Two approaches to development of new drugs for Chagas Disease

 James H. McKerrow Director, Sandler Center at Mission Bay University of California, San Francisco

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)

 Diversity or phenotypic screens: new uses for existing drugs Targets: proteases , cyp51(Podust), kinases(Taunton)

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.

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!!) 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.

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

NFkappaB(green) is sequestered on the surface of amastigotes in wt parasites

Wildtype NFkB signaling is interrupted including production of IL-12 Cruzain deficient Fails to establish infection Patricia Doyle de Engel

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

K11777 Ndao

et al

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

Selective labeling of parasite protease in host cell

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

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

Lead optimization chemistry: altering drug metabolism and enhancing oral bioavailability Scheme 1 N-demethylation, N-oxidation H 3 C HN+ Cl-

K11777

N C O N H CH 2 H N C O CH 2 CH 2 O S O hydroxylation HN+ Cl N C O N H CH 2 C O H N CH 2 CH 2

WRR497

O S O

Ascites following heart failure in CAI-72 T.cruzi infected mice

untreated treated Juan Engel and Patricia Doyle de Engel

untreated

treated

Troponin levels in untreated(#2) versus treated beagle dogs infected with T.cruzi

(TREATED DAILY x7d WITH 50mg/Kg po-Stephen Barr, Cornell)

From lead to clinical candidate

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

2 4 3 Plasma levels of K11777 and its major metabolites in male cynomolgus monkeys following an oral dose (200 mg/kg) 4 Male 15730 [K77]d1 [K77]d7 [norK77]d1 [norK77]d7 [K77Nox]d1 [K77Nox]d7 3 Male 14902 [K77]d1 [K77]d7 [norK77]d1 [norK77]d7 [K77Nox]d1 [K77Nox]d7 2 1 1 0 0 5 10 15 time (h) 20 25 0 0 5 10 15 time (h) 20 25

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

l

z (L/kg) C max (mg/L) Male CM Day 1 Male CM Day 7 Female CM Day 1 Female CM Day 7 CM (n=4) Day 1 CM (n=4) Day 7 22.4

25.5

23.0

34.3

22.7



9.5

29.9



9.1

22.7

27.6

23.3

34.7

22.9



0.80

31.2



8.6

9.9

7.6

10.1

6.1

10.0



4.2

6.8



2.0

3.3

5.6

4.2

3.1

3.9



0.4

4.4



2.2

47.2

57.1

64.7

25.3

56.1



28.0

41.3



22.0

2.75

3.00

3.25

4.60

3.00



0.88

3.80



1.38

Vinyl sulfone protease inhibitors

• • • • • • • • • • • • Orally bioavailable(20% in dogs) Maximum tolerated dose >150mg/Kg(reversible ALT elevation at 300mg/Kg

), NOAEL 50-100mg/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

(

The Scientist

19, #21,2005:”Pharmastart”)

Who is backing the effort?

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

High Throughput Structural Biology Pipeline

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

Screening for Novel Inhibitors by Molecular Docking Target structure Dock into site

Score by: electrostatics van der Waals solvation energy Brian Shoichet Rafaela Ferreiro

ZINC >2 million compounds Test high-scoring molecules Structure determination New inhibitor design

Virtual ligand libraries

ZINK ( http://blaster.docking.org/zinc/ )

Irwin and Shoichet, J. Chem. Inf. Model. 2005;45(1):177-82

project overview

NIH library (~198,000 compounds) High throughput screening (NCGC) Virtual screening (DOCK – UCSF) Experimental hits

COMPARISON

Computational hits

R O O O N H O

docking poses O N O O O O HN MeO Cl

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

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.

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 1980. 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 0.02-0.16, coefficient of variation range 13-28; Z' = ~ 0.55..

6. This protocol is

not constrained to a specific T. cruzi population or host cell

.

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

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

Indus trial and Government Collaborations Scienti c Advis ory Bo ard

Colley, Litterst, Roush

Communications Coordinator (So to ) Executive Board

DeRisi, McKerrow, Rosenthal, Sali, Wang, Wells

S andle r Ce nte r Org anizatio n Chart Project Manager (Robe rts o n) QB3 Re gulatory Affairs and Indus try Liais on (S axton) MedChem / SMDC

Renslo Technical Staff 2.5

Director

McKerrow

Co-Directors

DeRisi, Rosenthal

Geno mics /Pro teo mics

Burlingame, DeRisi Technical Staff 1.25

Bio info rmatics / Co mputatio n

Babbitt, Jacobson, Sali, Shoichet Technical Staff 1.5

Drug Metabolis m

Benet, Mirsalis (SRI) Technical Staff 1.0

S truc tural Biology / Crys tallog raphy

Brinen Technical Staff 1.0

Animal Mo dels /Culture S creens

Abdulla, Doyle, Engel, Rosenthal Technical Staff 2.5

Staff number only those supported by philantropy

HTS

Arkin Technical Staff 1.5

Bio chemis try / Protein Production

Caffrey, Craik, Sakanari Technical Staff 4.0

Micros co py and Imaging

Engel Technical Staff 1.0

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