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Two approaches to development of new drugs for Chagas Disease.

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Presentation on theme: "Two approaches to development of new drugs for Chagas Disease."— Presentation transcript:

1 Two approaches to development of new drugs for Chagas Disease

2 Two general approaches Target-based: study the biology of the organism to discover and target a weak link in metabolism or pathogenesis(eg HIV protease or RT inhibitors) Target-based: study the biology of the organism to discover and target a weak link in metabolism or pathogenesis(eg HIV protease or RT inhibitors) Diversity or phenotypic screens: new uses for existing drugs Diversity or phenotypic screens: new uses for existing drugs Targets: proteases, cyp51(Podust), kinases(Taunton)

3 Target Protease in T.cruzi- Biology Cruzain(cruzipain, gp57/51) is the major protease of T.cruzi, is expressed in all parasite stages, but is localized to unique compartments in each stage. Cruzain(cruzipain, gp57/51) is the major protease of T.cruzi, is expressed in all parasite stages, but is localized to unique compartments in each stage. In lysosome/endosome compartment of epimastigote(insect) stage. Helps to digest “stolen” blood meal in insect gut. In lysosome/endosome compartment of epimastigote(insect) stage. Helps to digest “stolen” blood meal in insect gut. May also be involved in epimastigote attachment to insect midgut(poster ontem!!) May also be involved in epimastigote attachment to insect midgut(poster ontem!!) At flagellar pocket of bloodstream trypomastigote stage. May play a role in generation of infective metacyclic tryps(Samuel Goldenberg) or cell interaction via kinin pathway activation(Julio Sharfstein). At flagellar pocket of bloodstream trypomastigote stage. May play a role in generation of infective metacyclic tryps(Samuel Goldenberg) or cell interaction via kinin pathway activation(Julio Sharfstein). On surface and in endosome compartment of intracellular amastigotes. May function in immune evasion. On surface and in endosome compartment of intracellular amastigotes. May function in immune evasion.

4 Amastigotes within mammalian cell(pH 7.4) Epimastigotes in insect vector (pH 5-6) Degrades NFkappa B Degrades hemoglobin

5 NFkappaB(green) is sequestered on the surface of amastigotes in wt parasites Wildtype Cruzain deficient NFkB signaling is interrupted including production of IL-12 Patricia Doyle de Engel Fails to establish infection

6 Searching for drug leads: “Get by with a little help from our friends” AMGEN ANANCOR-Anti- inflammatories ANORMED ARQULE ARRIS ASTRAZENECA AXYS CELERA-hit to lead CORVAS ESP GENZYME GSK ICONIX IMMUNEX KHEPRI KOSAN-17-DMAG MORVIS-DNDi NOVARTIS PFIZER PRAECIS PROTOTEK SEQUOIA TITAN

7 K11777 Ndao et al

8 Effect of protease inhibitor on intracellular parasites For mouse and dog studies see Barr et al, AAC;Engel et al JEM, Doyle et al AAC

9 Selective labeling of parasite protease in host cell

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11 Western blot of protease in parasite cells +/- inhibitor treatment

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13 Cocrystals of four vinylsulfone inhibitors(Bill Roush and Linda Brinen)

14 Lead optimization chemistry: altering drug metabolism and enhancing oral bioavailability

15 Ascites following heart failure in CAI-72 T.cruzi infected mice untreatedtreated Juan Engel and Patricia Doyle de Engel

16 untreated

17 treated

18 Troponin levels in untreated(#2) versus treated beagle dogs infected with T.cruzi (TREATED DAILY x7d WITH 50mg/Kg po-Stephen Barr, Cornell)

19 From lead to clinical candidate Metabolism and pharmacokinetics in primates Metabolism and pharmacokinetics in primates Safety studies in rodents, dogs and primates Safety studies in rodents, dogs and primates Calculation of equivalent human doses(HED) Calculation of equivalent human doses(HED) Confirmed human hepatocyte viability up to 100micromolar Confirmed human hepatocyte viability up to 100micromolar

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21 NONCOMPARTMENTAL PHARMACOKINETIC PARAMETERS for K11777 in CYNOMOLGUS MONKEYS AUC 0-24h (mg*h/L) AUC 0-  (mg*h/L) CL/F (L/h/kg) t ½ (h) V z (L/kg) C max (mg/L) Male CM Day Male CM Day Female CM Day Female CM Day CM (n=4) Day       0.88 CM (n=4) Day       1.38

22 Vinyl sulfone protease inhibitors Orally bioavailable(20% in dogs) Maximum tolerated dose >150mg/Kg(reversible ALT elevation at 300mg/Kg), NOAEL mg/Kg Projected therapeutic regimen 50mg/Kg Bid X15-28 days in mice, 5-10mg/Kg in humans Rescued mice from lethal acute T.cruzi infection(100%) Cured mice in model of late stage Chagasic heart disease(80%) Effective in immunodeficient mice Rescued dogs from lethal cardiac damage Effective against Nifurtimox resistant parasites Additive efficacy with benznidazole Also effective in mouse models of T.brucei, S.mansoni, and C.parvum infection Safety profile in rats, dogs and monkeys vastly better than current therapy, not mutagenic(SRI International, Chuck Litterst,NIAID) Application for Phase I trial in healthy volunteers now at US FDA

23 (The Scientist 19, #21,2005:”Pharmastart”)

24 Who is backing the effort? Funding: NIAID, Sandler Family Supporting Foundation, DNDi, MMV[Gates] Funding: NIAID, Sandler Family Supporting Foundation, DNDi, MMV[Gates] Medicinal Chemistry: Adam Renslo, Jim Wells(UCSF), Bill Roush(Scripps) Medicinal Chemistry: Adam Renslo, Jim Wells(UCSF), Bill Roush(Scripps) ADME: SRI International(Jon Mirsalis), Les Benet(UCSF), iOWH, NIAID ADME: SRI International(Jon Mirsalis), Les Benet(UCSF), iOWH, NIAID Process Chemistry: iOWH, now NIAID and Pharmastart(SRI) Process Chemistry: iOWH, now NIAID and Pharmastart(SRI) GMP synthesis, IND enabling: Phil Coyne, John Rogers and Beth Spinelli(NIAID)with Pharmastart GMP synthesis, IND enabling: Phil Coyne, John Rogers and Beth Spinelli(NIAID)with Pharmastart

25 High Throughput Structural Biology Pipeline 96-well format automated nano drop crystallization Automated imaging, tracking & strategies for crystal growth Robot driven screening of crystals in 96-sample cassettes at beam line

26 Screening for Novel Inhibitors by Molecular Docking ZINC >2 million compounds Structure determination Dock into site Test high-scoring molecules Target structure New inhibitor design Score by: electrostatics van der Waals solvation energy Brian Shoichet Rafaela Ferreiro

27 Virtual ligand libraries ZINK (http://blaster.docking.org/zinc/) Irwin and Shoichet, J. Chem. Inf. Model. 2005;45(1):177-82

28 project overview NIH library (~198,000 compounds)‏ NIH library (~198,000 compounds)‏ High throughput screening (NCGC)‏ High throughput screening (NCGC)‏ Virtual screening (DOCK – UCSF)‏ Virtual screening (DOCK – UCSF)‏ Experimental hits Computational hits COMPARISON

29 docking poses IC 50 = 11 uM, Rank 113 IC 50 = 1uM IC 50 = 260 nM Now in hit to lead chemistry

30 Two general approaches Diversity or phenotypic screens: new uses for existing drugs and development of high throughput screen for drugs targeting amastigotes in muscle cells. Diversity or phenotypic screens: new uses for existing drugs and development of high throughput screen for drugs targeting amastigotes in muscle cells.

31 HTS protocol to screen drugs against T.cruzi amastigotes in muscle cells-Juan Engel 1. To standardize the HTS protocol we used the T. cruzi cloned stocks CA-I/72 1,2 derived from an Argentine strain Cepa Argentina 1 (CA-I) isolated from a chronic Chagasic patient in However we tested and standardized the protocol for other T. cruzi populations from Brazil: Silvio-X10/7 3 cloned stock from the strain Silvio-X10 isolated from an infected insect and the Y strain 4 isolated from an acute case of human Chagas disease (Silva & Nussenzweig 1953). 2. To standardize the HTS protocol we used the primary cell line BESM (Bovine Embryo Skeletal Muscle Cells). We have Also tested and standardized the protocol for other cell lines: NIH- 3T3 cells a mouse embryonic fibroblast cell line, Huh7 cells a human hepatoma cell line and irradiated J774 mouse macrophage cell line. 3. We are using 96 well format. But it is possible to go to a 384 well format. 4. Reading method: is independent of the parasite strain used and is based on staining with DAPI the host nuclei versus the parasite’s kDNA. 5. Validation data: Standard deviation. Range , coefficient of variation range 13-28; Z' = ~ This protocol is not constrained to a specific T. cruzi population or host cell.

32 Image captured from untreated T. cruzi-infected cells using the IN Cell Analyzer 1000 with a 10x objective, 7 fields/well, exposure time 150 msec/field, 350/460 nm ex/em Partners for screening: ICONIX(completed drug library 16 parasite selective hits, 6 unique) Novartis Research Foundation-Rescreen of T.brucei and Leishmania hits

33 Natural products as a rich source of new leads  Phil Crews Roger Linington Scott Lokey (UCSC) Marine sponges and fungal metabolites  Now a six campus UC collaboration with SIO, Venter Institute, Amyris, UCB School of Natural Resources and Goldman Institute of Public Policy, 12 foreign sites  Phil Crews Roger Linington Scott Lokey (UCSC) Marine sponges and fungal metabolites  Now a six campus UC collaboration with SIO, Venter Institute, Amyris, UCB School of Natural Resources and Goldman Institute of Public Policy, 12 foreign sites

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35 Staff number only those supported by philantropy

36 A periodic table of the parasite posse Protein Structure, Assay Development, Biochemistry, Animal Models Cores


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