From protein folding to cognition: a serendipitous path of discovery

From protein folding to cognition: a serendipitous path of discovery


>>GOOD AFTERNOON, WELCOME TO THE NIH DIRECTOR WEDNESDAY AFTERNOON LECTURE SERIES. I’M THE CO-CHAIR OF THE PROTEOSTASIS SCIENTIFIC INTEREST GROUP WITH LEN ECERS. WE THE HOST FOR TODAY’S SPEAKER. IT’S MY PLEASURE TO INTRODUCE DR. PETER WALTER AN HHMI INVESTIGATOR AND PROFESSOR AND FORMER CHAIR OF THE DEPARTMENT OF BIOCHEMISTRY AND BIOPHYSICS AT THE UNIVERSITY OF CALIFORNIA SALVE. PETER WAS BORN IN WEST BERLIN, GERMANY AND HALF TOWARD HIS SUCCESS BEING IN THE 1960s WITH CURIOUS TINKERING WITH CHEMICALS IN HIS PARENTS’ DOG STORE AS A YOUNG BOY. CONTINUING ON THIS TRAJECTORY HERE, UNDER WHICH DEGREE FREE UNIVERSITY OF BERLIN IN CHEMISTRY, AND MASTERS DEGREE AT VANDERBILT UNIVERSITY. AND A Ph.D. IN BIOCHEMISTRY FROM ROCKEFELLAR UNIVERSITY. AS GRADUATE STUDENT HE EXPLORED HOW PROTEINS KNOW WHERE TO GO AND WHAT HE FOUND THE MOLECULAR MACHINERY KNOWN AS THE SIGNAL RECOGNITION — THAT BRINGS NASCENT LINEAR PROTEINS INTO THE FOLDING FACTORY OF THE ER, PROPELLED HIS ADVISOR TO 1999 NOBEL PRIZE IN PHYSIOLOGY OR MEDICINE. DR. WALTER MOVEED TO — FROM ROCKEFELLAR TO START HIS OWN LABORATORY AT UCSF WHERE HE EMBARKED ON HIS LIFE LONG JOURNEY OF CURIOSITY-DRIVEN RESEARCH WITH A BRAND NEW FOCUS SEARCHING FOR THE SIGNALS THAT TELL THE NUCLEUS WHERE PROTEIN FOLDING FACTORY, ER IS OVERLOADED. HIS GROUP IDENTIFIED IN ER RESIDENT TRANSPLANT BRAIN KINASE RIBONUCLEASE IN THE FOLDING CAPACITY OF THE ER LUMEN AND WHICH IS INITIATING A SIGNALING PATHWAY KNOWN AS AN FOLDING PROTEIN RESPONSE. THE CELLULAR FOR DISCOVERY OF UPR IN YEAST IS A SHANEING EXAMPLES HOW BAYING IS RESEARCH RELATE TO OUTCOMES WHICH ARE RELEVANT TO HUMANS. AND HUMAN DISEASE Z SINCE PROTEIN MISFOLDING SAL COMMON DENOMINATOR IN A VARIETY OF NOUN COMMUNICATABLE THE LASTER AWARD IN 2014 AND 2015. HE’S ALSO A MEMBER OF THE NATIONAL ACADEMY OF SCIENCES. OUTSIDE THE LAB, HE IS AN ACCOMPLISHED WOODWORKER AND HE IS A HIGHLY SUCCESSFUL MENTOR AND PROPONENT OF OPEN ACCESS PUBLISHING AND PAST PRESIDENT OF THE AMERICAN SOCIETY OF CELL BIOLOGY. LATELY-IDENTIFIES SMALL MOLECULES THAT MODULATE THE ACTIVITY OF EACH UPR RANGE IN MAMMALIANS IN THE HOPE THAT ONE DAY TWEAKK THE SYSTEM WILL HAVE PATIENCE. SO HE WILL DESCRIBE HIS UNCONVECTIONAL PATH TO DISCOVERY IN TODAY’S SEMINAR ENTITLED FROM PROTEIN FOLDING TO COGNITION, A SILENT PATH TO DISCOVERY. PLEASE JOIN ME TO WELCOME DR. WALTER TO NIH. [APPLAUSE] I ALSO WANT TO MENTION BEFORE RUNNING AWAY AT THE END OF THE SEMINAR, THERE WILL BE A RECEPTION SPONSORED BY FAES IN THE LIBRARY AFTER THE SEMINAR. IT WAS GREAT TO BE HERE AND BE INVITED TO GIVE THIS PRESTIGIOUS LECTURE. THANK YOU FOR SHOWING UP DESPITE THE FACT YOU CAN SEE IT FROM A SCREEN IN THE COMFORTED OF YOUR OFFICE. WHAT I WOULD LIKE TO DO TODAY IS TO TOLD YOU A STORY THAT IS REFLECTIVE OF THE FACT THAT DISCOVERY — THE PATH TO DISCOVERY IS NOT LINEAR, IT’S MEANERRING. THIS IS ONE OF THE WONDERFUL THINGS OF OUR CAREERS IS THAT WE GET CONSTANTLY EXPOSED TO NEW THINGS. AND FOR ME IT IS THIS ADVENTURE OF SUDDENLY ENDING UP WITH SOMETHING, YOU HAVE NO IDEA WHERE YOU — THAT YOU WOULD EVER GO THERE. THAT MAKES OUR PROFESSION SO REWARDING. AS YOU WILL SEE IN THIS TALK HERE WE ARE GOING TO GO ALL THE WAY FROM PROTEIN FOLDING TO THE PROCESS OF COGNITION. AND THAT PUTS US IN POSITIONS VERY OFTEN WE STUDY THINGS, WE HAVE NO IDEA YET ABOUT. AND WE HAVE TO LEARN NEW THINGS OR WE CAN TELL OTHERS IT’S NOT MY AREA SO MAYBE WE JUST PASS IT ON TO SOMEBODY ELSE. BUT I FIND THIS OPPORTUNIT TO CONSTANTLY LEARN NEW THINGS INCREDIBLE — INCREDIBLY REFRESHING. SO I WANT TO DO IS TELL YOU A LITTLE BIT ABOUT THE BASICS OF HOW WE STARTED AND YOU CAN FOLLOW, BRING YOU TO LATEST AS WE THINK ABOUT THIS PROCESS, BUT I WOULD ALSO LIKE TO PORTRAY TO YOU THIS PATH TO DISCOVERY AS WE GO ALONG AND HOW WE GOT WHERE WE ARE. I GAVE THIS TALK A SUBTITLE, THAT’S HOW I PHONE CALL CHANGE YOUR DAY. SO WE’RE IN THE MIDDLE OF THE TALK THIS WILL MAKE SENSE. SO THE STORY STARTS IN THE ENDOPLAIDS MIC RETICULUM WHICH IS THE COMPARTMENT WHERE PROTEINS THAT EVENTUALLY END UP ON THE CELL SURFACE BEGIN THEIR LIFE. THAT’S WHERE THEY GET FOLDED, FROM SUBUNITS, MODIFIED. THEN PACKAGED INTO VESICLES ON THE SECRETORY PATHWAY DISPLAYED AS MEMBRANE PROTEINS OR SECRETED PROTEINS ON THE CELL SURFACE. THIS MOVEMENT THROUGH THE SECRETORY PATHWAY IS A STEP OF QUALITY CONTROL FOR FIDELITY OF THE PROTEIN THE CELL IT IS PLAYS ON THE SURFACE TO COMMUNICATE WITH THE ENVIRONMENT. IT IS INCREDIBLY STRINGENT QUALITY CONTROL. SOME IMPORTANT SIGNALING MOLECULES IS THE RECEPTOR CYSTIC FIBROSIS, THE VAST MAJORITY OF THE POLYPEPTIDE CHAINS SYNTHESIZED NEVER LEAVES THE ENDOPLASMIC RETICULUM BUT NEVER MAKE TO CELL SURFACE BUT RATHER ARE DEGRADED. THIS IS ENORMOUS ENERGY EXPENSE FROM THE CELL BUT ILLUSTRATES BEAUTIFULLY THAT WHAT’S REALLY VALUED IS THE QUALITY, FIDELITY OF THE PROTEINS RATHER THAN CONSERVING ENERGY. THIS IS REFLECTED IN THE FACT IF YOU LOOK AT A CELL, PROFESSIONAL SECRETORY CELL IN THIS CASE THE PANCREATIC CELL IN THE BEAUTIFUL MICROGRAPH FROM GEORGE PALADES, THE CELL IS FILLED WALL TO WALL WITH THE COMPARTMENT THAT MAKES THIS FOLDING POSSIBLE, THE ENDOPLASMIC RETICULUM. THE CELL MAKES ITS OWN WEIGHT EVERY DAY IN SECRETORY PROTEINS SO THE SPECIALIZATION GOES HAND IN HAND WITH THE APPLICATION RESPONSIBLE FOR ASSEMBLING THEM. HOW DOES HE KNOW HOW MUCH ENDOPLASMIC RETICULUM IT NEEDS? YOU CAN THINK ABOUT THAT IN PRINCIPLE IN TWO WAYS. ONE WHICH IS THAT THE CELL IN DEVELOPMENT RECEIVES THE SIGNAL THAT TELLS US YOU’RE GOING TO BECOME A SECRETORY CELL AND THE CELL RESPONDS BY BUILDING THE APPROPRIATE MACHINERY TO DO SO. OR COULD BE CELL RECEIVES SIGNALS THAT INCREASES THE PRODUCTION OF SECRETORY PROTEINS, THEY END IN THE ENDOPLASMIC RETICULUM BUT REACH LIMIT OF PROTEIN FOLDING CAPACITY AND CREATES A FEEDBACK SIGNALNAL THAT TELL IT IS GENE EXPRESSION PROGRAM TO MAKE MORE ORGANELLE ACCORDING TO NEEDS OF CELL. THE UNFOLDEDDED PROTEIN RESPONSE IS SIGNALING OF THE CYTOKINE, WHERE WE HAVE COMMUNICATION FROM THE LUMINAL END OF ENDOPALACE IN THIS CASE RETICULUM TO THE GENE EXPRESSION PROGRAM THAT ADJUSTS ABUNDANCE OF THE ENDOPLASMIC RETICULUM. IT’S CALLED THE UNFOLDEDDED PROTEIN RESPONSE BECAUSE IT INITIATES BY OVERLOAD OF PROTEINS ENTERING THE ENDOPLASMIC RETICULUM, THESE PROTEINS THEN NOT BEING ABLE TO FOLD PROPERLY, UNFOLDEDDED MISFOLDED PROTEINS ACCUMULATE AND THEY ACT ADS AGONISTS FOR A SET OF RECEPTOR MOLECULES SENSORS THAT MONITOR CONDITIONS AND TRANSMIT THAT INFORMATION ACROSS THE BILAYER OF THE MEMBRANE. IN MET ZONE CELLS THERE’S THREE PRINCIPLE BRANCHES OF SIGNALING NETWORK. EACH OF THESE LEADS TO PRODUCTION OF CHARACTERISTIC TRANSCRIPTION ACTIVATORS THAT THEN ACTIVATE DOWNSTREAM UPL TARGET GENES. THESE TRANSCRIPTION FACTORS ARE PRODUCED BY ACTIVATION OF THE SENSORS BY VERY UNCONVENTIONAL SIGNALING PATHWAY PATHWAYS. IAE 1 USES A UNIQUE MECHANISM OF mRNA SPLICING WE DISCOVERED IN YEAST BUT ALSO THE PRINCIPLES ARE THROUGH MAMMALIAN CELLS ALSO. ATF 6 IS A TRANSCRIPTION FACTOR SIMILAR TO BONN AND GOLDSTEIN’S CHOLESTEROL BIOSYNTHESIS TRANSCRIPTION FACTOR SABB MADE AS TRANSMISSION PROTEIN AND WHEN UNFOLDEDDED PROTEINS ACCUMULATE IN THE ENDOPLASMIC RETICULUM IT IS MOVED BY MECHANISMS WE DON’T UNDERSTAND AT ALL CURRENTLY, THROUGH THE GOLGI APPARATUS WHERE IT MEETS UP WITH TWO PROTEASES THAT RELEASES FRAGMENT WHICH IS NOW SEVERED FROM THE MEMBRANE GOES TO NUCLEUS AND BE THE TRANSCRIPTION FACTOR, TO COLLABORATE WITH OTHER GUYSES AND FINALLY RIGHT IN THE MIDDLE IS A TRANSMEMBRANE KINASE THAT USES A MECHANISM OF TRANSLATIONAL CONTROL TO PRODUCE THIS TRANSCRIPTION FACTOR. THE GOAL OF ACTIVATING THESE PATHWAYS IS TO BRING THE SYSTEM WHICH HAS AN IMBALANCE BETWEEN LOAD AND CAPACITY OF THE FOLDING MACHINERY IN THE ER BACK INTO HOMEOSTASIS TO ADAPT. IF THIS CANNOT BE ACHIEVED, HOWEVER, THE CELL MAKES AN ACTIVE DECISION TO UNDERGO NETWORK CELL FATE. RATHER THAN PRODUCING POTENTIALLY MISFOLDING PROTEINS AND PUTTING ON THE CELL SURFACE, THE ENTIRE CELL DECIDES TO REMOVE ITSELF FROM ITS ORGANISM. IT’S THIS DECISION THEN THAT THE UNFOLDEDDED PROTEIN RESPONSE MAKES FOR CELLS, LIFE DEATH DECISION THAT PUTS IN THE CENTER OF MANY DIFFERENT HUMAN DISEASES. MOST INTUITIVE ONES FAMILIAL MUTATIONS RETINITIS PIGMENTOSA, THE RHODOPSIN MOLECULES CODED WITH MUTATION AND WHEN THESE PROTEINS ENTER THE ENDOPLASMIC RETICULUM, IT’S FRUSTRATED BY HAVING MISFOLDED PROTEINS IT CAN’T DO ANYTHING ABOUT AND THE CELL KILLS ITSELF. SO THE ENTIRE RETINA IN THIS INHERITED FORM OF BLINDNESS DEGENERATES BY APOPTOTIC CELL DEATH. SIMILARLY IN TYPE 2 DIABETES, BETTER CELLS IN PANCREAS ARE ASKED TO MAKE EVER INCREASING AMOUNTS OF INSULIN EXCEEDING THE SECRETORY PROTEIN FOLDING CAPACITY IN THE ENDOPLASMIC RETICULUM AND THE CELLS DIE APOPTOTIC CELL DEATH. ONE CELL DIES THE ONE NEXT DOOR IS ASKED TO MAKE MORE INSULIN AND BECOMES A DEGENERATIVE SPIRAL. SEEMINGLY CONTRADICTORY IN OTHER INSTANCES, THE UNFOLDEDDED PROTEIN RESPONSE CAN ALSO KEEP CELLS ALIVE THAT WOULD RATHER HAVE DIED. EXAMPLE EXAMPLES OF THAT IS TO USE THE PATHWAY TO ENVELOPE TO VIRUSES TO MAKE ENOUGH MEMORY PROTEINS FOR THAT. SO IF YOU CAN DEVELOP SOME PHARMACEUTICAL THAT WOULD BIAS THIS LIFE DEATH DECISION YOU COULD POTENTIALLY MAKE EFFECTIVE ANTIVIRALS FROM WHICH THE VIRUS COULDN’T MUTATE AWAY BECAUSE IT’S A CELLULAR FUNCTION THAT WE ARE AFFECTING HERE. THEN YOU HAVE CANCER, WE HAVE ALL CANCERS HAVE IN COMMON THE FACT THAT THEY EVOLVE TO THEN GROW AND — IN OUR BODY AND PLACES WHERE THEY SHOULD NOT GROW. THIS MUTATIONAL PROCESS THAT TAKES PLACE IN THEM, CAUSED AND LEADING TO INCREASING GENOMIC INSTABILITY LEADS TO MANY MUTATIONS THAT ACCUMULATE, NOT ONE DRIVER MUTATION BUT PASSENGER MUTATIONS EVOLUTIONIST DIRECTED SO THE PROTEOME IN THESE CELLS HAS MANY MISTAKES, THIS PAST RACE NOT JUST UNFOLDEDDED PROTEIN RESPONSE BUT PASS RATES ACTIVATED IN THESE CELLS AND PROVIDE PROTECTIVE MECHANISMS. AND WE ARE ENGAGED IN NICE COLLABORATION WITH GENERAL ANYONE TECH TO EXPLORE — GENENTECH TO EXPLORE PATHWAYS AND WHETHER WE CAN MANIPULATE THEM IN THE CANCER SETTING. AS I JUST TOLD YOU, THE UNFOLDEDDED PROTEIN RESPONSE CAN BE BOTH GOOD AND BAD AT THE SAME TIME. THE CHALLENGE REALLY IS FOR US TO FIGURE OUT AT THIS POINT WHETHER WE CAN COME UP WITH A THERAPEUTIC WINDOW THERE BY MANIPULATING DIFFERENT PATHWAYS WE CAN ACTUALLY DO MORE GOOD THAN HARM IN A CLINICAL SETTING. SO COUPLE OF YEARS AGO WE ENGAGED IN A PROJECT FUNDED BY HOWARD HUGHES MEDICAL INSTITUTE WHERE WE SCREEN FOR SMALL MOLECULES THAT WOULD THEN FUNCTION AS TOUR COMPOUNDS TO AFFECT DIFFERENT BRANCHES OF THE PATHWAY. I’M HAPPY TO REPORT WE HAVE FOR EACH OF THESE BRANCH WE HAVE BOTH INHIBITORS AND ACTIVATORS AT VARIOUS STAGES OF REFINEMENT SO THIS PROJECT IS WORKING QUITE NICELY. I WILL TELL YOU TODAY ABOUT PROGRESS WE MADE IN ONE OF THESE BRANCHES AND THAT IS THE PERK PATHWAY IN THE MIDDLE HERE. PERK USES A MECHANISM OF TRANSLATIONAL CONTROL IS THAT AFFECTS PRODUCTION OF TRANSCRIPTION FACTOR ATH 4. THE PROJECT I WILL TELL YOU ABOUT IS INITIATED AND CARRIED IN THE LAB BY SINGLE POST-DOCTORAL FELLOW. THE STEP THAT IS AFFECTED BY PERK SIGNALING IS A SIMPLE STEP IN PROTEIN TRANSLATION THAT IS OPERATING IN EVERY CELL AND LEADS TO TRANSLATION INITIATION AT PRETTY MUCH EVERY AGE IN THE TRANSCRIPTOME. THE EIF-2 IS A GENERAL TRANSCRIPTION FACTOR, TRIMERIC PROTEIN THAT EXISTS AS A GTPASE AND IS IN ACTIVE STATE IT’S BOUND TO GDP. THEN WHEN GDP IS EXCHANGED TO GTP IT’S ACTIVE AND BINDS TO INITIATED T RNA AND THEN INITIATES TRANSLATIONS AT ALL INITIATED OPEN READING FRAMES. AS FOR MANY GTPASES THIS REACTION IS NOT SPONTANEOUS BUT CATALYZED BY A GDP EXCHANGE FACTOR WHICH IS CALLED IEF 2B BUT SINCE — SUCH AN AWFUL KNOW NOMENCLATURE, JUST GOING TO REFER TO IT AS EXCHANGE FACTOR OR THE GEF OF THIS REACTION. THIS EXCHANGE IS REGULATED BY THE FACT THAT EIF-2 CAN BECOME PHOSPHORYLATED AT SINGLE SERIN RESIDUE AT THE ALPHA SUBUNIT AND THIS PHOSPHORYLATION EVENT MAKES IT STICK TIGHTLY TO EXCHANGE FACTOR. THE EXCHANGE FACTOR IS LIMITING IN CELLS AND IS TITRATED OVER HERE, EFFECTIVELY PHOSPHORYLATION THEN BLOCKS THIS EXCHANGE REACTION FROM HAPPENING. SO WE GOT INTO THIS STUDYING THE PERK PATHWAY. IT PHOSPHORYLATES AT THE SERIN RESIDUE HERE SENSING ER STRESS. BUT THERE ARE OTHER KINASES IN THIS CELL, PKR HRI THAT SENDS DIFFERENT STRESSES ACTIVATED BY DIFFERENT CONDITIONS. SUCH AS DOUBLE STRANDED RNA INDICATIVE OF VIRAL INFECTION ON THIS KINASE HERE, AMINO ACID STARVATION, NUTRIENT AVAILABILITY DCN 2, REDOX STATE INDICATED BY INDEFICIENCY HERE IN THIS KINASE. ALL THESE KINASES COME TOGETHER AND INTEGRATE THE FORMATION AT THE SINGLE SERIN RESIDUE HERE WHERE PHOSPHORYLATION TAKES PLACE, BECAUSE OF THIS INTEGRATION POINT THIS RESPONSE IS GOT — REFERRED TO AS INTEGRATED STRESS RESPONSE. SUBNETWORK OF THE UNFOLDEDDED PROTEIN RESPONSE IN THIS CONTEXT. THE CONSEQUENCES ARE AS I TOLD YOU, THE GENERAL DIVISION OF TRANSLATION BUT AGAIN SEEMINGLY PARADOXICALLY WE ALSO HAVE THE SELECTIVE TRANSLATION OF SOME mRNAs. INCLUDING OUR TRANSCRIPTION FACTOR ATF 4 THAT FUNCTIONS IN PRETTY MUCH EVERY CELL BUT ALSO SELECTIVE THAT ARTICULATE SPECIFICALLY EXPRESSED SUCH AS THIS PROTEIN OPHN 1 NEURONALLY EXPRESSED. HOW THIS COMES ABOUT WAS ELEGANTLY WORKED OUT BY IN THE YEAST SYSTEM INITIALLY. WHERE WE HAVE EIF 2, TRANSLATION IS UP IF NOT PHOSPHORYLATED BUT THERE ARE SOME mRNAs THAT GET TURN LITTLE UPSTREAM READING FRAMES AND WHEN THESE READING FRAMES ARE TRANSLATED THE RIBOSOME GAUZEN’T MAKE DOWN TO AUG SO ATF 4 IS NOT PRODUCED. BUT WE’RE NOW PHOSPHORYLATION SETS IN, TRANSLATION GENERALLY IS BLOCKED BUT NOW RIBOSOMES ARE ALLOWED TO ACCESS TO THIS AUG RESIDUE DOWNSTREAM OF THIS OPEN READING FRAME AND ATF 4 IS PRODUCED UNDER CONDITIONS OF LIMITED INITIATION CAPACITY IN THE CELL. SO WHAT WE DID VERY SIMPLY LOOK FOR SMALL MOLECULE, BEREPLACE THIS LUCIFERASE READING FRAME AND WE ADDED A REPORTER CELL LINE AND SCREENED A SMALL MOLECULE LIBRARY. WE INDUCE ER STRESS SO NOW LUCIFERASE IS PRODUCED UNDER THESE CONDITIONS. LIGHT GOES ON, YOU ADD VARIOUS COMPOUNDS, THE LIGHT GOES OFF AND YOU HAVE YOUR INHIBITOR. THAT SOUNDS VERY EASY TOOK US A COUPLE OF YEARS TO DO THAT AND IN THE END WE HAD SORTED THROUGH HUNDRED THOUSAND COMPOUNDS SORTED THEM AND SECONDARY SCREENS THAT THEY’RE ONLY P HITTING THE PERK BRANCH OF THE UNFOLDEDDED PROTEIN RESPONSE THAT THEY ARE AFFECTING ENDOGENOUS PROTEINS, NOT JUST REPORTER PROTEINS. SO FORTH. IN THE END WE ENDED UP WITH ONE COMPOUND. AND OUR SCREENING FACILITY DECIDED TO THROW IT AWAY. IT DIDN’T MEET TRIAGE CRITERIA BECAUSE IT WASN’T SORTED ENOUGH FOR THEM TO THINK IT WOULD AMOUNT TO ANYTHING. THIS BEAUTIFUL LITTLE COMPOUND, WE CALL IT ISRIB. EVERYTHING ELSE I WILL TELL YOU FROM NOW ON IS BASICALLY DUE TO THE FACT THAT CAMELL,A SADROWSKI DECIDED TO PULL THE COMPOUND OUT OF THE GARBAGE CAN AND SAY I WANT TO STUDY ANYWAY. SO SHE DECIDED TO IGNORE THE ADVICE OF THE EXPERTS AND TELL LATER WHY THIS IS THE CASE. SO NEVER EVER THINK OUTSIDE THE BOX. REMINISCENT OF A FEW GRANT REVIEWS YOU GOT BACK YOURSELF. SO THE COMPOUND SINCE I HAVE STARTED MY CAREER IN CHEMISTRY IS A BEAUTIFUL COMPOUND, THERE’S BEAUTY IN SYMMETRY, TWO FOLD SYMMETRIC THESE TWO ARMS ARE THE SAME. IN THE MIDDLE YOU HAVE THIS CYCLOHEXANE RING WHICH CAN BE SUBSTITUTED THROUGH VARIOUS ON TRANS, THE TRANSCOMPOUND IS ACTIVE, THE SIS COMPOUND IS COMPLETELY INACTIVE SO WE HAVE VIRTUALLY CHEMICAL MATTER THAT PROVIDES NICE CONTROL. WE HAVE MADE NUMEROUS MUTATIONS SOME 200 DERIVATIVES OF THIS MOLECULE, AND WE HAVE PRETTY MUCH ONLY MADE IT WORSE. SO RIGHT OUT OF THE SCREEN IT SEEMED TO BE QUITE NICE YOU CAN MAKE SOME SUBSTITUTIONS HERE AT THIS RING THAT MAKE IT A LITTLE BIT BETTER. AND THEN IF YOU DO THE SAME ON THE OTHER RING IT MAKES IT A LITTLE BETTER STILL. IF WE MAKE SOME SUBSTITUTION IN THIS RING THAT MAKE IT WORK WORSE WE DO THE SAME ON THE OTHER RING IT MAKES IT WORSE STILL. SO IN OTHER WORDS, THIS LITTLE KEPT POUND SEEM TO WANT TO TELL US I WANT TO BE SYMMETRIC. THAT’S GOING TO COME THROUGH THE REST OF THE TALK. HERE IS WHAT IT DOES. YOU TAKE CELLS, YOU LABEL THEM AS S — ME THIONEINE, WHEN PROTEINS ON THE GEL, YOU GET NICE SYNTHESIS, NO ATF FORCE BEING PRODUCED. WE INDUCE ONE KINASE SHOWN FOR THE REST OF THE PERK KINASE, THERE’S PHOTO SYNTHESIS SEVERELY DOWN-REGULATED AND NOW ATF-4 IS BEING PRODUCED. YOU ADD THE ISRIB TO THE CELLS AND YOU SEE THAT NOW IT’S THE AFFECT OF THE STRESS IS ENTIRELY REVERSED. SO IT’S A VERY EFFECTIVE WAY. THERE ARE DIFFERENT WAYS YOU CAN READ OUT INHIBITION OF TRANSLATION INITIATION. ANOTHER WAY OF DOING IT IS TO LOOK AT THE mRNAs THAT WHEN THEY’RE COVERED WITH RIBOSOMES AND TRANSLATING ACTIVELY, THEY ARE PREVENTED FROM P. CXFCR. CXFCN. CXFC BINDING PROTEINS ONCE INITIATION IS INHIBITED RNA BINDING PROTEINS HELP UNDER THEM AND LOW COMPLEXITY SEQUENCES GET DRAGGED INTO THE RNA GRANULES STRESS GRANULES WHICH YOU CAN FOLLOW BY FLUORESCENCE. YOU SEE HERE YOU HAVE CELLS IN WHICH WE INDUCE ER STRESS, YOU WILL SEE STRESS GRANULES FORM, IF WE DO THE SAME IN THE PRESENCE OF THE ISRIB YOU WILL SEE THAT THE STRESS GRANULE FORMATION IS ENTIRELY PREVENTED. THIS IS — YOU CAN PREFORM THE STRESS GRANULES WE CAN THEN ADD THE COMPOUND AND WITHIN TWO MINUTES THEY ARE GONE. SO THESE GRANULES ARE REVERSIBLE STORAGE FORMS IN WHICH THE MRNA IS PUT ASIDE FOR A WHILE AND FROM WHICH IT CAN BE RETRIEVED INCREDIBLY FAST ONCE PROTEIN SYNTHESIS IS GOING AGAIN. SO LONG STORY SHORT, THIS IS CELL BASED SCREEN, WE HAVE NO IDEA WHERE ON THE PATHWAY DOES OUR COMPOUND ACT. WE ARE MOST INTERESTED IN THE UNFOLDEDDED PROTEIN RESPONSE. TURNS OUT THAT IT DOESN’T AFFECT PERK ACTIVITY, PERK STILL AUTOPHOSPHORYLATES AND IL 2 IS STILL PHOSPHORYLATED. SO THESE TAKE PLACE, BLOCKS TRANSLATION DOWNSTREAM, ALL THESE DIFFERENT KINASES THAT WE HAVE TESTED. AND IT REVERSES ALL THE PHOSPHORYLATION INDUCED TRANSLATIONAL CHANGE THAT WE CAN MEASURE WHICH WE TRIED TO DO CAREFULLY WITH NICK AND RIBOSOME FOOTPRINTING SO HE’S ONE OF THE INVENTORS OF THIS TECHNIQUE AND JONATHAN WISEMAN’S LAB AT UCSF. U SHOWED YOU IT REVERSES STRESS GRANULE FORMATION. SO ISRIB BLOCKS DOWNSTREAM. OF THIS PHOSPHORYLATION EVENT. IT MAKES CELLS IMMUNE TO THIS PHOSPHORYLATION. IT AS IF IT NEVER HAPPENED. IT IS A REMARKABLE COMPOUND. RIGHT OUT OF THE SCREEN WE GOT IN CELL EFFICACY OF FIVE NANOMOLAR. NORMALLY WHEN YOU DO THESE THINGS YOU’RE HAPPY IF YOU END IN THE MICROMOLAR RANGE AND IT TAKES CHEMISTRY TO REFINE THE AFFINITIES AND MAKE IT BETTER. THIS STUFF WORKED FIVE NANOMOLAR. WHICH EXPLAINS WHY THE SCREENING FACILITY WAS TRIAGING AGAINST IT BECAUSE WE CANNOT WORK VERY FAR LIMITED SOLUBILITY BUT NANOMOLAR YOU DON’T NEED MUCH IN SOLUTION. IT WAS PERFECTLY FINE. WE MADE IT TENFOLD BETTER BUT AS I TOLD YOU PUTTING IT SUBSTITUTIONS ON THE SIDE WINGS HERE SO THE HIGH PICA MOLAR RANGE NOW. RIGHT OUT OF THE SCREEN IT HAS GOOD-BYE AVAILABILITY IF WE INJECT INTO THE — HALF TIME IN CIRCULATION IS ABOUT EIGHT HOURS. IT HAS GOOD BLOOD BRAIN PENETRANTS, AGAIN, PROPERTY NORMALLY NEED TO WORK ON WHEREAS IT WAS JUST GIVEN TO US FROM THE PRIMARY SCREEN. IT HAS — IT IS ONLY AVAILABLE IN THE ANIMALS AND MOST SURPRISINGLY IT SHOWED NO — TO DATE HAS SHOWN NO OVERT TOXICITY, WHICH IS VERY SURPRISING SINCE THE PATHWAY EXISTS IN EVERY CELL IN OUR BODIES. SO WE HAVE INJECTED MICE FOR WEEKS AND SEEN NO ADVERSE EFFECTS. SO WHAT DOES IT BIND TO? ALL THE SEXY TECHNIQUES WE TEACH TO OUR STUDENTS PUTTING BIOTIN HANDLES ON CROSS LINKING REACTIONS CLICK CHEMISTRY, SO ON SO FORTH, ONLY PUT OUT CRAP THAT NOT ALLOW US TO IDENTIFY TARGET OF THIS. THE ONLY CLUE WE HAVE THAT THE STRUCTURE ACTIVITY ANALYSIS INDICATED SOME IMPORTANCE OF SOME TWO FOLD SYMMETRY. SO WHAT IS THE MOLECULAR TARGET? THE FIRST CLUE CAME FROM AN EXPERT, EVERYTHING ELSE HAVING FAILED THAT WE DID TOGETHER IN COLLABORATION WITH JONATHAN WISEMAN’S LAB. WE DID SH RNA SCREEN TO BASEICALLY ASK WHICH GENE IN THIS CELL WHEN KNOCKED DOWN WILL MAKE CELLS MORE OR LESS SENSITIVE TO THE ACTION OF THE DRUG? THIS SCREEN IDENTIFIED TWO SUBUNITS OF THE EXCHANGE FACTOR THAT IMPAIRED THIS REACTION. THEN AT THE SAME TIME TWO PAPERS APPEARED THAT SHOWED IN CELLS THIS EXCHANGE FACTOR IS NOT JUST THE HEADER PENTAMER BUT EXISTS IN EQUALIBRIUM AS A DEKRAMERRIC FORM. SO A DIMER OF THE PENTAMERS. THE QUESTION WHENEVER YOU FORM A DIMER OF SOMETHING YOU BUILD — YOU HAVE THE OPPORTNITY OF FORMING A TWO FOLD SYMMETRIC INTERFACE. WHAT THIS SUGGESTED THEN IS A MODEL MAYBE HERE IN THE MIDDLE AND THEN POSES EQUILIBRIUM FROM THE PENTAMERRIC FORM TO THE DECAMERIC FORM. SO JORDAN SIGH IN THE LAB TESTED THE DIRECTORY BY PUTTING CELL EXTRACTS TREATED OR NOT TREATED WITH THE DRUG ON SUCROSE GRADIANTS AND SIZE OF THE COMPLEX. I THINK IN VITRO HERE. HERE IS WHERE THE PENTAMER IN THE GRADIANTS FILL MENNATION FROM LEFT TO RIGHT. THESE ARE CONTROLLED PROTEINS ON THE GRADIANTS. IF WE MAKE A CELL EXTRACT FROM CELLS TREATED WITH DRUG YOU SEE THE SUBUNITS OF THE EXCHANGE FACTOR SHIFT IN THE GRADIANT INTO THE POSITION OF DECAMER WHERE THE OTHER PROTEINS STAY PUT ON THE GRADIANT. I THINK THE EXPERIMENT BEAUTIFULLY ILLUSTRATES PENTAMER DECAMER CONVERSION DUE TO ACTION OF THIS DRUG. THIS WAS A BIAS EXPERIMENT, WE ONLY SEE THE BANDS WE WANT TO SEE BY PROBING ANTIBODY ON THE WESTERN BLOT SO WE DID THE SAME EXPERIMENT BUT BY MASS SPEC PROFILED THROUGH THE PEAK OF THE DECAMER AND CORRELATED ALL THE PROTEINS WE IDENTIFY WITH ONE ANOTHER AND HERE AT THE TOP OF CORRELATION STAND OUT ALL THE SUBUNITS OF THE EXCHANGE FACTOR. THIS MEAN THERE’S NO OTHER PROTEIN THAT HOPS ON TO THIS COMPLEX OR PROVIDES DECAMERIIZATION, BUT MOST LIKELY PENTAMERRIC FORM. TO SUMMARIZE THE ENTAMO LOGIC WORK TAKING THE ISRIB AND ADDING IT INCREASESES SPECIFIC ACTIVITY EXCHANGE ACTIVITY OF THIS ENZYME BY THREE TO FIVE FOLD SO ENZYME ENZYMATIC ACTIVATOR OF THIS ACTIVITY AND IT LESSENS BUT NOT DOES NOT BLOCK COMPLETELY INHIBITION BY LIMITING AMOUNTS OF PHOSPHORYLATED FORM OF THE F 2B. THINK WHAT THIS SAY, IF YOU ADD ENOUGH INHIKINGTOR YOU CAN STILL BOCK EXCHANGE ACTIVITY ENTIRELY. I THINK WHAT THIS MAY TELL US IS IT PROVIDES A NICE EXPLANATION WHY THIS COMPOUND IS NOT TOXIC IN CELLS, THAT AT HIGH CONCENTRATIONS OF THE INHIBITOR, WHEN UNFOLDEDDED PROTEIN RESPONSE IS FULLY TURNED ON THIS PATHWAY STILL WORKS AND PROVIDE PROTECTIVE EFFECTS OF UNFOLDEDDED PROTEIN RESPONSE BUT WHAT WE HAVE IS NOW A DIFFERENT DOSE RESPONSE CURVE PROBABLY DUE TO DIMERIZATION WHERE YOU CAN PUT IN ALLOSTERIC REGULATION, NOT JUST NEEDING ONE BUT NOW TWO INHIBITORS SO CHANGE IN HYPERBOLLIC INHIBITION CONDITION SIGMOIDAL CURVE AND ALLOWS CELLS TO REGULATE SOMETHING IN THE MIDDLE BUT KEEP THE END TO PROTECTIVE EFFECTS INTACT AT THE END. SO WE ARE PRETTY SURE THAT ERF-2 EXCHANGE FACTOR IS THE TARGET OF THE DAC AND THAT WAS INDEPENDENTLY VALIDATED BY OUR SOMETIMES COLLABORATOR, SOMETIMES COMPETITOR, DAVID RYAN WHO IDENTIFIED SOMATIC CELL MUTATIONS THAT MAKE CELLS INSENSITIVE TO THE ACTIONS OF ISRIB. AND THESE MUTATIONS MAPPED INTO ONE OF THE SUBUNITS OF THE EXCHANGE FACTOR. SEEING IS BELIEVING, TWO FANTASTIC GRADUATE STUDENTS IN MY LAB SET OUT AND PURIFIED THE EXCHANGE FACTOR AND ERF 2 WHICH WE CAN NOW HAVE RECOMBINANTLY IN LARGE AMOUNTS. JUST TO GIVE AN EXAMPLE, IF WE PULL OUT THE EXCHANGE FACTOR FROM HELO CELLS YOU GET FIVE NANOGRAMS FROM A LITER OF CELL CULTURE, THIS IS RECOMBINANT SYSTEM IN E. COLI YOU GET MILLIGRAMS SO IT WAS NECESSARY TO PRODUCE THESE PROTEINS, IT WAS VERY DIFFICULT. AND THE TRICK WAS WE HAVE TO EXPRESS ALL FIVE SUBUNITS IN THE SAME CELL IN ORDER TO GET THE PARTICLE ASSEMBLED. IF WE TRIED TO EXPRESS SEPARATELY WE COULD NEVER PUT IT TOGETHER. WITH THAT MATERIAL IN HAND WE COLLABORATED WITH ADAM FROST AND WE HAVE BY CRYO-EM NOW A STRUCTURE, FIRST STRUCTURE OF A HUMAN ERF 2B EXCHANGE FACTOR AND YOU CAN SEE IN THE SHAPE HERE LOOKS VERY MUCH LIKE A CRYSTAL STRUCTURE DETERMINED IN YEAST. THIS IS WHAT THE STRUCTURE LOOKS LIKE, IN HIGH RESOLUTION NOW. 3.4-ANGSTROM. THERE IN THE MIDDLE IS OUR DRUG. THIS IS PHOTO TOE SHOPPED. THE TECHNIQUE IS AMAZING WHAT YOU CAN DO. DENSITY FITS PERFECTLY, YOU CAN CHASE THE TRAIN SPACE ON THE ANALOGY TO THE STRUCTURE SO WE NOW HAVE A VERY NICE MOLECULAR MODEL OFF BINDING SITE OF THE DRUG SMACK IN THE CENTER BRIDGING TWO HETERO PENTAMERS LIKE WE HAVE DRAWN BEFORE. AND THIS RESIDUE INDICATED HERE OTHER RESIDUES THAT WERE MUTATIONS MAP THAT MAKE THIS ERF 2 INSENSITIVE. SO THIS IS ALL THIS IS BASICALLY TO TELL YOU YOU KNOW A LOT OF HOW THIS WORKS, VERY HIGH AFFINITY, BINDS IN THE MIDDLE AND DOTS CONNECT BEAUTIFULLY. SO SOME DOING THIS WORK, I CALLED UP A COLLEAGUE OF MINE AT MCGILL AND THE PURPOSE OF THE PHONE CALL WAS BASICALLY TO ASK HIM IF HE’S BEEN STUDYING TRANSLATION INITIATION FOR HIS CAREER. IF THERE’S ANYTHING IN THE FREEZERS THAT WOULD BE USEFUL FOR US FOR REAGENTS. HE OFFERED EVERYTHING HE HAD BUT DIDN’T HAVE MUCH SO THIS WAS IN PREVIOUS WORK. BUT WHAT HE POINTED US TO IS RESULTS FROM HIS LAB WHICH WE HAD LINKED THIS VERY PATHWAY HERE INTEGRATED STRESS RESPONSE THROUGH THE MEMORY CONSOLIDATION. THIS IS AN OLD CONCEPT THAT WENT ALL THE WAY BACK TO LEARNING IN APPELLEE SHAH REQUIRES ONGOING PHOTO SYNTHESIS AND THEN AFFECTS SYNAPTIC PLASTICITY. AND RESULTS FROM HIS LAB EXPERIMENTS WERE THAT IF YOU TAKE A HETEROZYGOUS MOUSE IN WHICH THE SINGLE PHOSPHORYLATION SITE IN ELF 2 IS CHANGED FROM SERENE TO ALANINE SO NO LONGER PHOSPHORYLATED, THEN THE MICE LEARN BETTER. THE HOMOZYGOUS MOUSE IS DEAD. DOESN’T DEVELOP BECAUSE OF PANCREATIC FAILURE. THE HETEROZYGOUS MOUSE ONLY MAKING HALF AS MUCH AS PHOSPHORYLATED FORM EIF 2 IS PERFECTLY FINE AND LEARNS SIGNIFICANTLY BETTER. SO WE SEND SOME OF OUR DRUG WHICH PROTECTS THE VERY SAME PATHWAY THAT MUTATIONAL GENETIC ANALYSIS PIN DOWN AND HE PUT IT INTO THAT BEHAVIORAL ASSETS. THE IMPORTANT THING HERE IS AT THIS POINT IN THE STORY WE DIDN’T KNOW THAT THIS COMPOUND HAD ANY BIOAVAILABILITY. OR THAT IT WOULD CROSS THE BLOOD BRAIN BARRIER. ADVENTUROUS ENOUGH TO DO THE EXPERIMENT ANYWAY. HERE THE RESULTS ARE AMAZING. WE TESTED IN THIS MORRIS WATER MAZE WHERE YOU BASICALLY DEMOLISH — THE MOUSE IS PUT INTO MURKY SOLUTION TO FIND THIS HIDDEN PLATFORM, IT ORIENTS ITSELF BY VISUAL CLUES AROUND THE ROOM VERY MUCH THE SAME WE WOULD ORIENT OURSELVES IN THE BUILDING. THEN YOU JUST MEASURE TIME IT TAKESES TO FIND HIDDEN PLATFORM. THEY TRAIN THE MOUSE LIGHTLY ENOUGH THAT WE STILL HAVE ROOM FOR IMPROVEMENT UNDER THE CURVE. THE PLATEAU AT THIS PERFORMANCE LEVEL BUT IF THEY HAVE BEEN INJECTED WITH THE DRUG THEY LEARN SUBSTANTIALLY BETTER. THESE PARALLEL FROM THE GENETIC HOMOZYGOUS COMPARED TO WILD TYPE. WE CAN USE DIFFERENT ASSAYS SUCH AS CONTEXTUAL LEARNING WHERE THE MOUSE ASSOCIATES THIS PARTICULAR ENVIRONMENT WITH ELECTRIC FOOT SHOCK AND NEXT TIME IT ENCOUNTERS THIS ENVIRONMENT IT STARTS FREEZING AND YOU MEASURE THE FREEZING ABILITY OF THE MOUSE. HAVING THE DRUG INJECTED MAKES NO DIFFERENCE SHORT TERM, SHORT TERM MEMORY BUT AFTER 24 HOURS MEMORIES ARE MOVED FROM SHORT TERM MEMORY INTO LONG TERM STORAGE, YOU SEE A SIGNIFICANT DIFFERENCE. IN THIS CASE THE DRUG WAS INJECTED AFTER THE TRAINING IT’S NOT PERCEPTION BUT SHORT TERM MEMORY INTO LONG TERM MEMORY. AND THE SAME IN AUDITORY FIELD LEARNING AND RATS IN THIS CASE THE ANIMAL ASSOCIATES A NASTY SOUND WITH THE FOOT SHOCK AGAIN, NO DIFFERENCE BETWEEN DRUG TREATED ANIMALS OR CONTROL ANIMALS IN SHORT TERM REGIME BUT AFTER 24 HOURS ANIMALS INJECTED LEARN SIGNIFICANTLY BETTER. SO WHAT WE HAVE DONE HERE THEN IS WE HAVE INJECTED THIS COMPOUND INTO A WILD TYPE ANIMAL AND WE HAVE IMPROVED THEIR PERFORMANCE IN THESE ASSAYS. THIS TELLS US THE STRESS RESPONSE VIA PHOSPHORYLATION EVENT MUST INHERENTLY LIMIT MEMORY CONSOLIDATION SO BREAK WITH THE COMPOUND OR GENETIC MANIPULATION WE CAN REALLY SET BREAK. SO THERE’S EXTRA CAPACITY TO BE TACKED — TAPPED INTO. I FIND THAT VERY FASCINATING. IT BASICALLY MEANS IF THE SOLUTION HASN’T OPTIMIZED THE PROCESS BUT RATHER PROBABLY GOOD REASONS WE CAN COME BACK TO IN DISCUSSION, BUT BASICALLY A RESERVOIR IN WHICH WE CAN TAP INTO. HOW COULD THAT BE THE CASE? Y’ALL KNOW THAT MEMORY CONSOLIDATION AND LEARNING IS ASSOCIATED WITH CHANGING THE STRENGTHS OF SYNAPTIC TRANSMISSION, HAVING A POST SYNAPTIC GLUTAMATE GATED CHANNELS THE AND THAT RECEPTORS. THE STRINGS CAN CHANGE TWO DIFFERENT WAYS YOU CAN HAVE LONG TERM POTENTIATION WHICH STRENGTHENS THE SYNAPSE BY INCREASING NUMBER OF AM PA RECEPTORS OR WE CAN HAVE THE REVERSE, LONG TERM DEPRESSION WHICH WEAKENS THE SYNAPSE BY DECREASING CONCENTRATION. OF AMPA RECEPTORS. WE COLLABORATED WITH (INDISCERNIBLE) AT BAILOR AND THAT’S THE POST-DOC WHO BROUGHT THESE BEHAVIORAL STUDIES INTO MALCOLM ZONENBERG’S LAB AND NOW HAS HIS OWN AT BAILOR. HE HAS FOUND IN THE GENETIC MODEL HE DEVELOPED, IT IS THE PROCESS OF LONG TERM DEPRESSION THAT IS BEING AFFECTED BY THIS PHOSPHORYLATION EVENT. WHAT YOU’RE SEEING HERE IS A SYNAPSE STIMULATED AND REPORTED FROM RECORDED FROM WHEN YOU TREAT TO INDUCE THIS PROCESS OF LONG TERM DEPRESSION, YOU WILL SEE THAT CURRENTS GO DOWN AND DON’T RECOVER TO THE SAME LEVEL DUE TO THE FACT CONCENTRATION OF AMPA RECEPTORS IS REDUCED NOW. THIS IS WHAT HAPPENS, THIS IS CONTROL, THIS IS WHAT HAPPENED IN WILD TYPE ANIMALS. IF YOU DO THE SAME IN THE HETEROZYGOUS ANIMAL YOU SEE THIS WEAKENING OF THE SYNAPTIC CONNECTION IS ENTIRELY BLOCKED. SO IN OTHER WORDS, THE SYNAPSE STAYS STRONGER. YOU CAN DO THE SAME AS A DRUG, IN THIS WORK YOU GET PARALLEL RESULTS. SO AGAIN, LONG TERM DEPRESSION, IS BLOCKED UNDER THESE CONDITIONS. WE CAN LOOK AT IMMUNOSTAINING OF THE SYNAPSES. SO YOU START AT ONE HERE YOU THEN WILD TYPE ANIMAL, YOU PHARMACOLOGICALLY INDUCE LONG TERM DEPRESSION. THE REMOVAL OF AMPA RECEPTORS BY ENDOCYTOSIS. YOU HAVE ABOUT 40% LEFT AFTER YOU’RE DONE WITH THIS. IF YOU DO THE SAME IN THE HETEROZYGOUS ANIMAL THIS REDUCTION OF AMPA RECEPTORS DOES NOT TAKE PLACE. YOU CAN DO THE SAME AS OUR DRUGS, START OUT AT ONE, INDUCE LONG TERM DEPRESSION, GO DOWN TO 40%. YOU DO IT IN THE PRESENCE OF THE DRUG, NO REDUCTION TAKES PLACE, YOU DO IT WITH INACTIVE ANALOG OF THE DRUG, NOTHING HAPPENS. SO WHAT WE HAVE HERE IS WE HAVE A PERFECT MATCH BETWEEN THE GENETICS AND THE PHARMACOLOGY OF THIS COMPOUND WHICH GIVES US IN GREAT CONFIDENCE THAT THIS STUFF IS ACTUALLY ACTING ON THE TARGET THAT WE ASSUME. SO NOW WE WENT ONE STEP FURTHER AND HE IDENTIFIED IN NEURONS, THIS mRNA ENCODING THE PROTEIN OPHN 1 WHICH IS NEURONALLY EXPRESSED. AND IS NOW TRANSLATIONALLY UPREGULATED IN THE PROCESS OF LONG TERM DEPRESSION AND WAS PREVIOUSLY IMPLIED TO BE INVOLVED IN THE PROCESS OF AMPA RECEPTOR DOWN-REGULATION BY ENDOCYTIC EVENT. SO ROGUE GDPASE MODULATOR. THIS mRNA CONTAINS THE SAME READING FRAMES AS THE ATF-4 MRNA SO UNDER IDENTICAL SIMILAR TRANSLATIONAL CONTROL AS WE KNOW FOR OUR TRANSCRIPTION FACTOR. IN THE UNFOLDEDDED PROTEIN RESPONSE. HE’S GONE — UNFOLDED H RNA SHOWING YOU CAN BLOCK THIS REACTION DOWN REGULATION OF THE RECEPTORS ENTIRELY. SO WHAT WE THINK THEN IS THAT P THAT THE ACTION OF THE DRUG IS BY PREVENTING THESE ACTIONS DOWNSTREAM. SO IT’S A REASONABLE WORKING HYPOTHESIS I THINK THAT ALLOWS US TO CONNECT THE DOTS. WHAT DO WE DO WITH THIS? I’M — I WAS REALLY AMAZED HOW QUICKLY ONE CAN SLIP FROM CURIOSITY-DRIVEN BASIC RESEARCH INTO ISSUES THAT ARE ACTUALLY ETHICALLY QUITE COMPLICATED. WE HAVE IDENTIFIED A COGNITIVE ENHANCER AND WE DON’T WANT IT TO FALL INTO THE HANDS OF THE TIGER MOMS WHO GIVE IT TO THEIR DAUGHTERS THE DAY BEFORE EXAM SO THEY CAN PERFORM BETTER. UNFOLDED UNFOLDED — MEANINGFUL CLINICAL APPLICATIONS. SO THE TASTE KIDS LOVE, CAN WE PLEASE HAVE SOME MORE. THE MOST IMMEDIATE CLINICAL CONNECTION IS PROBABLY A DISEASE CALLED (INDISCERNIBLE) WHITE MATTER DISEASE IN WHICH IT’S AUTOSOMAL RECESSIVE SO THE CHILDREN INHERIT PARTIALLY DEFECTIVE ALLELES FROM BOTH PARENTS. AND THEN IT LEADS TO SEVERE NEUROLOGICAL DELINEATION AND COGNITIVE IMPAIRMENT. 95% IMPAIRMENT IN THIS DISEASE HAVE MUTATIONS IN THE TRANSLATION FACTOR. THESE SMART OVER SUBUNITS OF EXCHANGE FACTOR SO MOST LIKELY HYPOMORPHIC AND REDUCE THE ACTIVITY OF THIS ENZYME, AND THEREFORE, LEAD TO VARIOUS DEGREES OF CLINICAL CONSEQUENCES. HERE WE HAVE AN ENZYME ACTIVATOR. SO I THINK WE HAVE A GOOD CHANCE OF PERHAPS MAKING A DIFFERENCE TO THESE PATIENTS. IT’S A VERY RARE DISEASE BUT THE KIDS CURRENTLY DON’T HAVE ANY HOPE. SO THIS MIGHT BE A NICE INDICATION. AND CAMILLE SADROWSKI HAS MOVED ON AND HER PROJECT TO SEE TO BUILD THE MOUSE MODEL AND TO TEST THE USEFULNESS. THERE ARE MANY OTHER COGNITIVE FUNCTIONS COGNITIVE COGNITION MODELS. ALL THE NEURODEGENERATIVE DISEASES YOU CAN THINK OF FALL INTO THIS CATEGORY. WE OF COURSE HAVE NO HOPE HERE TO CURE THESE DISEASES WHICH HAVE VERY DIFFERENT ORIGIN BUT MAYBE WE CAN MAKE THE LIFE OF THE PATIENTS A LITTLE BIT BETTER BY IMPROVING — TAPPING INTO THE EXTRA RESERVOIR OF COGNITION. FINALLY AS IT’S BECOMING INCREASESSINGLY APPARENT TO ME, WE ALSO HAVE DECREASE COGNITIO DUE TO NORMAL AGING SO HOPEFULLY THIS WILL MOVE FAST ENOUGH FOR ME. ALL OF THESE MODELS HOWEVER NEED TO GIVE THIS COMPOUND CHRONIC ALLY. THERE’S ALSO ACUTE COGNITION MODELS POST-OPERATIVE COGNITIVE DECLINE, WHERE AFTER SEVERE SURGERY LIKE HIP REPLACEMENTS, YOU WILL GET — INITIATE DUE TO TISSUE TRAUMA CYTOKINE STORM THAT LEADS TO BRAIN INFLAMMATION AND THE NEXT DAY YOU WON’T BE QUITE AS SMART. FOR MOST PATIENTS, IT MAKES NO DIFFERENCE, IT DOSE GOES AWAY, IT CLEARS ITSELF BUT IN PARTICULAR FOR ELDERLY PATIENTS IT LEADS TO PERMANENT DEMENTIA. AND MAYBE WE CAN PREVENT SOMETHING THERE. THEN WE HAVE TRAUMATIC BRAIN INJURY MODELS WHERE MECHANICAL INSIGHTS TO THE BRAIN LEADS TO INFLAMMATION AND COGNITIVE LOSS. I WANT TO CLOSE THIS TALK BY SHOWING RESULTS IN THE LADDER MODEL OF TRAUMATIC BRAIN INJURY. WE COLLABORATED WITH ANNA ROSSI AT THE TRAUMA CENTER AT UCSF AND THEY BUILT THIS MODEL WHERE YOU INFLICT MECHANICAL INSULT CONTROLLED MECHANICAL INSULT TO THE BRAIN OF A MOUSE AND THEN TEST THESE MICE IN THESE BEHAVIORAL ASSAYS. SO IN THIS CASE HERE WE HAVE A SIMILAR TEST I SHOWED BEFORE IN CARTOON VERSION BUT NOW WE HAVE THESE RADIAL ARMS HERE, THERE’S A CONTROL MOUSE, IT HAS LEARNED TO FIND HIDDEN PLATFORM HERE, ALL IN ITSELF BY VISUAL CLUES AROUND THE ROOM. DOWN HERE IS A TRAUMATIC BRAIN INJURY MOUSE WHICH BASICALLY IN A RANDOM PATTERN. IT DOESN’T LEARN. THIS MOVIE GOES ON FOREVER. EXPERIMENTAL DESIGN OF THE DATA I WILL SHOW YOU IS SAID WE INFLECT THE INJURY, IN THIS CASE DIRECT INSULT CONTUSION ON THE BRAIN AT THIS POINT IN TIME. WE THEN LET ANIMALS RECOVER FOR FOUR WEEKS. AFTER FOUR WEEKS THE FIRST TIME THEY SEE THE ISRIB INJECTION. THEY’RE TRAINED THE NEXT DAY, THEY INJECT AGAIN IN THE EVENING, TRAIN ONE MORE DAY, INJECT AGAIN THEN THE NEXT DAY WE PERFORM A MEMORY TEST AND THEN NO MORE INJECTION AND A WEEK LATER ANOTHER MEMORY TEST. THIS IS WHAT THE DATA LOOKED LIKE, NO DRUG IN THIS EXPERIMENT YET. HERE ARE ANIMALS LEARNING HOW TO MAKE FEWER ERRORS IN THIS DAY ONE, SO FOUR WEEKS AFTER THE INJURY AND ANIMALS THAT CONTROL MICE, THESE ARE THE MICE THAT RECEIVE INJURY AND AS YOU SEE THEY DON’T GET BETTER, CONTROL MICE, THE NEXT DAY NEXT BLOCK OF LEARNING REMEMBER WHAT THEY HAVE DONE THE DAY BEFORE AND THEY GET BETTER AND BETTER UNTIL THEY MAKE HARDLY ANY MISTAKE ANY MORE, A DAY LATER THEY REMEMBER AND THEN A WEEK LATER THEY START SLOWLY FORGETTING BUT SO WOULD WE. THIS IS THE DATA RISEN DRUG INJECTION. SUPERIMPOSED HERE, THEY INJECT ISRIB TO THE INJURED ANIMALS ALREADY ON THE FIRST DAY YOU NOW HAVE A POSITIVE FLOW. THEY START LEARNING SOMETHING. IN THIS CASE THERE’S NO DIFFERENCE BETWEEN THE CONTROL MICE OR CONTROL MICE INJECTED WITH DRUG BECAUSE IT IS A STRONG TRAINING REGIME THAT THERE’S NO ROOM DOWN HERE TO IMPROVE MUCH. NEXT DAY THE MAGIC HAPPENS. ABSOLUTELY MAGICAL THE INJURED MICE RED DASH LINE BECOME INDISTINGUISHABLE FROM THE CONTROL GROUP. A DAY LATER THEY REMEMBER, A WEEK LATER THEY REMEMBER. S? SMALL NUMBERS OF ANIMALS BUT I THINK LOOKS INCREDIBLY PROMISING. AND THE STATISTICS ARE QUITE NICE INDIVIDUAL ANIMAL PERFORM IN THIS ASSAY. SO SIT’ NOT STATISTICALLY SIGNIFICANT I THINK YOU SEE A SLIGHT TREND HERE THE DRUG INJECTED MATURE MICE ALSO IMPROVE THEIR LEARNING AND MAKE FEWER ERRORS. SO WE TRIED TO MAKE IT LAST DATA PIECE HERE. WE TRIED TO MAKE IT A LITTLE BIT MORE DIFFICULT FOR THEM. AND THIS IS ANOTHER MEMORY TEST WE HAVE A TABLE WITH 4 HOLES AND THE MOUSE HAS TO REMEMBER ONE — ONLY ONE OF THE HOLES HAS ESCAPE HATCH UNDERNEATH, THEY DON’T HAVE THE STRESS OF SWIMMING, THEY ARE PUT IN THE MIDDLE, THEY DON’T LIKE TO BE EXPOSED TO BRIGHT LIGHT SO THEY FIND IT A PLACE TO HIDE. USING VISUAL CLUES AROUND THE ROOM, THE TBR MOUSE BASICALLY WALKS RANDOM PATH TO FIND THIS HOLE. THEN THE NEXT DAY WE DO THE SAME TEST AGAIN BUT CHANGE THE POSITION OF THE ESCAPE HATCH. SO THE MOUSE HAS TO FORGET WHAT IT LEARNED BEFORE, HAS TO RELEARN THE TASK. BUT AT THE SAME TIME ALSO LEARNS HOW TO LEARN. SO IT GETS FASTER AND FASTER. SO IT’S LIKE FINDING YOU CAR IN THE PARKING GARAGE. GARAGE IS THE SAME BUT YOUR CAR IS IN A DIFFERENT SPOT. ADMITTEDLY MY PATH LOOKS LIKE THIS. HERE ARE THE DATA. WE DO THIS AGAIN, VERY SIMILAR PROTOCOLS, COUPLE OF WEEKS AFTER THE INJURY AND YOU SEE THAT BY DAY THREE OF THE LEARNING PARADIGM, THE INJURED ANIMALS HERE WITHOUT THE DRUG STAY FLAT WHEREAS THE INJURED ANIMAL THAT HAS RECEIVED INJECTION NOW START LEARNING AND REMEMBERING THE PLATFORM BECOMING STATISTICALLY INDISTINGUISHABLE FROM THE CONTROL GROUPS. HERE YOU ALSO SEE FOR THE CONTROL GROUP THE DRUG INJECTION THEY REACH THIS LEVEL OF MAXIMUM PERFORMANCE ON DAY TWO MUCH EARLIER THAN RECEIVE THE DRUG SO YOU HAVE IMPROVEMENT ON THE UNINJURED ANIMAL. SO THIS IS WHERE WE ARE. GOOD NEWS FOR THE MICE. WE HAVE NO IDEA HOW ANY OF THIS WILL TRANSLATE INTO HUMAN SITUATION. MOUSE BRAINS AND HUMAN BRAINS ARE DIFFERENT MANY STUDIES FAILED BEFORE BUT WE ARE VERY OPTIMISTIC AND DEFINITELY WILL PURSUE. SO HERE IS A THE ATTITUDE OF PEOPLE IN MY LAB. NEVER MIND WHAT’S HOLDING UP, KEEP HAMMERING. THIS IS MY ROLE HERE. HOW STUDENTS AND POST-DOCS, NOT JUST BRINGING IN THE MATERIAL AND WRITE THE GRANTS AN STUFF BUT IT’S ALSO TO PROJECT CONFIDENCE THAT EVENTUALLY WILL REACH OVER HERE. TO SUMMARIZE WHAT I TOLD YOU TODAY, IS THAT IF IDENTIFIED THIS BEAUTIFUL LITTLE MOLECULE HERE, ADS A POTENT FIRST DRUG LIKE MOLECULE TO BLOCK INTEGRATIVE STRESS POINTS. THAT ISRIB BINDS TO AND ACTIVATES BY DIMERIZATION, YOU NOW HAVE A CLEAR DEFINITION OF THE BINDING SITE. WHICH BRINGS UP THE QUESTION DOES A BINDING SITE LIKE THAT EVOLVE FOR SYNTHETIC COMPOUND LIKE THAT OR IS THERE NORMALLY SOME METABOLITE OR CELL OR CONSTITUENT THAT BIND AND AFFECTS THIS DIMERIZATION REACTION IN A PHYSIOLOGICAL SETTING? WE HAVE SHOWN ISRIB EFFICACY HAS EFFICACY AS COGNITIVE ENHANCER. WITH THAT WE IDENTIFIED THE INTEGRATIVE AS A DRUGGABLE PASS AND INHIBITION HAS PROMISING AVENUE TO COMBAT COGNITIVE DISORDERS. IT HAS AMAZING EFFICACY IN THE MODELS OF ACUTE TRAUMATIC BRAIN INJURY AND DATA I WON’T SHOW YOU BUT WE HAVE SIMILARLY PROMISING RESULTS FOR THE POST-OPERATIVE COGNITIVE DECLINE ESSAYS. AND THIS YOU WILL SEE AT UCSF. WHAT I’M MOST PROUD OF, WHAT ANGELA ALREADY ALLUDED TO IN THE INTRODUCTION, WE HAD NO IDEA WE WERE GOING THERE. WE COULD HAVE NEVER HITTEN THE GROUND ON A PROJECT LIKE THAT AND SAID WE WERE GOING TO STUDY NEURAL COGNITION. BUT ALL THIS EMERGED BY WALKING DOWN THIS MEANDERING PATH CURIOSITY BASED DRIVEN RESEARCH. WE ARE NOW AT A STAGE WE ARE TRANSLATING THINGS THAT WE DEFINITELY DIDN’T SET OUT TO — THE IDEA OF KNOWING WHAT MIGHT BE TRANSLATABLE WHEN WE GOT THERE. BASED ON THIS THE MAGNITUDE OF DIFFERENT TECHNIQUES AND EXPERTISE, THERE HAS BEEN A MASSIVELY COLLABORATIVE INVESTIGATION WHICH AS I TOLD YOU HAS BENEFITED GREATLY FROM OPEN COMMUNICATION AND THESE ARE THE PEOPLE IN MY LAB, CAMILLE MOVEED TO A POSITION, MOVED THE DRUG TO THIS COMPANY AND HOPEFULLY THEY WILL PUT IT INTO CLINICAL TRIALS. SO ALL THESE PEEP HERE CONTRIBUTED IN A VERY MAY JR. WAY AND THANKS TO NAHOME MAHURE AND MANY OTHERS. WITH THAT, THANK YOU. [APPLAUSE] I’M HAPPY TO ANSWER QUESTIONS.>>I WAS WONDERING WHETHER THE EFFECTS OF THE ISRIB IN THE TBI SYSTEM HOW IT WAS LINKED TO CHANGES IN THE LEVEL OF AMPA RECEPTORS AS YOU SHOWED WITH SYNAPTIC PLASTICITY.>>Q. WE WANT TO DO THAT. WE DON’T KNOW THAT YET. WE ARE LOOKING AT MANY — WE NEED GOOD READ OUTS FOR TARGET ENGAGEMENT LIKE AMPA RECEPTOR OR SPINE DYNAMICS. SO MANY OF THIS WORK IS ONGOING. WE DON’T KNOW YET.>>CONGRATULATIONS FOR EXCITING WORK. WE DEFINITELY NEED MORE DRUG MOLECULE FOR DEGENERATION AND OTHER ISSUES SO LOOKING AT THIS STRUCTURE, I SEE YOU HAVE TWO PEPTIDE BONDS. OF ARE THEY SUSCEPTIBLE TO DEGRADATION, AFTER YOU MENTIONED INTERPERITONEAL INJECTION.>>OTHER PEPTIDE BONDS HYDROLYZE, WE KNOW NOTHING ABOUT CATABOLISM, METABOLISM OF THE DRUG AT PRESENT.>>HOW WOULD THE PARTITION — OBVIOUSLY YOU NEED SOME GOOD PARTITION CO-EFFICIENT.>>IT SEEMS TO EQUILIBRATE FREELY, IT DOES — THE CONCENTRATIONS ARE A BIT LOWER THAN IN CIRCULATION BUT WE CAN ACHIEVE TENFOLD OVER THE C 50 BY JUST IP INJECTION.>>OF COURSE YOU ARE CONTINUING THE MOUSE RELEVANCE GOING TO HUMAN SO YOU MENTION DEMYELINATION. SO YOU THINK THIS MIGHT HAVE BEEN IMPLICATIONS FOR ALL THE OTHER DISEASE INCLUDING SPINAL CORD IN OTHER AREA?>>I HAVE NO IDEA. YOU TELL ME.>>GOOD LUCK.>>THANK YOU.>>SO PETER, THERE WAS A RECENT REPORT FROM GRAND PAVID IDENTIFYING A NEW ASPECT OF THIS WHOLE ISR WHERE THE GEF IS CONVERTED INTO AN INHIBITOR. WHEN WHEN IF 2 IS PHOSPHORYLATED IT BINDS AND KNOCKS OFF THE TRNA. IT’S NOT KNOWN WHIT USES DIMER OR MONOMER, SO WONDERING IF IT POSSIBLE YOUR COMPOUND –>>COULD INFERENCE AT THAT STAGE.>>IF IT HAS TO WORK AS AN INHIBITOR AS MONOMER, THAT COULD BE A ANOTHER WAY IT COULD WORK.>>WHAT WE ALL HAVE IS YEAST ENVY AT THIS STAGE IN THE GAME. BECAUSE WE KNOW SO MUCH ABOUT THESE THINGS IN YEAST, THEN IN MAMMALIAN CELLS WE DON’T KNOW WHERE THE EQUILIBRIUM LIES. IN THE CELL ITSELF IS A PENTAMER, IS A DECAMER WHAT DRIVES THIS EQUILIBRIUM. THERE’S SO MUCH TO BE DONE. WHAT ARE YOU DOING NEXT WEEK?>>PETER, THE COMPOUND SO POTENT AND SPECIFIC, IS IT A NATURAL PRODUCT>>IT COMES OUT OF PUBLICLY AVAILABLE LIBRARY, IT WAS TRIAGED IN EVERY SCREEN THAT HAS BEEN DONE BEFORE. WAS OF THE SOLUBILITY. I HAVE TRIED TO TRACE IT BACK WHO PUT IT IN LIBRARY, WHO SYNTHESIZED IT AND WHY AND IT’S UNTRACEABLE.>>IF YOU PUT A BULKY RESIDUE IN ONE-HALF OF THE SYMMETRY, WILL IT FORM INHIBITOR OF GEF DIMERIZATION?>>NO, HALF A MOLECULE DOESN’T WORK.>>TOO LOW AFFINITY.>>YEAH. BUT THESE ARE ALL THINGS THAT NOW KNOWING AS MUCH AS WE KNOW, WE COULD GO BACK AND SCREEN FOR AGAIN. ALL RIGHT? RECEPTION TIME.

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