAL ANIMAL SPECIES WORKING ON FORMULATION. THE GOAL IS TO APPLY TO THE FDA IN A FEW MONTHS TO BEGIN WHAT’S KNOWN AS INVESTIGATIONAL NEW DRUG PROCESS TO ENABLE PHASE 1 SAFE TRIAL AND THEN EFFICACY ASSESSMENT AND THE GOAL IS TO ACTUALLY VIEW THE PROOF OF CONCEPT WITH NCI SUPPORT WITH THE ROAD BLOCK BEING THE CLINICIAN. SO KIND OF SWITCHING GEARS. I TALKED ABOUT THE DISCOVERY OF SINGLE MODULATORS, SMALL MOLECULE INHIBITORS, ETCETERA. WE KNOW WELL THERE’S VALUE IN FINDING DRUG COMBINATIONS. RESISTANCE TO TACKLE VARIOUS DISEASE SCENARIOS. TWO OR THREE DRUGS. THERE’S VARIOUS EXAMPLES OF SUCH COMBOS FROM THE HIV WORLD. HOW DO YOU GO THROUGH RAPID DISCOVERY OF COMBO. IF YOU LOOK AT DRUG A AND DRUG B MANUALLY AND USING SEMI AUTOMATED EQUIPMENT THE NUMBERS WILL OVERWHELM YOU AND IT WILL BE SO EXPENSIVE AND SLOW YOU WON’T BE ABLE TO TEST ANYTHING. WE WORKED PRETTY HARD IN THIS SPACE TO FIRST DEVELOP VARIOUS MOLECULES AND APPROVE DRUGS AND A COUPLE OF THOSE WITH EMERGING TECHNOLOGIES IN THIS CASE THIS COULD ALLOW YOU TO LITERALLY SPOT COMBOS OF TWO OR MORE AGENT PRETTY RAPIDLY WITHOUT USING INTERMEDIATE SOLUTIONS IN THE TIME SPAN OF A COUPLE HOURS FOR SEVERAL THOUSANDS OF COMBINATIONS. AND IN PARALLEL DEVELOP INFORMATICS PLATFORM TO PROCESS ALL THAT DATA AND CALCULATOR? SYNERGISM AND DISSEMINATION BY THE SCIENTISTS. YOU CAN SEE WHERE THIS IS IMPORTANT THEN RESISTANCE AFFECT THE ONCOLOGY FIELD, CANCER AND THE INFECTIOUS DISEASE FIELD. WHY? BECAUSE IT’S A FUNCTION OF DOUBLING TIME FOR A CELL AND MUTATION RATE AND WHEN YOU START MAPPING VARIOUS SPECIES, CANCER CELLS AND VARIOUS INFECTIOUS AGENT THEY’RE ALL MAPPING TOGETHER. YOU CAN GET RESISTANCE NOT JUST IN MALARIA OR OTHER INFECTIONS BUT RESISTANCE IN CANCER. SO SOLVING THIS PROBLEM IS AWAY OF SIRN — SYNERGISTIC COMBINATIONS THAT CAN SOMETIMES SEEM NOT TO HAVE ANYTHING IN COMMON. WE HAVE SHOWN THE PROOF IN CONCEPT IN ONCOLOGY APPLICATION IN THIS RARE BLOOD TUMOR. SEVERAL YEARS AGO WE SHOWED ON THE LOWER LEFT BUT AGAIN IT’S JUST A PAPER. SO WHY AM I SHOWING IT? IT’S BECAUSE OF THE BULLETS ABOVE IT. WE MADE THE PROTOCOLS PUBLICLY AVAILABLE BECAUSE IT’S TO ENABLE OTHERS TO PREFACE THIS OPERATION IN THEIR OWN LABS WITHOUT HAVING TO KNOCK ON THEIR DOOR FOR EVERY LITTLE DETAIL. ON THE INFECTIOUS DISEASE SIDE, ONE EXAMPLE OVER THE LAST FEW YEARS, THIS IS WORK WITH PEOPLE AT THE CLINICAL CENTER HERE. YOU PROBABLY HEARD THE STORIES OF MULTIDRUG RESISTANT OUTBREAKS A COUPLE YEARS AGO AT THE CLINICAL CENTER THAT KILLED SEVERAL PATIENTS. MITS CLOSE TO HOME — HITS CLOSE TO HOME, RIGHT SO WHEN IT’S MULTIDRUG RESISTANT HOW CAN YOU TACKLE THAT SO WE TRIED TO DEVELOP THIS START A SINGLE AGENT AND THE PROVED DRUGS AND DRUG CANDIDATES AND THEN COMBINE THEM IN CONCENTRATIONS A VERSUS B. WE CAME UP WITH SEVERAL SYNERGISTIC COMBOS. GREEN IS THE COMBO, BLUE AND BLACK THE INDIVIDUAL AGENTS. THE WHOLE PROFESSIONAL WAS PUBLISHED IN THIS PAPER. GROUP TOOK THIS ONE STEP FURTHER AND TESTED THREE DRUGS. P PROGRESSING FROM SINGLE AGENT TO TRIPLE AGENT COMBOS BECAUSE YOU CAN’T TEST ALL THE AGAINST EACH OTHER AND CAME UP WITH SEVERAL COMBOS THAT WERE HIGHLY EFFECTIVE AGAINST PRETTY MUCH ALL COMMON DRUG RESISTANT ISOLATES. WE’RE THOUGHT THERE YET BUT THE DREAM IS YOU CAN ONE DAY SET UP A SCREENING OPERATION SO RAPID AND EFFICIENT YOU CAN TAKE A SAMPLE AND RUN THE TESTS AND IN LESS THAN 12 HOURS DELIVER ACTIONABLE SUGGESTION OR LIST OF DRUGS THAT CAN BE COMBINED TO TACKLE THE INFECTIONS OR AFFECT THE TUMORS. I MENTIONED WE’RE NOT VERY GOOD AT PREDICTING EFFICACY IN HUMANS TYPICALLY WE USE HELA CELL MODELS IN PLATES AND WHEN WE GET TO HUMANS WE DON’T KNOW WHY IT’S NOT WORKING SO WE HAVE MODELS OF HEALTHY AND DISEASE MODELS. WE ARE FAMILIAR WITH THE CONCEPT OF DESTROYEDS AND MAY HAVE HEARD — STEROIDS AND YOU MAY HAVE HEARD OF MICROFLUIDIC CHIPS OR OTHER PROCESSES THAT CONTAIN THEM AND AS YOU MOVE FROM LEFT TO RIGHT YOUR PHYSIOLOGICAL COMPLEXITY INCREASES. AN ORGAN ON A CHIP IS PRETTY COMPLEX. THE NEXT THING ON THE RIGHT NOT SHOWN IS THE ANIMAL AND HUMAN. ON THE LEFT WE HAVE A WAY TO TEST MANY SAMPLES. WE CAN TEST MILLIONS OF SAMPLES WITHIN LESS THAN ONE WEEK. CLEARLY THERE’S A TRADE-OFF. WE’RE WORKING ON THE THING IN THE MIDDLE. TRYING TO ESSENTIALLY BUILD A TRIAGE FUNNEL TO PRESENT LEVELS OF NEXT OR TRIAGE BETWEEN EASY TO EXECUTE SCREENS AND THE LIMITED THROUGHPUT OF ORGANS ON CHIP FOR ANIMAL OR HUMAN EXPERIMENT. THE THING IN THE MIDDLE IS 3-D TISSUE BIOPRINTING. THIS IS A VERY NAIVE CARTOON SHOWING HOW IT’S SUPPOSED TO WORK IN A BASIC LEVEL. CELLS ARE SPENDED IN A MATRIX AND LOADED AND THE PRINTER IS ESSENTIALLY A 3-D PRINTER WITH PRECISE X, Y, Z CONTROL AND THE EXTRUSION DEVICE SO CELLS ARE ESSENTIALLY EXTRUDED CONTROLLED BY CAD SOFTWARE, LAYERED IN VARIOUS WAYS. THE CONSTRUCT IS INCUBATED AND EVENTUALLY YOU GET A PIECE OF TISSUE. IT’S NOT AS SIMPLE AS IT SOUNDS AND A LOT CAN GO WRONG. CELLS DON’T JUST STAY WHERE YOU PLACE THEM SO THERE’S A LOT OF WORK ON THE TISSUES SO WE’VE BEEN WORKING ON SEVERAL TISSUES MORE THAN WHAT’S SHOWN HERE BUT THESE ARE THE MOST PROMINENT RETINA, BLOOD VESSEL, SKIN, WALL, WE BEGAN COLLABORATING WITH TISSUE EXPERTS UTILIZING THE TECHNOLOGY STRENGTHS AND TISSUE EXPERTISE AND YOU FIRST BUILD THE WILD TYPE AND LEARN HOW TO LAYER ON TOP OF IT DISEASE MODELS. THE SKIN IS THE FIRST TISSUE FROM THE SKIN THAT IS STAINED. RIGHT SIDE SHOWED THE 3-D SKIN FROM OUR GROUP AND WE HAVE OTHER WAYS TO QUALIFY THE SKIN. IT’S BARRIER FUNCTION AND OTHERS AND TECHNOLOGY DEVELOPMENT HAS TO GO TO IMAGE THE BIOLOGY IN THE TISSUES. YOU CAN’T JUST STICK THE PIECE OF TISSUE THAT IS SEVERAL MILLIMETERS IN SIZE UNDER A MICROSCOPE AND SEE WHAT’S INSIDE SO A GROUP HAS WORK OND TISSUE CLEARING — WORKED ON TISSUE CLEARING TECHNIQUES YOU CLEAR THEM WITH CLEARING AGENTS AFTER YOUR EXPERIMENT TO REMOVE FATTY ACIDS AND OTHER COMPONENTS AND ALLOW LIGHT TO PENETRATE THE TISSUE AND THEN THE IMAGING SYSTEM WILL SEE OTHER WISE IT WOULD BE MASKED BY THE FATTY ACIDS AND OTHER COMPONENTS. YOU CAN BEGIN TO REBUILD TISSUE DEPENDING ON WHAT MARKERS YOU EMBEDDED. WHETHER IT’S INFUSION PROTEINS OR OTHERS OR WHETHER IT’S STAINED WITH ANTIBODY. HOW DO YOU FILL THESE BIOPRINTERS WITH ENOUGH CELLS? YOU CAN’T JUST BUY PRIMARY CELLS FOR EVERY TYPE OF TISSUE. WE’RE WORKING PRETTY HEAVILY ON STEM CELL TECHNOLOGIES. ABOUT TWO YEARS AG WE WORKED ON THE BORING PART. HOW DO YOU SCALE UP CELL CULTURES AND HOW DO YOU AUTOMATE THAT AND MAKE THE SCALE CHEAPER. HOW DO YOU MAKE THE IPS DIFFERENTIATION PROTOCOLS INTO MATURE LINEAGES OF MIOCYTES OR VARIOUS CELLS OR NEURONALCYTES AND EVERY DAY WE SEE MORE PROTOCOLS GET PUBLISHED AND THEY’RE EXPENSIVE. SO THE LAB IS ACTUALLY ABLE TO FOLLOW CELLS AT THE SINGLE CELL OR POPULATION LEVEL TO CHARACTERIZE THEM AND DEVELOP BETTER MARKERS FOR WHAT CONSTITUTES A MATURE LINEAGE. IN PARALLEL IS WORKING TO IMPROVE THE PROTOCOLS TO IMPROVE THE CELL TYPES AND WE HAVE SEVERAL SUCCESS STORIES THAT ARE PUBLISHED I DON’T HAVE TIME TO TALK ABOUT THEM TODAY. ALL SORTS OF FREE AGENT WERE BASED ON CHEAP SMALL MOLECULES ABLE TO REPLACE EXPENSIVE PROTEIN FREE AGENT LIKE TRANSCRIPTION FACTORS AND MEDIA COMPONENTS. THAT’S THE ONLY WAY TO ENABLE 3-D MODELS USING STEM CELLS OF SORTS FOR THE CELLS THAT WILL GO INTO THESE MODELS. OTHERWISE THE MATH JUST DOESN’T ADD UP. YOU CANNOT FINANCE SUCH PROJECTS WITH PHARMA AND ACADEMIA. I WANT TO SWITCH GEARS ONE MORE TIME AND TALK ABOUT THIS SORT OF OTHER PROBLEM IN DRUG DEVELOPMENT WHICH IS HARD TO PREDICT TOXICITY WHICH REARS ITS HEAD AND CONTRIBUTE TO HEALTH. A THIRD OF THE LATE-STAGE DRUG FAILURES. THAT’S A PRETTY BIG PROBLEM. HOW COME WE CANNOT PREDICT SOMETHING WILL BE TOXIC IN HUMANS BASED ON CELL ASSAYS. THERE’S ANOTHER ANGLE TO THIS PROBLEM OF PREDICTING TOXICITY AND IT’S SHOWN IN THIS OTHER BULLET DOWN BELOW. THERE’S ABOUT 80,000 CHEMICALS SO THOSE ARE USUALLY NOT DRUGS. THEY’RE OTHER KINDS OF CHEMICALS IN COMMERCE, WORLDWIDE AND MOST HAVE NOT TOXICOLOGICAL DATA. THERE’S NO AGENCY AROUND THE WORLD THAT DEMANDS SUCH TOXICOLOGY EVALUATION. AND THESE ARE MOLECULES TO WHICH WE’RE ALL EXPOSED BY TOUCHING PIECES OF PLASTIC, DRINKING WATER THROUGH PLASTIC BOTTLES, ETCETERA, ETCETERA. REVIEW HAVE BEEN CHARACTERIZED AND EXTENSIVE STUDIES ARE VERY EXPENSIVE. NOBODY WILL TEST THESE CHEMICALS AND PREPARE THESE PACKAGES, PERIOD. NO ONE FORCES INDUSTRY TO DO THAT. SO ALSO THERE’S PRESSURE TO MINIMIZE ANIMAL TESTING AND USE OF ANIMALS IN COSMETIC DEVELOPMENT, ETCETERA. SO THERE’S DRUG DISCOVERY ANGLE AND THERE’S ENVIRONMENTAL SAFETY AND PRIORITIZATION ANGLE. SO WOULDN’T IT BE NICE TO COME UP WITH A PANEL OF CELL-BASED RELATIVELY CHEAP THROUGHPUT ASSAYS TO REPRESENT MAJOR HALLMARKS OF TOXICITY, TEST THE CHEMICALS AND DRUG CANDIDATES AND ENVIRONMENTAL CHEMICALS IN THEM AND BUILD PREDICTIVE MODELS FOR HOW THE NEXT CHEMICAL WILL FARE AND WHETHER THEY’LL BE TOXIC IN HUME JON — HUMANS OR NOT AND YOU CAN PREDICT WHETHER CUR CANDIDATE WILL FAIL OR NOT. SO IT’S BASICALLY THE MOTHER OF ALL SYSTEMS BIOLOGY PROJECT. IF YOU THINK ABOUT IT, CAN YOU DE CONSTRUCT IT INTO PATHWAYS AND USING THAT IN VITRO CELL BASED BETA REBUILD THE HUMAN OR ANIMAL. PRETTY AMBITIOUS. WE STARTED THIS AS A PARTNERSHIP WITH SEVERAL GOVERNMENT AGENCIES WITH E.P.A. AND PART OF NIH IN NORTH CAROLINA AND LATER OTHERS JOINED THE PARTNERSHIP AND THIS FLOW CHART ON THE TOP SHOWS THE PROJECT IN A VERY BASIC NAIVE WAY. YOU HAVE TO DEVELOP A COLLECTION OF CHEMICALS. WHAT ARE YOU GOING TEST? YOU HAVE TO COME UP WITH THE ASSAYS REPRESENTING SOMETHING BAD THAT HAPPENS TO AN ORGANISM OR A CELL. YOU HAVE TO GENERATE VERY HIGH QUALITY DATA AND HOPEFULLY USING THAT DATA BUILD PREDICTIVE MODELS. INITIALLY, THOSE MODELS WILL PREDICT ASSAY LEVEL ACTIVITY AND LATER ON HOPEFULLY IF YOU HAVE ENOUGH PATHWAYS INTERROGATED, MAYBE YOU’LL BE ABLE TO REBUILD A HUMAN ONE DAY. SO WE BUILT ABOUT 10,000 CHEMICALS OF SOME PROVED AND SOME FAILED DRUGS AS WELL AS KNOWN CHEMICALS, TEPESTICIDES
AND A WIDE RANGE OF MOLECULES. IT WAS UNIQUE BECAUSE WE HAD TO ACTUALLY DEVELOP QUITE A FEW NEW PROCESSES FOR HANDLING THE MOLECULES AND THERE’S THE COMPOUND. BECAUSE THERE WERE SO MANY WEIRD MOL COULD IN THIS COLLECTION IT COULD NOT BE QC’D USING THE SAME STANDARD METHOD. AND YOU NEVER SEE THAT IN DATA SETS. IT WILL SHOW IT SEPARATELY. IN THIS CASE WE SHOW THE ACTIVITY OF THE COMPOUNDS AND THE QUALITY IS TESTED AND THEN THERE’S STORAGE TO BEGIN TO UNDERSTAND THE EFFECT OF STORAGE ON BIOACTIVITY. OKAY THE CELL ASSAY SIDE, WE RAN OVER SCREENING CAMPAIGNS. THE COLLECTION WAS 15 DOSES IN TRIPLE SHOWING DATA POINTS IN EACH SCREEN. SO WHAT WAS SCREENED ACTUALLY? LOW HANGING FRUIT,s TROE GEN RECEPTOR — ESTROGEN RECEPTOR ASSAYS AND RECOGNIZING THE ENDOCRINE RECEPTORS WERE BACK. WE RAN SEVERAL VERSIONS OF THE ESTROGEN RECEPTOR ASSAY. SEVERAL OTHER NUCLEAR HORMONE RECEPTOR ASSAYS, MITOCHONDRIAL MEMBRANE AND THE DNA DAMAGE RESPONSE PATHWAYS, ETCETERA. ALL IN THE PUBLIC DOMAIN AND AGAIN, I DON’T HAVE TIME TO GO INTO DETAIL BUT TO DATE WE’VE DEPOSITED ALMOST 600 MILLION DATA POINTS IN THE PUBLIC DOMAIN. SO WHAT, RIGHT? INTERESTINGLY, THE MIDDLE BULLET POINT IS THE CELL POINT. PEOPLE ALL OVER THE WORLD HAVE USED THE DATA TO BUILD PREDICTIVE MODELS USING MACHINE LEARNING AND SHARING THEIR MODELS AND AS A RESULT SOME MODELS ARE 90% ACCURATE IN PREDICTING WHETHER A NEW STRUCTURE WILL BE ACTIVE OR INACTIVE IN THE ESTROGEN RECEPTOR ASSAY. THE EPA IS USING THE ASSAY FOR REGULATORY PURPOSES AND WE’RE HOPING OTHERS WILL FOLLOW AND OTHERS LIKE PUBLIC PRIVATE PARTNERSHIPS IN THE EUROPEAN UNION AND BIG PHARMA USING THE DATA FOR BUILDING THEIR OWN MODELS. SO I’M GOING WRAP UP BY JUST SHOWING YOU THIS SLIDE YOU MAY FIND OF INTEREST WITH FURTHER READING. I GAVE A NUMBER OF EXAMPLES BUT THE OVERALL-THEME IS WE’RE TRYING TO IMPROVE THE PROCESSES. IF THEY DON’T RELATE TO YOUR WORK YOU PROBABLY WOULDN’T CARE SO THIS IS ACTUALLY WHERE WE PUT A LOT OF THE KNOW-HOW, THE GUIDANCE. WE CALL THIS ASSAY GUIDANCE MANUAL THAT STARTED ABOUT 13 YEARS AGO AS PARTNERSHIP WITH ELI LILLY THE BIG PHARMACEUTICAL COMPANY THAT RELEASED THE EARLY SCREENING IN PUBLIC DOMAIN AND WORKED WITH THEM OVER THE YEARS TO ESSENTIALLY ENHANCE THE CON ENT AND MAKE IT MORE USER FRIENDLY AND NOW WE’RE APPROACHING 1500 PRINTED PAGE EQUIVALENT OF THIS ELECTRONIC BOOK. IT’S COULD BE — CONSTANTLY REFRESHED AND UPDATED. VEE CHAPTERS ON ALL ASPECTS OF EARLY DRUG DISCOVERY STARTING FROM ASSAY DESIGN SOURCES OF ART ACTS, DATA ANALYSIS AND MOVING TOWARDS PHARMACO KINETICS AND DRUG ME METABOLISM, ETCETERA. ON THE RIGHT WE’RE DOING SOMETHING ELSE WHICH IS TO HAVE A SERIES OF WORK SHOPS WHERE THERE’S LECTURES ON THE VARIOUS TOPICS. IF YOU WANT TO WATCH THE VIDEOS THEY’RE ALL AVAILABLE AND IT SHOWS THE RETRIEVAL. THIS IS A PRETTY POPULAR RESOURCE FOR THOSE INTERESTED IN WORKING IN THIS SPACE, HOW TO DESIGN AN ASSAY THAT IS BETTER THAN JUST USING THIS OR IF YOU USE IT WHAT SHOULD YOU PAY ATTENTION TO. WHAT IS THE PRIME FACTOR AND STANDARD DEVIATION. WE’RE HOPING TO ADD CONTENT IN TERMS OF STEM CELL BASED SCREENING MODELS OR ACTUALY REGULATORY AFFAIRS, CLINICAL CONSIDERATIONS, ETCETERA. I’LL END WITH SOMETHING FORWARD LOOKING I SHOWED OUGHT — AUTOMATION AND ROBOTICS. I DIDN’T TALK ABOUT CHEMISTRY. VERY LITTLE HAS CHANGED IN THE CHEMISTRY LAB OVER THE PAST CENTURY. MAYBE PEOPLE ARE MORE COMPLIANT IN TERMS OF SAFETY GOGGLES AND GLOVES. CHEMICAL SYNTHESIS IS STILL A MANUAL OPERATION. WE’RE BEGINNING TO WORK IN THE SPACE YOU CAN READ OUR WHITE PAPER WE PUBLISHED LAST YEAR UTILIZING MACHINE LEARNING TO PREDICT OUTCOMES AND DESIGN THE ROUTES FOR CHEM SYNTHESIS AND TO VALIDATE AUTOMATION MODULES TO PHYSICALLY CONDUCT THE CHEMICAL REACTIONS SEPARATION AND STEPS IN AN AUTOMATED FASHION. CATALOG AS MANY REACTIONS AS WE CAN AND PUT IT IN THE PUBLIC DOMAIN AND ESSENTIALLY DEMYSTIFY ORGANIC SYNTHESIS. SO PEOPLE CAN USE THEIR BRAINS AND NOT SO HANDS SO HEAVILY IN THE AREA OF MET CAM. I’LL STOP HERE AND BE HAPPY TO ANSWER QUESTIONS. THANK YOU. [OFF MIC]>>WELL, IF A COMPANY INITIATES THE TRIAL THEY’RE NOT GOING ASK US AHEAD OF TIME WHAT’S GOOD AND WHAT’S NOT GOOD. REMEMBER, THERE’S TUMOR SEQUENCING FROM OTHER OTHER TYPES OF EXPERIMENTAL PLATFORMS TO GUIDE YOU TOWARDS A PARTICULAR COMBINATION. ONE CHALLENGE I DIDN’T GO OVER IN THE SPACE IS GOING OVER THE TUMOR TO FILL IT AND RUN THE TEST AND YOU CAN HAVE ENOUGH MATERIAL TO RUN THE COMBOS AND WITH SOLID TUMORS AND THE CELLS WILL LIKELY NOT DIVIDE. IT’S ACTUALLY PRETTY CHALLENGING WHERE YOU CAN’T JUST FAKE THE BYPASS AND SNAP YOUR FINGERS AND GROW THE CELLS AND COME UP WITH COMBOS. AS MORE AND MORE SOLID TUMORS BECOME DEMYSTIFIED AS IN PEOPLE FIND WAYS TO GET THE CELLS FROM THE SOLID MASS AND FIND WAYS TO GROW THEM AND THROUGH RNA SIC THEY RETAIN THE TUMOR SIGNATURES AND BECOME SOMETHING ELSE, IRRELEVANT, WE CAN ACCESS MORE AND MORE MODELS AND RUN MORE SCREENS AND ACTUALLY WE DO THIS WITH NCI INVESTIGATORS ALL THE TIME I WOULD SAY. IT’S HARD TO DO IT WITH SOMEBODY ELSE’S ORGANIZATIONAL TRIAL BECAUSE OF THE ISSUE OF PEOPLE WILL DO WHAT THEY WANT TO DO FOR THEIR OWN PURPOSES. SCALEABILITY ISSUES FROM BEING ABLE TO GROW UP EVERY TYPE OF TUM TUMOR IS WHAT’S COMING DOWN FIELD RIGHT NOW. FOR RARE DISEASES WE DON’T FOCUS ON SMALL MOLECULES BUT ALL THERAPIES INCLUDING GENE THERAPY. IT’S LATE STAGE AND DISEASES ARE SINGLE MUTATION BASED AND THEY’RE A PRIME CANDIDATE FOR GENE THERAPIES WHERE YOU CAN ACTUALLY CURE THE DISEASE. SO WE ARE DEALING WITH AN INTERESTING CASE WHERE WE’VE WORKED WITH A COMPANY IN THE SPACE OF RARE DISEASE PARTICULARLY THE AMINO ACID DEFICIENCY. WHERE PART IS INJECTING THE TLEEN THERAPY AND — GENE THERAPY AND VECTOR INTO THE SPECIFIC PART OF THE GRAIN OF VERY YOUNG CHILDREN. HOPEFULLY WE CAN CATCH THEM BY AGE OF 1 OR 2. SO IT JUST GOES THERE. IT’S A SINGLE INJECTION SPACE AND WORKS THERE AND THE RESULTS ARE PRETTY AMAZING. NORMALLY THE NATURAL HISTORY SHOWS THEY’LL BE DEAD BY THE AGE OF 8 AND FAIL ALL DEVELOPMENTAL MILESTONES. THEY CAN’T WALK, THEY CAN’T SPEAK. THEY’RE SORT OF IN A VEGETATIVE STATE. THOSE WHO GET TREATED WITH THE THERAPY ARE PRETTY MUCH LIKE NORMAL KIDS BY AGE OF 3. IT’S LIKE NIGHT AND DAY. THE SMALL SCALE WAS RAN IN TAIWAN. THE ORGANIZATIONAL CHALLENGE, WILL FDA ACCEPT THAT DATA. THAT’S WHERE WE DID THE HEAVY LIFTING PREPARING THE DATA ANALYSIS AND THE PACKAGE FOR THE FDA. THEY ACCEPT THE CLINICAL TRIAL FROM TAIWAN AND WE’RE PROCEEDING WITH THE APPLICATION TO QUALIFY A MANUFACTURER AND BEGIN THE CELL THERAPY TO DEPLOY IT. THERE’S NO REQUIREMENT FOR U.S.-BASED TRIALS WHICH SAVES AN ENORMOUS AMOUNT OF RESOURCES AND HOPEFULLY WILL GET TO A DEPLOYMENT SOON.>>ONE CAN SET UP COMPOUNDS THAT WILL FACILITATE THE SKIPPING. THIS DISEASES ACTUALLY ORIGINATE FROM SUCH A PROBLEM. IT JUST HAPPENS TO BE DIFFERENT PARTS BUT THE IDEA IS TO FIND THE COMPOUND THAT ALLOW THE SKIPPING OF THAT AND CONTINUE TO CULTURALLY GENERATE A GENE PRODUCT AND EVENTUALLY A PROTEIN THAT IS SEMI FUNCTIONAL. IT MAY HAVE A MUTATION THERE OR SOMETHING THAT IN SOME OF THESE DISEASES EVEN A 5% IMPROVEMENT IN TERMS OF GENE PRODUCT IS ENOUGH TO DRAMATICALLY ACTUALLY EFFECT THE NATURAL HISTORY. SO YOU DON’T NEED TO CORRECT 14 100% IN MANY CASE. 5% TO 10% MAY BE ENOUGH TO ACHIEVE SIGNIFICANT THERAPEUTIC BENEFIT. THANK YOU.

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