>> OKAY, GOOD EVENING. MY NAME IS ED SAUSVILLE, I HAVE THE PLEASURE OF TALKING TO YOU TONIGHT IN YOUR CONTINUING COURSE IN CLINICAL PHARMICOLOGY ABOUT THE OVERALL SUBJECT OF DRUG DISCOVERY. I FOR MANY YEARS ACTUALLY WORKED AT THE NIH AND THROUGH 2004 SERVED AS DIRECTOR OF NCI'S DEVELOPMENTAL THERAPEUTICS PROGRAM. SINCE THEN I'VE BEEN AT THE UNIVERSITY OF MARYLAND IN THEIR CANCER CENTER AS PROFESSOR OF MEDICINE. SO IN LOOKING OVER THE SYLLABUS THAT YOU FOLKS HAVE BEEN EXPLORING OVER THE PAST SEVERAL WEEKS AND MONTHS, YOU THOUGHT A LOT ABOUT THE HANDLING OF DRUGS, THE WAY THAT THE BODY DISPOSES OF IT AND HOW THEY INTERACT. THIS STARTS A SERIES OF LECTDURES THAT MOVING THING A BIT BACK AND IT PROVIDES FOR YOU AN OVERVIEW OF HOW THE MOLECULES THAT EVENTUALLY END UP AS IN SOME CASES CLINICAL PRODUCTS GET THEIR START AND TO GIVE SOME SENSE OF THE DIFFERENT PESTS TO GETTING TO THE CLINIC. SO THE--LET'S SEE. HOW DO I DO THIS? I NEED SOME TECHNICAL SUPPORT HERE. THANK YOU. UTILIZING THE WRONG TOOL. SO AS AN OVERVIEW OF WHERE WE'RE GOING TO BE HEADED TONIGHT, FIRST A BIT OF A GENERAL INTRODUCTION, A DISCUSSION OF HOW WE DEFINE A POTENTIAL DRUG TARGETS, HOW WE GENERATE DIVERSITY IN THE SMALL MOLECULES THAT MIGHT MANIPULATE THESE TARGETS, HOW LEAD STRUCTURES ARE DEFINED AND HOW THESE ARE QUALIFIED TO TRANSITION TO EARLY TRIALS. I WILL SAY AT THE OUTSET THAT I'M DEFINITELY SOMEWHAT CANCER HEAV CENTRIC GIVEN MY ORIGINS AND PAST ACTIVITIES BUT THE GENERAL DRIFT WILL BE TRUE ACROSS A VARIETY OF DIFFERENT DISEASE AREAS. SO DRUG DISCOVERY CAN BE THOUGHT OF AS A SUCCESSION OF STYLES WITH--UNTIL RELATIVELY RECENTLY IN THE LAST CENTURY, MANY OF THE INITIAL EFFORTS USING MIXTURES OF NATURAL PRODUCTS IN BIOASSAYS THAT REPRESENT WHAT WAS THE DESIRED OUTCOME OF THE DRUGS AND THESE WERE USEFUL GIVING RISE TO A VARIETY OF ANTIINFECTIVE CARDIOVASCULAR AND CERTAINLY CANCER DIRECTED THERAPEUTICS. FROM THE 1930S TO THE PRESENT TIME PURE COMPOUNDS VERSES THE SAME BIOASSAYS WERE USED AND THESE GENERATED A VARIETY OF AGENTS THAT ARE USED IN THE CLINIC TO THE PRESENT TIME. THE CONCEPT OF HAVING COMPOUNDS VERSES PURE ENZYMES OR PURE MOLECULAR TARGETS OF DRUG ACTIONS IS RELATIVELY MORE RECENT INNOVATION, BUT THESE HAVE YIELDED ACTUAL AGENTS IN BLOOD PRESSURE INFECTED AND METABOLIC AREAS. AND MORE RECENTLY AS WE'LL DESCRIBE, SO CALLED COMBINATTORRIAL METHODS HAVE SOUGHT TO BRING MIXTURES OF COMPOUNDS, SOMETIMES AGAINST A VARIETY OF TARGETS IN THE SAME ASSAY WITH THE GENERAL GOAL OF INCREASING EFFICIENCY OF RECOGNIZING NEW MOLECULAR TYPES THAT MIGHT BE ACTIVE AS THERAPEUTIC CLASSES. IT'S IMPORTANT TO EMPHASIZE THAT THIS IS AN INTRINSICALLY INEFFICIENT PROCESS. WANT THE REASONS FOR FAILURE ULTIMATELY HAVE A COMPOUND RELATE TO TOXICITY OR LACK OF EFFICACY IN THE DISEASE MODEL, POOR BIOPHARMACEUTICAL PROPERTIES IS A PROMINENT SOURCE OF FAILURE. SLOW DOWN OR NEEDS FOR RETOOLING DURING THE PROCESS CAN RELATE TO THE NEED TO WORK OUT A SYNTHETIC ROOT THAT IS REASONABLE AND EFFICIENT DEALING WITH LOW POTENCY AND DEALING WITH AMBIGUOUS TOXICITY FINDINGS IN PRECLINICAL STUDIES AND AGAIN OVERCOMING POOR BIOPHARMACEUTIC PROPERTIES. IN--SO WHEN YOU LOOK AT THE OVERALL OF A PARTICULAR SCREEN THAT MAY BE ACTIVE IN--THAT MAY QUERY COMPOUNDS, AGAIN I'M LOSING THIS. OKAY, WHERE'S THE BEST POINTER FOR THIS? RIGHT ARROW. BUT IS THERE A POINTER I CAN USE. THE MOUSE. MOUSE IS NOT FUNCTIONING HERE. SO TO REITERATE THE RESULTS OF THIS IS THAT IT--THERE'S A SUCCESSFUL WINDOWING OF POTENTIAL HITS OR LEADS IN SCREENS THROUGH PRECLINICAL DEVELOPMENT SO THAT THERE MAY BE 1 LICENSED OR APPROVED COMPOUND FOR IN THE ORDER OF A HUNDRED TO THOUSANDS OF POTENTIAL HITS OR LEAD STRUCTURES. SO IN CONSIDERING THE WAYS WE DEFINE DRUG TARGETS, THE--2 CONTRASTING PHILOSOPHIES MIGHT BE ALLUDED TO. SO CALLED IMPERICAL EFFORTS RECOGNIZE THE INITIAL DRUG LEAD BY A FUNCTIONALLY USEFUL EFFECT SO THAT FOR EXAMPLE PENICILLINS AND VARIOUS ANTIINFECTIVE AGENTS WERE RECOGNIZED BY AN INTRINSIC ANTIBACTERIAL EFFECT. RAUWOLFIA WAS ANTIHYPER TENSESSIVE ALLOWED THE DEFINITION OF MANY CHEMO THERAPEUTIC AGENTS AND OF COURSE HISTORICALLY DIGOXIN AND CARDIO TONS WERE RECOGNIZED IN THE DISEASE EFFECT THAT THEY WERE DESIGNED TO DEAL WITH. SO CALLED RATIONAL APPROACHES RECOGNIZES THE DRUG BY A DESIGN OR SCREEN AGAINST THE DRUG TARGETS FUNCTION WHERE THE DRUG TARGET HAS BEEN VETTED BY A KNOWLEDGE OF BIOLOGY TO BE IMPORTANT IN THE DISEASE PROCESS. THE FOR EXAMPLE, HIV PROTEASE INHIBITORS WERE ACTUALLY SCREENED BY EFFECTS AS ANTIPROTEOLYTIC ENZYME AGENTS. THE TOPOL, EMERGED AS A ANTIHYPER TENSIVE WITH STRUCTURE OF A PRIOR ACTIVITY RELATIONSHIPS AGAINST THE BETA CATACOLLIC RECEPTOR. METHOTREKSATE, ALTHOUGH A CYTOTOXIC AGENT WAS DESIGNED TO INTERFERE WITH FULLY METABOLISM UNDERSTANDING THE IMPORTANCE OF FIELD FUNCTIONSET IN THE REPLICATION OF NUCLEOTIDES CLAYIC ACIDS. SO THE PROBLEM IS HOW TO RECOGNIZE DISEASE RELEVANT TARGETS. AND IN THE CANCER SPACE, WE RELY AS IN OTHER AREAS HEAVILY ON THE BIOLOGY OF THE DISEASE, SUCH AS CYTOGENETICS TO DEFINE BREAK POINTS TO DEFINE SPECIFIC MOLECULES THAT ARE ACTIVA AS ONCA GENES AND THESE HAVE GIB RISE TO A NUMBER OF SCREENING CAMPAIGNS THAT HAVE YIELDED ACTIVE MOLECULESMENT 1 CAN RETROFIT ACTIVE MOLECULES IN A KNOWN BIOASSAY SO THAT FOR EXAMPLE, BINDING PARTNERS TOO AGENTS THAT ARE IMPERICALLY ACTIVE SUCH AS GELDANAMICEIN OR RAPAMYCIN OR FUMAGILLIN, ARE IN A CLASS, COMPUTATIONAL ALGORITHMS TO MINE THE ACTIVITY OF PARTICULAR MOLECULES HAVE BEEN DEFINED GAINS THE EXPRESSION OF TARGETS IN A SCREENING SYSTEM, USUALLY CELLS, EXAMPLES WOULD INCLUDE THE MULTICELL LINE PANELS, MAINTAINED IN THE PAST BY NCI AND CURRENTLY BY A NUMBER OF PRIVATE AND ACADEMIC CONCERNS. SO CALLED CLASSICAL APPROACHES WILL SEEK TO USE A KNOWLEDGE OF CELLULAR METABOLISM AND BIOCHEMISTRY TO SUGGEST SINGLE TARGETS AND THEN MUCH IN IN THE WAY THAT WE DEFINE A FEW SLIDES AGO, LOOK FOR AGENTS THAT WILL INHIBIT FOR EXAMPLE, ACTIVE ENZYMES. THIS CLASSICAL APPROACH IS SOMEWHAT INEFFICIENT BUT WHERE IT CAN BE PURSUED, IT'S USEFUL BECAUSE MEDICINAL CHEMISTRY AROUND LEAD STRUCTURES IS READILY POSSIBLE. MORE RECENTLY AND WHAT WE WON'T TALK ABOUT MUCH IN DETAIL TONIGHT AGAIN USES SO CALLED CHEMICAL GENETICS TO DEFINE ORGANISMS THAT HAVE BEEN ALTERED TO HAVE PARTICULAR PHENOTYPES AND LOOK AT LIBRARIES OF MOLECULES THAT MIGHT MODULATE THOSE PHENOTYPES. AN EXAMPLE OF A BIOLOGICALLY ORIENTED APPROACH TO DEFINING VALUE HAS BEEN PROMINENTLY REPRESENTED BY VARIOUS APPLICATIONS BY INTERFERING RNA TECHNOLOGY. IT'S POSSIBLE TO TRANSFECT PLASMIDS BEARING A PARTICULAR RNA THAT CAN ULTIMATELY DEGRADE THE RNA FOR TARGET MOLECULES TO ALLOW ALTERED PHENOTYPE OF CELLS EXPRESSING THE SHRNA TO THEN SERVE AS A BASIS FOR SCREENING, TO LOOK FOR GENES WHO CAN BE DELETED AND BECOME IN THE CONTEXT OF THAT DELETION A BASIS FOR ACTIVITY OF AGENTS TO PREVENT GROWTH, TO MODULATE THE SUSCEPTIBILITY TO KNOWN CISE O TOXICS BY THE CAPACITY TO DEGRADE SPECIFIC RNAs. AND THEN, IT'S ALSO POSSIBLE TO USE THIS IN TARGET VALIDATION, TO USE AN ALTERED CELL LINE TO DEFINE EITHER AN AUGMENTATION OR LOSS OF EFFECT AFTER ADDITION OF A CANDIDATE AGENT. SO THIS HAS BEEN A BASIS FOR DESIGNING SCREENS ULTIMATELY PREDICATED ON TARGETS BY SiRNAs. ANOTHER WAY OF LOOKING AT BIOLOGICAL TIEWPTS IS TO CHARACTERIZE IN THE CASE OF CANCER, THE EXPRESSION OF PARTICULAR TRANSCRIPTS IN CELLS AND TO THEN CATALOG THE PRESENCE OR ABSENCE OF GENES IN THE DIFFERENT TUMOR TYPES AND WHAT 1 GETS IS AN INFORMATIONAL ARRAY OF GENES THAT ARE OVEREXPRESSED IN A CANCER CELL COMPARED TO A NORMAL CELL FOR EXAMPLE OR ARE ABSENT IN THE CANCER CELL COMPARED TO NORMAL CELLS AND THERE ARE DATABASES AVAILABLE IN BOTH A PUBLIC DOMAIN AND IN CORPORATE CIRCLES THAT PROVIDE A BASIS FOR DEFINING GENES THAT MAY BE RELEVANT TO DERIVING PUTATIVE CANCER TARGETS AND THIS IS AN EXAMPLE, AGAIN OF A DATABASE ORGANIZED BY THE NIH. AN EXAMPLE OF RETROFITTING AN ACTIVE MOLECULE BY DEFINING ITSELF TARGET IS PROVIDED BY THE GELDACIN, AMICEINS WANTS THESE WERE COMPOUNDS ISOLATED FROM BACTERIA THAT HAD ANTIPROLIFERATIVE EFFECTS AND THE COMMON CHEMISTRY THAT WAS PRESSENT IN A NUMBER OF SUCH AGENTS WAS A SO CALLED ANSA-RING WITH A BRIDGING A BENZOQUINONESPAY EMOTEY AND THIS WAS CONDUCTED BY COLLABORATORS IN JAPAN THAT REVEALED THAT THESE MOLECULES COULD REVERSE THE PHENOTYPE, SRC-TRANSFORMED CELLS, IT DECREASED PHOSPHORYLATION OF THE TARGET BUT DID NOT INHIBIT THE ACTIVITY DIRECTLY AND IT WAS POSITIVE TO ALTER THE QUOTE INTRACELLULAR ENVIRONMENT OF THE ONCA PROTEIN. SUBSIDIARY KEPT STUDIES SHOWED A DECREASE IN THE STEADY STATE LEVEL, LEVEL OF THE ONCA PROTEIN. SO INVESTIGATORS IN THIS BUILDING ACTUALLY LED BY LYNN METHERS IN THE EARLY 90S KNOW THE PHARMACOLOGIC FEATURES OF GELDACIN, AMICEINS WERE ABLE TO CREATE A DERIVATIVE WHERE GELDANAMICEIN WERE LINKED TO A BOO ED AND WERE ABLE TO DEFINE A STRONG APPROXIMATELY 90 KILLA DALTON SIZED BINDER TO BOUND THAT THE GELDANAMYCIN--THAT HAD 2 PRINCIPLE ROLES, FIRST TO INDUCE THE CORRECT FOLDING OF MOLECULES, PROTEIN MOLECULES EMERGING FROM THE ENDOPLASMID RETIC LUMTO ASSUME A PROPER CONFIRMATION TO ALLOW ACTIVITY. IF HSP 90 WAS ABSENT OR INHIBITED IN THIS CASE AFTER BYPASSING THE ANTIBIOTIC THE PROTEINS OF INTEREST WERE DEGRADED AND THEIR LEVELS DECREASED. IT WAS ALSO KNOWN THAT HSP 90 SERVED AS A SCAFFOLD IN WHICH CERTAIN MOLECULES WERE MAINTAINED IN A CORRECT CONFIRMATION AND IT WAS FOUND THAT THE--THIS WAS TRUE FOR A NUMBER OF STEROID HORMONE BINDING MOLECULES. SO BIOLOGICALLY IT WAS POSSIBLE TO CHARACTERIZE A NUMBER OF SO CALLED PROTEINS FOR HSP 90 AND AMONG THEM WERE MANY ONCA PROTEINS AS WELL AS STEROID HORMONE RECEPTORS AND IMPORTANT MOLECULES IN THE GROWTH OF CELLS. AND THAT WE COULD GO THROUGH EXAMPLES OF WHERE FOR EXAMPLE, RAPAMYCIN WAS ACTUALLY THE BASES FOR DISCOVERYING THE SO-CALLED MAMMALIAN TARGET OF RAPAMYCIN AND AGAIN A DRUG CLASS THAT HAS PROVED TO BE VERY ACTIVE IN A NUMBER OF DIFFERENT NEOPLASTIC CONTEXT. SO ONCE YOU HAVE A PUTATIVE TARGET HOW DO YOU GENERATE A DIVERSITY OF POTENTIALLY NEW STRUCTURES THAT WOULD BE VALUABLE IN ADDRESSING THE ACTIVITY OF THE TARGET? SO AGAIN, HISTORICALLY, SO CALLED NATURAL PRODUCTS, ENTITIES DERIVED FROM PLANTS, ANIMALS AND BACTERIA OFTEN AS EXTRACTS HAVE BEEN A RICH SOURCE OF NOVEL STRUCTURES AND THESE CAN BE PRESENT EITHER AS PURE COMPOPPED COLLECTIONS, AS EXTRACTS OF VARIOUS ORGANISMS OR ACTUALLY PRODUCED IN VARIOUS FORMS BY GENETICALLY MODIFIED PRODUCER ORGANISMS. AND WHEN--WHEN 1 LOOKS AT OVERALL DRUGS ACTIVE IN A VARIETY OF SPACES, APPROXIMATELY 30% ARE EITHER NATURAL PRODUCTS THEMSELVES OR DERIVATIVES OF NATURAL PRODUCTS IN CONTRAST TO THE VARIOUS OTHER CLASSES SHOWN HERE INCLUDING SYNTHETIC STRUCTURES. THE REASON WHY SO CALLED NATURAL PRODUCTS ARE THOUGHT TO BE POTENTIALLY USEFUL AS INITIAL DRUG LEADS IS THE FACT THAT NATURE CREATES A FAIRLY RIGID STEREO CHEMICALLY PRECISE SCAFFOLDS THAT HAVE AN ARRAY AND A STEREO CHEMICAL MAINTENANCE OF PROPERTIES BETWEEN HYDROPHILIC AND HYDROPHOBIC AND ACID BASIC. AND IT'S THIS UNIQUE SEROCHEMMISTRY THAT IS THE BASIS FOR HAVING ACTIVITY IN THE CELLULAR ENVIRONMENT. SO AN EXAMPLE OF SUCH A SOURCE FOR A NONCAPSER TYPE OF INDICATION WAS THE DISCOVERY ULTIMATELY OF LIDOCAINE. AS IT TURNED OUT A NUMBER OF YEARS - IN THE LAST CENTURY IT WAS OBSERVED THAT CERTAIN CENTRAL ASIAN CAMELS REFUSED TO EAT A CERTAIN TYPE OF REED. GRAMMINE WAS FOUND TO BE PRESCRIBINGINENT THESE PLANT PRODUCTS THAT DISCOURAGED THE ANIMALS FROM INGESTING IT AND UPON FURTHER EXAM NATION, NUMNESS, UPON THE EXPERIENCE OF TASTE DELIVER SUGGESTED IT AS A LEAD FOR ANESTHETIC DEVELOPMENT WHICH INDEED IS IT'S CURRENT USE. SO WHEN 1 IS GOING TO APPROACH NATURAL PRODUCTS SOURCES AS THE POTENTIAL BASES FOR NEW DRUGS, THERE IS CLASSICALLY THEN A SCREEN OF THE EXTRACT AND THEN IF POSITIVE, THE PARTICULAR EXTRACT IS PROMISING, THE CHALLENGE IS TO ISOLATE A PURE COMPOUND WHICH CAN THEN RELIAISONNABLY REPRODUCE THE EFFECT OF THE EXTRACT. AND THIS IS WHERE IT CAN BE VERY DIFFICULT BECAUSE DIFFERENT EXTRACTS GATHERED AT DIFFERENT TIMES OF THE YEAR OR FROM DIFFERENT SPECIES OF A PUTATIVE ORGANISM OR SUBSPECIES OR A DIFFERENT ENVIRONMENTAL ZONE WHERE THE ORGANISM EMERGED, CAN GIVE DIFFERENT RESULTS IN THE BASES FOR CONFUSION A NUMBER OF DIFFERENT AGENTS ULTIMATELY DERIVE FROM NATURAL PRODUCTS. AND THIS IS TO DEFINE SO CALLED CHEMICAL COMPOUND LIBRARIES, THAT MAY BE TARGET NONSELECTED IN THE SENSE THAT THEY ARE DERIVED BY--WITHOUT ANY PARTICULAR TARGET IN MIND OR ARE TARGET DIRECTED IN THE SENSE THAT THEY ARE--DIRECTED AGAINST THE--A STRUCTURE THAT IS KNOWN TO BE A BASIS FOR POTENTIAL ACTIVITY. LOOKING AT HOW FOR EXAMPLE, PEPTIDE COMPOUND LIBRARIES CAN BE FORMED, YOU CAN CONSIDER THAT IF YOU HAVE A TRI-PEPTIDE, THAT THERE ARE ESSENTIALLY 64 POTENTIAL TRI PEPTIDES WHERE THE SELECTION IS LIMITED TO THESE 4. IF 1 LENGTHENS THE PEPTIDE AND STILL CONSIDERS THE POTENTIAL LIMITED SUBSTITUTION WITH THE AMINO ACIDS, BY THE TIME YOU GET TO 8 THERE IS A RAPID DECRETION OF MILLIONS OF COMBINATIONS. IN PRACTICAL TERMS IT'S WHEN THIS APPROACH IS TAKEN, 1 LOOKS TO HAVE AN APPROXIMATELY NO MORE THAN A THOUSAND TO 10,000 PEPTIDES OF THIS GENERAL LENGTH IN EACH BATCH AND THE BASIS FOR THAT IS THAT A--IF YOU CONSIDER A SINGLE ACTIVE COMPOUND AT THE 1 NANO MOLAR LEVEL, THEN WHEN REPRESENTED IN THE--AT APPROXIMATELY 10,000 BASED LIBRARY, ITS AFFINITY WOULD BE SHIFTED APPARENTLY OF THE MIXTURE BY 5-6 FOLD AND THIS IS WHERE WHEN 1 BEGINS TO REACH THE LIMITS OF SOLUBILITY IN MOST CONVENTIONAL ASSAYS. SO, WHAT ARE THE ADVANTAGES OF SYNTHETIC COMBINATTORRIAL MIXTURES. WELL IN COMPARISON TO NATURAL PROJECT EXTRACTS IN BOTH CASES YOU CAN DIRECTLY SCREEN COMPOUND MIXTURES FOR ACTIVITY, CAN YOU DISCOVER COMPOUNDS THAT ARE ACTIVE AS YOU SAW, AS LOW AS POTENTIALLY IN A NANO MOLAR BASIS EASILY. THE PROBLEM IS THAT IN NATURAL PRODUCTS, YOU REALLY DON'T HAVE AN UNDERSTANDING OF THE CONCENTRATION RATIO OF THE ACTIVE COMPOUND TO OTHERS IN THE MIXTURE, THE CHEMICAL STRUCTURES ARE NOT KNOWN, SYNTHETIC PATHWAY IS NOT KNOWN AND IT'S IN TERMS OF FERRETING OUT THE STRUCTURE ACTIVITY RELATIONSHIP, IT'S HARDER WHEREAS THESE CAN ALL BE CONTROLLED FOR WHEN 1 USES COMBINATTORRIAL MIXTURES. IT'S ALSO POSSIBLE TO DERIVE AN ANALOGOUS THINKING TO DERIVE NONPEPTIDE FUNCTIONALITIES THAT AGAIN ARE SYNTHESIZED TO BE ABLE TO EXPRESS DIFFERENT FUNCTIONAL GROUPS DEFINED POSITIONS. THE WAY THESE ARE DESIGNED IS TO TRY AND STACK THE DECK THAT THE COMPOUNDS THAT WILL EMERGE WILL HAVE FAVORABLE PROSPECTS FOR ULTIMATELY BEING USEFUL AS A DRUG AND THE SO CALLED RULE OF 5, FROM THE LIST KEY IS USEFUL IS COMPOUNDS THAT AVOID HAVING MORE THAN H1 DONORS OR HIGHER MOLECULAR WEIGHT THAN 500 HAVE A VERY PROMINENT OIL BASED SOLUBILITY AND HAVE A LIMITED NUMBER OF HYDROGEN BOND ACCEPTORS. EXAMPLES OF THE TYPES OF LIBRARIES CREATED INCLUDE THIS TYPE OF STRUCTURE WHERE WITH R1, R2, R3 ARE CONTROLLED YOU CAN CREATE VARIETIES OF BATCHESS OF DIFFERENT COMPOUNDS. ONCE YOU HAVE THE LIBRARIES, YOU CAN APPLY THE ASSAYS TO THE VOLUNTEERSUBLE RECEPTORS, AND YOU CAN CERTAINLY USE THEM, DIRECTED AGAINST CELLS OR MICRO ORGANISMS AND EXAMPLES OF THE LIBRARY THAT IS REPRESENTED IN THE NONPEPTIDE EXAMPLE THAT WE JUST CONSIDERED, IF YOU CONSIDERED ACTIVITY WITH DIFFERENT FUNCTIONALITIES IN THE R1 POSITION AND THE R3 POSITION AND THE R2 POSITION, THERE'S ACTIVITY AS A CAPPA RECEPTOR ANTAGONIST FOR THE OPIATES THAT IS MANIFEST IN MOLECULES THAT HAVE A DEFINED R3, R2 AND R1 POTENTIAL POSITIONS AND YOU CAN PUT FURTHER VERSIONS TOGETHER THAT MAXIMIZE THE LIKELIHOOD OF THE BEST BINDING OF THIS CASE TO THE CELL SURFACE RECEPTOR. SO ONCE YOU HAVE A--A SET OF MOLECULES CONTAINING LIKELY HITS TO A--IN AN ASSAY, HOW DO YOU ACTUALLY LOOK AT SELECTING LEAD STRUCTURES. YOU CAN USE A SCREEN THAT IS OPTIMIZED FOR ITS BIOCHEMICAL PROPERTIES SO THAT THE ACTUAL MOLECULE THAT YOU'RE POSTULATING TO TARGET IS PRESENT IN--IS THE ACTUAL TARGET, THE ADVANTAGE OF COURSE IS THAT YOU'RE GOING TO GET A STRUCTURE THAT IS ACTIVE IN A PURE BIOCHEMICAL SENSE. THE DISADVANTAGE IS THAT THIS IS LOOKING AT THE TARGET OUT OF ITS CELLULAR CONTEXT, IF 1 USES A SOLE CALLED CELL BASED SCREEN, THE ADVANTAGE IS THAT THE READ OUT OCCURS IN A SO CALLED LIVING SYSTEM OR A SYSTEM THAT IS CLOSER TO WHERE IT'S GOING TO BE ACTIVE IN THE ORGANISM. THE DISADVANTAGE IS THAT A POSITIVE RESULT IN A CELL BASED SCREEN MUST ULTIMATELY BE DECONVOLUTED OF THE MECHANISM OF THE EFFECT ITSELF TIES TO THE CHECK EXPECTED BIOCHEMICAL ACTIVITIES. SO WHAT WOULD BE SOME EXAMPLES OF CASE STUDIES WHERE THESE APPROACHES HAVE BEEN TAKEN. I MENTIONED ORCHGA PROTEINS AS AN IMPORTANT TARGET IN THE CANCER SPACE. ONCA GENES EXPRESS PROTEINS THAT ARE OVEREXPRESSED OR ACTIVATED IN CANCER CELLS. IN MANY CASES IT HAS ENHANCED CAPACITY TO BE RELATED TO CANCER CAUSATION BY MUTATION OR ITS ACTIVITIES ALTERED BY TRANSLOCATION TO OVEREXPRESS MOLECULES THAT ACTUALLY PROMOTE CANCER DEVELOPMENT IN A VARIETY OF CASES OVEREXPRESSION IS ASSOCIATE WIDE ADVANCED STAGE INFERIOR PROGNOSIS. SO THESE ARE REPRESENTED IN THE KNOWN FAMILIES OF THE ONCA PROTEIN FAMILIES, THE ENZYMATIC STEP TO ALL KINASES IS THE TRANSFER OF THE GAMMA PHOSPHATE OF ATP TO THE HYDROXYL OF A TYROSINE THROUGH THE INTERMEDIATE IS THE VARIETY OF CASES IS FAIRLY SIMILAR AMONG DIFFERENT TYROSINE KINASES. THIS LED TO SCREENS OF NATURAL PRODUCTS AND CHEMICAL COLLECTIONS THAT INITIALLY SOWED A NUMBER OF COMMON STRUCTURAL THEMES IN CLASSES OF AGENTS THAT EMERGED IN THIS REVIEW FROM DR. LEVITSYC'S WORK AND YOU NOTICE IN 1 EXAMPLE IT VAGUELY LOOKS LIKE A TO ROSINEI AND IN THIS CASE AND THE CASE OF LAVENDOUSTIN, IT'S BI-PH ENYL. THIS IS PATHOGENIC FOR CHRONIC MOO MILLEOG NOWS LEUKEMIA, THERE'S A VARIETY OF MOTIFS THAT'S POSITIONED. THE PROTEIN IN ITS ABILITY TO BIND TO INTRACELLULAR COMPONENTS AND IT'S HEART IS A VERY ACTIVE KINASE THAT DRIVES CELLULAR PROLIFERATION. SO IN THOSE INITIAL TYROSINE DIRECTED FAMILY CLASSES. I SHOWED YOU A FEW SLIDES AGO, THIS COMPOUND EMERGED AAG957 AS A COMPOUND THAT WAS STRUCTURALLY RELATED TO THE HERB STATIN AND WENT INTO AN EFFORT FOR DEVELOPMENT BUT IT ULTIMATELY FAILED TO PHARMACOLOGIC SUSCEPTIBILITY DEGRADATION. IN CONTRAST THE COMPOUND SHOWN HERE STI571 AND SELECTED TO MIMIC THE BINDING FEATURES TO THEN INITIAL CRYSTAL STRUCTURES OF MODEL PROTEIN KINASES, EMERGED FROM THE COMPANY AND ITS PROPERTIES WERE VERY USEFUL IN THAT IT HAD--WHEN STUDIED IN EITHER INTERPERO TON EEL OR ORAL ADMINISTRATION FORMS THE CAPACITY TO DECREASE PHOSPHORYLATION OF THE BCL ABLE TARGET AND WITH MICE, BEARING TUMORS THAT EXPRESSED THAT TARGET, IT PROLONGED THEIR SURVIVAL IN COMPARISON TO MICE BEARING TUMORS THAT DID NOT HAVE THE BCR ABLE ONCA PROTEIN SUGGESTING THAT ITS ACTIVITY WAS UNIQUELY RELATABLE TO THE BCL KINASE. AND IN THIS CASE WHEN ADVANCED TO THE CLINIC, IT SHOWED EVIDENCE OF PROMINENT CAPACITY TO INHIBIT WHITE CELL PROLIFERATION IN PATIENTS WITH MILEOG NOWS LEUKEMIA IN INITIAL PHASE 1 SHOWING PROMINENT DECREASE IN CELL COUNT AND IN LOOKING AT CELLS THAT WERE PRESENT IN THE MARROW THAT ACTUALLY HAD THE TRANSLOCATION SHOWING PROMINENT DECREASE IN THEIR PREVALENCE IN THE GREAT MAJORITY OF PATIENTS. AND THIS WENT ON TO BECOME KNOWN AS AMAT NIB AND IS NOW A STANDARD TREATMENT FOR ACUTE MILEOG NOWS LEUKEMIA. THE THIS WAS IN A TRIAL WHERE IT WAS COMPARED TO INTERFERON, THE STANDARD AT THE TIME AND YOU CAN SEE THAT IMAT NIB HAS A MUCH HIGHER DEGREE OF THE CYTOGENETIC RESPONSE. UNFORTUNATELY, PATIENTS IN NOT ALL CASES WILL BE RESPONSIVE INITIALLY, OR THERE WILL BE PATIENT WHO IS RELAPSE FOLLOWING A PERIOD OF TIME RECEIVING THE AGENT. THERE ARE 2 SUBTYPES OF SOPHISTICATED CALLED MYELOID BLAST CRISIS AND WHEN THE BCR ABLE ONCA PROTEINS WERE CHARACTERIZED WITHIN THESE RESISTANT CELL TYPES IT WAS FOUND THERE WAS A VERY SPECIFIC ACQUISITION OF THE--A 39 LUNG CANCER SIN MUTATION WHICH DISRUPTED THE CAPACITY OF AMAT NIB TO BIND TO THE ONCA PROTEIN AND AS CAN YOU SEE IN THE RESISTANT BCR ABLE THERE WAS VERY LITTLE EFFECT IN BEING ABLE TO INHIBIT PHOSPHORYLATION IN CONTRAST TO THE WILD-TYPE. BUT THE VALUE OF THE EXISTENCE OF COMMONLY ACQUIRED CLINICAL MUTATIONS WAS ABLE TO SERVE AS A BASIS FOR DEFINING MOLECULES THAT WOULD HAVE THE PROPERTY OF BEING ACTIVE IN MUTATIONS WHERE IMAT NIBS CAPACITY TO INHIBIT THE ENZYME HA BEEN LOST AND THIS ALLOWS THE DEFINITION OF DESAT NIB BEING INACTIVE AGAINST MORE IMATINIB MUTATIONS. SO THAT'S THE CASE WHERE ONCE HAVE YOU A COLLECTION OF POTENTIAL INTERACTORS OF USING THE EFFECT ON THE DISEASE RELATED TARGET ONCA PROTEIN, TO MAXIMIZE THE SELECTION OF ACTIVE MOLECULES AND TO FURTHER USE THAT INFORMATION TO SELECT MOLECULES ACTIVE IN RESISTANT VARIANTS. NEXT CASE IS TO SET UP A SCREEN WHERE 1 MODULATES USING THE RNA INTERFERON TECHNOLOGY, THE TARGET OF THE POTENTIAL DRUG IN A WAY THAT WOULD LEAD TO THE FACILE RECOGNITION OF ACTIVITY. SO THIS WOULD REMIND YOU AS WE SAID AT THE BEGINNING THAT IN AN SiRNA RELATED SCREEN THAT THE RNAi, CREATES CELLS THAT HAVE HAD A PARTICULAR TARGET REMOVED AND IN THAT CONTEXT, IT'S POSSIBLE TO DEFINE COMPOUNDS THAT ARE ACTIVE IN THE STATE OF HAVING THE TARGET BEING ABSENT. SO WHY THIS WAS USEFUL WAS THE SELECTION OF POLYADP RIBOSE INHIBITORS, IT WAS KNOWN THAT POLYADPPOLYMERASE IS AN IMPORTANT PARTICIPANT IN DNA REPAIR MECHANISMS. IT WAS KNOWN THAT THE BRACKA 1 AND 2 PROTEINS OPERATED IN THE SAME PATHWAY AND THE PREDICTION WOULD BE THAT IN A BRCAC1 AND 2 RESISTANT OR LACKING CELLS, WOULD BE PARTICULARLY SELECTIVE AND INHIBITED BY CANDIDATE PART INHIBITORS. AND WITH THAT, THERE WAS THE DEMONSTRATION OF A HARP INHIBITOR THAT HAS GONE ON TO CLINAL APPROVAL IN BRCAC1 AND 2 DELETED CARCINOMAS. A THIRD TYPE OF SCREEN IS TO AGAIN UTILIZE THE BIOCHEMICALLY EXPRESSED TARGET THAL CASE, THE CDC 25 PHOSPHATASES, CDC25 PHOSPHATASE IS OVEREXPRESSED IN MANY CANCER CELL TYPES. OVEREXPRESSION HAS BEEN DETECT INDEED A NUMBER OF CLINICAL VARIETIES AND IT COOPERATES WITH DIFFERENT ONCA PROTEINS IN THE DEVELOPMENT OF SO CALLED TRANSFORMED FOCI, THE KEY IS TO REMOVE THE PSYCHE LOW KINASE TO ALLOW CELL CYCLE PROGRESSION BY THE PROPERLY ACTIVATED PSYCHE LYNN DEPENDENT KINASE, SO IN ORDER TO IDENTIFY CDC 25 PHOSPHATASE, INHIBITORS, COLLEAGUES AT THE UNIVERSITY OF PITTSBURGH CREATED A BIOCHEMICAL ASSAY WHERE A FLUOROSEEN WAS LINKED TO A PHOSPHATE AND HAD AN ALTERED FLUORESCENCE ONCE THAT PHOSPHATE WAS REMOVED. USING THAT APPROACH, IT WAS POSSIBLE TO DETERMINE FAMILIES OF MOLECULES THAT HAD RELATED STRUCTURES AS SHOWN HERE RELATED TO A BEN ZOHAR QUINN OWN AND WHEN THESE COMPOUNDS WERE INCUBATED WITH CDC 25 IT WAS POSSIBLE TO SHOW THEY WERE SELECTIVE AND MORE POTENT IN INHIBITING CDC25 AND OTHER PHOSPHATASES SO THAT A BASIS FOR HAVING RELATIVE SELECTIVITY TO CELL CYCLE REGULATION COULD BE DEVELOPED AND IT COULD BE PROVEN THAT THE COMPOUNDS DO FUNCTION AGAINST THE CBC 25 BY USING A TEMPERATURE SENSITIVE CDK MANIPULATED CELL TYPE SO THAT AT THE PERMISSIVE TEMPERATURE, YOU HAD A FUNCTIONAL CDK THAT ALLOWED CELLS TO EXIST BOTH IN G1, AND SAND G2, AT THE NONPERMISSIVE HIGH TEMPERATURE, HAVE YOU NO FUNCTIONAL CDK AND THEREFORE THE CELLS ACCUMULATE IN THE G2 PHASE. SO WHAT WAS POSSIBLE TO SHOW IS TO VALIDATE THAT THE COMPOUND SERIES SELECTED BIOCHEMICALLY RECAPITULATED THE ABILITY TO CAUSE G2 M ARREST AT THE--WHEN THE CELLS ARE AT THE PERMISSIVE TEMPERATURE SO THAT IN THE ABSENCE OF ANY COMPOUND, HAVE YOU DISTRIBUTION ACROSS THE CELL CYCLE, A POSITIVE COTTER VOWEL BLOCKER PRODUCES A BLOCK, HOWEVER COMPOUND 5 AND INCREASING CONCENTRATIONS RECAPITULATES THE CAPACITY TO CAUSE A BREAK IN CELL CYCLE PROGRESSION, NOT REPRESENTED BY OTHER COMPOUNDS IN THE SERIES, SO THIS IS AN EXAMPLE OF USING BOTH THE BIOCHEMICAL AND THE CELLULAR READ OUT TO DEFINE A COMPOUND OF FURTHER INTEREST AND DEVELOPMENT. THE LAST CASE THAT WE'LL CONSIDER IS WHERE 1 INCORPORATES KNOWLEDGE OF THE STRUCTURE THAT 1 WISHES TO MODIFY AND FURTHER DEFINING LEADS. LET YOU CAN DO THIS IF YOU HAVE FOR EXAMPLE X-RAY CRYSTALLOGRAPHY INFLAMMATION AND PARTICULARLY IF YOU CAN CO CRYSTALLIZE A DRUG WITH A TARGET OR OF GREATER UTILITY HAS BEEN SO CALLED NMR BASED FRAGMENT AFFINITY APPROACHES. THIS IS EXEMPLIFIED OR COME SAYS FROM THE FACT THAT THE MOLECULES EXIST WITH POTENTIAL BINDING POCKETS ON THEIR SURFACES. OF GREAT INTEREST RECENTLY IS TO GENERATE RAF ISHT MEDIATES AS A WAY OF DEALING WITH THE VERY PROFOUND CHALLENGE OF FINDS RAF RELATED THERAPEUTICS. AND IN ESSENCE 1 USES THE COMPUTATIONAL MODELS OF A TARGET STRUCTURE TO PROVIDE AN UNDERSTANDING OF SO CALLED POCKET SPACE THAT IS DEFINED BY CONFIRMATIONS THAT THEN CAN BE COMPUTATIONALLY LINKED TO CANDIDATES, SMALL MOLECULES AND THEN 1 CAN USE THE NMR TO PROVE THAT A PARTICULAR COMPOUND IS ACTUALLY ACTIVE. USED FOR KINASE ANTAGONIST GENERATION AND CAN ALSO--HAS BEEN USED PROMINENTLY IN THE DERIFFATION OF BCL 2 RELATED FAMILY ANTAGONIST. THE REASON NMR BASED SCREENING IS OF VALUE IS THAT YOU CAN CHARACTERIZE NOT ONLY THE EXISTENCE OF BINDING AND INTERACTION BUT YOU CAN ALSO DEFINE A PORTION OF THE MOLECULE TO WHICH THEY CAN BYPASSED. QUITE SIGNIFICANTLY THE TECHNIQUE WORKS BEST AT RELATIVELY NONPOTENT WEAK AFFINITYS SO THAT YOU DON'T HAVE TO HAVE A GOOD IDEA OF WHAT A GOOD BINDER LOOKS LIKE AND THEN YOU CAN MATURE THROUGH THE SCREENING. YOU CAN ALSO LABEL THE PROTEIN OF INTEREST WITH ISOTOPE SENSITIVE TO LIGAND EFFECTS AND THEN UTILIZE THIS PROTURBATION OF THE DRUG TO PROVE THAT YOUR DRUG IS ACTUALLY FINDING THE SURFACE THAT IT WANTS. SO TO EXEMPLIFY THIS, THIS IS JUST--IT WILL REMIND US THAT WHEN A SMALL MOLECULE BINDS TO ITS TARGET, THE CAPACITY OF THE BOUND PORTION TO INTERACT OR WITH WATER MOLECULES IS DECREASED SO THE MOTION IS RESTRICTED AND AS A RESULT THERE IS AN ALTERATION OF ITS NMR PEAK SO THIS IS AN EXAMPLE OF BLEOMICEIN AND WHEN BOUND TO DNA AS AN ALTERATION OF ITS REG ILLEGALS RESONANCES BY ATTENUATION OF ABSORPTION WITHOUT MUCH EFFECT ON THE MOLECULE, IT'S KNOWN THAT IT BINDS THROUGH DNA THROUGH THAT PART OF THE MOLECULE. SO IF YOU WANT TO BUILD A DRUG LEAD, YOU WOULD TAKE YOUR TARGET THAT HAS DIFFERENT POCKETS THAT YOU ALREADY DEFINED AND MIGHT HAVE CANDIDATE ABILITY TO INTERACT WITH SMALL MOLECULES. INTRODUCE YOUR LEAD: AND SHOW EVIDENCE OF BINDING OF THE LEAD BY ALTERATION OF ITS--OF IT RESIDENCES. YOU CAN THEN DETYPE IN A SECOND LIBRARY OF COMPOUNDS THE CAPACITY TO BIND TO ANOTHER PART OF THE MOLECULE AND THEN IF YOU PUT THESE 2 BINDING DOMAINS TOGETHER, YOU COULD POTENTIALLY HAVE A MULTIPLICATION OF THAT AFFINITY ALLOWING OF THE BUILDING OF A MORE POTENT BINDING MOLECULE AND THIS WAS EXEMPLIFIED BY COMPOUNDS THAT WERE DIRECTED POTENTIALLY TO INHIBIT THE FUNCTION OF THE ONCA PROTEIN BCL XL, AND THE APOPTOTIC PROTEIN FROM THE BCL 2 TO ONCA PROTEIN FAMILY, AS YOU CAN SEE, THERE ARE DIFFERENT FRAGMENT STRUCTURES THAT HAVE DIFFERENT CAPACITIES TO INTERACT WITH RECOMBINANT BCL EXCEL AND WHEN THESE WERE PUT TOGETHER, YOU CAN SHOW IN BCL-XL THAT WAS LABELED WITH NITROGEN OR HYDRO GEN OR AMINO ACIDS AT SITES THAT COULD BEING PERTERBED AND PROVE THAT THESE WERE EFFECTED BY BINDING TO THESE 2 DIFFERENT AREAS AS SHOWN HERE. AND WHEN THESE WERE PUT TOGETHER, ULTIMATELY RESULT INDEED A CLINICAL CANDIDATE AND THESE HAVE BEEN POTENTIALLY VALUABLE IN A VARIETY OF DISEASE STATES INCLUDING CHRONIC LYMPHSIDIC LUNG CANCER KEEP CAIA. SO IN THE LAST PART OF PRESENTATION WE'LL TALK ABOUT ONCE YOU HAVE A LEAD STRUCTURE AND PROVEN ITSELF AND OPTIMIZED HOW DO YOU QUALIFY THAT FOR TRANSITION TO EARLY CLINICAL TRIALS. IN GENERAL, 1 WISHES TO OPTIMIZE EVIDENCE OF ACTIVITY, RELATE THE ACTIVITY TO THE CONCENTRATIONS AND DURATIONS OF DRUG EXPOSURE AND EXEMPLIFIED HERE ARE BENZ OIL FEN ILLEGALSEUREAS AT NCI THAT CAME FROM A CORPORATE DONOR AND THESE WERE BROADLY ACTIVE IN SCREENING TESTS IN DIFFERENT CELL TYPES AS SHOWN HERE. THE CAPACITY TO ACT AS INHIBITOR OF CELL GROWTH WAS ESTABLISHED AND FOUND TO BE RELATED TO THEIR INTERACTING WITH MICROTUBIALS IN A DIFFERENT WAY. THESE WERE STUDIED IN A VARIETY OF HUMAN TUMOR XENOGRAFTS ON A SCHEDULE THAT WOULD MIMIC POTENTIAL USE IN THE CLINIC. IT CAN BE SHOWN THAT AGAIN 1 OF THESE COMPOUNDS HAD EVIDENCE OF ACTIVITY IN A VARIETY OF DIFFERENT MODELS WITH INHIBITION OF TUMOR GROWTH. PHARMACOLOGY SUGGESTED IN A SERIES THAT THERE WAS THE CAPACITY TO HAVE A RELATIVELY LONG HALF LIFE AS A FUNCTION OF THE METHYLATION OF A SIDE CHAIN AND THIS ALLOWED SELECTION OF A MOLECULE THAT WOULD BE POTENTIALLY VALUABLE AS A CLINICAL CANDIDATE. TO DEFINE A SAFE STARTING DOSE IN HUMAN CLINICAL TRIALS, CURRENT REQUIREMENTS REQUIRE FOR DRUGS, 2 SPECIES, 1 RODENT AND 1 NONRODENT ASSAYED FOLLOWED THE CLINICAL ROUTE AND SCHEDULE IN THE CLINICS, PHARMACOKINETICS INFORMATION NEEDS TO BE INCORPORATED AND IN THE CASE OF BIOLOGICALS IT'S SOMEWHAT DIFFERENT IN THAT THERE'S ONLY 1 REQUIRED FOR MOST RELEVANT SPECIES TO THE BIOLOGICAL BUT AGAIN USING CLINICAL AND ROUTE SCHEDULE. SO WHEN THESE BENZOYLPH ENYLUREA, SHOWED A MAXIMUM TOLERATED DOSE SHOWN HERE IN THE RATS AND DOGS WERE SENSITIVE, THE DOSE LIMITING WAS A REFLECTION OF BONE MARROW AND GI TRACT AND SO, THE INITIAL HUMAN DOSE WAS SELECTED TO BE 1 10th OF THE MOST SENSITIVE SPECIES IN WHICH THIS WAS THE CASE OF THE DOG AT 2 KILOGRAMS PER METERS SQUARED. AND WHEN THE PROBLEM WITH USING THE SO CALLED MAXIMUM TOLERATED DOSE DRIVEN END POINT SYSTEM THAT I'VE SHOWN YOU IN DRUGS THAT ARE DIRECTED AGAINST REGULATORS OF ONCA GENIC GROWTH, THESE MIGHT NOT BE WELL REPRESENTED IN SO CALLED NORMAL TISSUES AND SO WHETHER DOSING BEYOND ITS EFFECT ON THE DESIRED TARGET IS OF VALUE IS NOT CLEAR AND THEREFORE A DISTINCTION DESIRE TO LOOK FOR A BIOLOGICALLY EFFECTIVE DOSE AS OPPOSE TO MAXIMUM TOLERATED DOSE HAS BEEN AN AREA OF INTEREST, WITH A USE OF THE BIOLOGIC RATHER THAN TOXIC END POINTS IN CLINICAL TRIALS. ANOTHER WAY TO THINK ABOUT THIS IS THAT RETURNING TO THE CONCEPT OF RATIONAL DRUG DISCOVERY, IF YOU KNOW WHAT YOUR TARGET IS WHEN YOU SELECT THE MOLECULE, YOU KNOW WHAT YOUR TARGET IS IN PROVING THAT THE MOLECULE IS POTENTIALLY OFA VALUE IN VIVO MODELS THAT IT'S IMPOSSIBLE TO INCORPORATE ITS EFFECT ON THE TARGET EITHER IN TOXICOLOGY AND CERTAINLY IN INITIAL PHASE 1 TO INFORM HOW TO ESCALATE DOSE AND SELECT A DOSE FOR SUBSEQUENT STUDIES. SO WHEN THIS APPROACH WAS VALUABLE, IT WAS IN THE MOLECULE SHOWN HERE. THIS IS 1 OF A NUMBER OF BORONIC ACIDS THAT WERE SUBMITTED BY THE PRO SCRIPT COMPANY, IT HAS THE RELATIVELY UNIQUE FEATURE OF THE BORONIC ACID AND THESE WERE ACTIVE IN A VARIETY OF PROLIFERATIVE SCREENS. AND THIS ENTITY HERE PS341 WAS ACTIVE AND ALL THESE MOLECULES THAT HAD BEEN DEVELOP INDEED PROTEOSOME RECEPTORS AND INFLAMMATORY AGENTS HAD AN EXACT CORRELATION BETWEEN THEIR CAPACITY TO ACT AS A PROTEOSOME INHIBITOR AND THEIR CAPACITY TO INHIBIT CELL GROWTH AND INHIBITING TUMOR GROWTHEN BETWEEN .3 AND 1 MILLIGRAM PER KILOGRAM AND WHEN 1 LOOKED AT THE INHIBITION BY THE COMPOUND OF PROTEOSOME ACTIVITY IN CIRCULATING WHITE BLOOD CELLS IN THE MOUSE AND IN THE TUMORS THAT THEY BORE, YOU COULD SEE THAT BY THE 0.3 TO 1 MILLIGRAM DOSE RANGE THERE'S ABOUT 80% INHIBITION OF PROTEOSOME ACTIVITY IN THE CIRCULATING WHITE BLOOD CELLS AND ABOUT A 60-70% INHIBITION OF THAT ACTIVITY IN THE TUMOR CELLS. AND WHEN THIS WAS--THIS LEVEL OF INHIBITION IN THE WHITE CELLS WAS COMPARED TO THE DOSES THAT WERE APPARENTLY AT THE MTD OF ACROSS SPECIES, YOU CAN SEE THAT LEGAL THERE WAS A CONSIDERABLE DIFFERENCE BETWEEN MOUSE, RAT AND DOG HUMAN PRIMATE AND THE DOSES THAT CAUSED TOXICITY THESE ALL TRACKED VERY CLOSELY TO A SIMILAR DEGREE OF INHIBITION OF THE PROTEOSOME IN PERIPHERAL WHITE BLOOD CELLS SO THAT LED TO AN INITIAL HUMAN EXPERIENCE WHERE THERE WAS ESCALATION OF DOSE, ONLY TO A LEVELED INHIBITED PROTEOSOME ACTIVITY IN THE PATIENTS CIRCULATING WHITE CELLS BY 80% AND THIS WAS IN A NUMBER OF DIFFERENT CENTERS, THE COMPANY AND NCI SPONSORED TRIALS AND WITHOUT GOING ABOVE THAT LEVEL OF INHIBITION, THERE WAS INITIAL EVIDENCE OF PATIENTS PARTICULARLY TREATED UNIVERSITY OF NORTH CAROLINA IN MULTIPLE MYELOMA AND THE DRUG HAS GONE ON TO APPROVAL FOR AND ACTUALLY PROTOTYPIC IN A SERIES OF AGENTS APPROVED FOR ACTIVITY AND MULTIPLE MYELOMA. LET SO TO SUMMARIZE PRECLINICAL DRUG STUDIES FROM DRUG DISCOVERY THROUGH DEVELOPMENT, SCREENING AND ULTIMATELY TOXICOLOGY STUDIES ARE ALL DIRECTED AT AIDING AND PROMOTING DLINICAL TRIALS TO ASSURE SAFETY AND VALUE OF AN INITIALLY EXPLORED REGIMEN AND PROVIDE SCIENTIFIC BASES FOR ASSESSING THE CLINICAL EFFECTS OF THE AGENT. AND I THINK AS THE FIELD MOVES FORWARD, THERE WILL CONTINUE TO BE GREAT INTEREST IN CORRELATING THE MOLECULAR EFFECTS OF AGENTS ON THEIR INTENDED TARGETS ALONG WITH USUAL PHARMACOLOGICAL AND TOXICOLOGICAL END POINTS AND TYING THESE BACK TO THE MOLECULAR MECHANISMS BY WHICH THEY ARE POSITIVE WORK. SO WITH THAT I THANK YOU IF ARE YOUR ATTENTION AND WILL BE HAPPY TO TAKE ANY QUESTIONS. [ APPLAUSE ]