Two approaches to development of Two approaches to development of new drugs for Chagas Diseasenew drugs for Chagas Disease

James H. McKerrowJames H. McKerrow

Director, Director,

Sandler Center at Mission BaySandler Center at Mission Bay

University of California, San FranciscoUniversity of California, San Francisco

Two general approachesTwo general approaches

Target-based: study the biology of the Target-based: study the biology of the organism to discover and target a weak organism to discover and target a weak link in metabolism or pathogenesis(eg HIV link in metabolism or pathogenesis(eg HIV protease or RT inhibitors)protease or RT inhibitors)

Diversity or phenotypic screens: new uses Diversity or phenotypic screens: new uses for existing drugsfor existing drugs

Targets: proteases, cyp51(Podust), kinases(Taunton)

Target Protease in T.cruzi- BiologyTarget Protease in T.cruzi- Biology

Cruzain(cruzipain, gp57/51) is the major protease of T.cruzi, is Cruzain(cruzipain, gp57/51) is the major protease of T.cruzi, is expressed in all parasite stages, but is localized to unique expressed in all parasite stages, but is localized to unique compartments in each stage.compartments in each stage.

In lysosome/endosome compartment of epimastigote(insect) stage. In lysosome/endosome compartment of epimastigote(insect) stage. Helps to digest “stolen” blood meal in insect gut.Helps to digest “stolen” blood meal in insect gut.

May also be involved in epimastigote attachment to insect May also be involved in epimastigote attachment to insect midgut(poster ontem!!)midgut(poster ontem!!)

At flagellar pocket of bloodstream trypomastigote stage. May play a At flagellar pocket of bloodstream trypomastigote stage. May play a role in generation of infective metacyclic tryps(Samuel Goldenberg) role in generation of infective metacyclic tryps(Samuel Goldenberg) or cell interaction via kinin pathway activation(Julio Sharfstein).or cell interaction via kinin pathway activation(Julio Sharfstein).

On surface and in endosome compartment of intracellular On surface and in endosome compartment of intracellular amastigotes. May function in immune evasion.amastigotes. May function in immune evasion.

Amastigoteswithin mammalian cell(pH 7.4)

Epimastigotes ininsect vector(pH 5-6)

Degrades NFkappaB

Degrades hemoglobin

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

Wildtype Cruzain deficientNFkB signaling is interrupted including production of IL-12

Patricia Doyle de Engel

Fails to establish infection

Searching for drug leads: “Get by with a little Searching for drug leads: “Get by with a little help from our friends”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 Effect of protease inhibitor on intracellular parasitesintracellular parasites

For mouse and dog studies see Barr et al, AAC;Engel et al JEM, Doyle et al AAC

Selective labeling of parasite Selective labeling of parasite protease in host cellprotease 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

HN+ NH3C C NH

O

C

HN

O CH2

CH2

CH2

K11777

Cl-

S

O

O

Scheme 1

N-demethylation,N-oxidation

hydroxylation

HN+ N C NH

O

C

HN

O CH2

CH2

CH2

Cl-

S

O

O

WRR497

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

untreated treatedJuan Engel and Patricia Doyle de Engel

untreated

treated

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

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

From lead to clinical From lead to clinical candidatecandidate

Metabolism and pharmacokinetics in Metabolism and pharmacokinetics in primatesprimates

Safety studies in rodents, dogs and Safety studies in rodents, dogs and primatesprimates

Calculation of equivalent human Calculation of equivalent human doses(HED)doses(HED)

Confirmed human hepatocyte viability up Confirmed human hepatocyte viability up to 100micromolarto 100micromolar

Plasma levels of K11777 and its major metabolites in male cynomolgus monkeys

following an oral dose (200 mg/kg)

time (h)

0 5 10 15 20 25

[K7

7]

ug

/mL

0

1

2

3

4

[K77]d1

[K77]d7

[norK77]d1 [norK77]d7 [K77Nox]d1 [K77Nox]d7

time (h)

0 5 10 15 20 25

[K77

] u

g/m

L

0

1

2

3

4

[K77]d1

[K77]d7

[norK77]d1 [norK77]d7 [K77Nox]d1[K77Nox]d7

Male 15730 Male 14902

NONCOMPARTMENTAL PHARMACOKINETIC PARAMETERS for

K11777 in CYNOMOLGUS MONKEYSAUC0-24h

(mg*h/L)

AUC0-

(mg*h/L)

CL/F

(L/h/kg)

t ½

(h)

Vz

(L/kg)

Cmax

(mg/L)

Male CM

Day 122.4 22.7 9.9 3.3 47.2 2.75

Male CM

Day 725.5 27.6 7.6 5.6 57.1 3.00

Female CM

Day 123.0 23.3 10.1 4.2 64.7 3.25

Female CM

Day 734.3 34.7 6.1 3.1 25.3 4.60

CM (n=4)

Day 1

22.7

9.5

22.9

0.80

10.0

4.2

3.9

0.4

56.1

28.0

3.00

0.88

CM (n=4)

Day 7

29.9

9.1

31.2

8.6

6.8

2.0

4.4

2.2

41.3

22.0

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?Who is backing the effort? Funding: NIAID, Sandler Family Supporting Foundation, Funding: NIAID, Sandler Family Supporting Foundation,

DNDi, MMV[Gates]DNDi, MMV[Gates] Medicinal Chemistry: Adam Renslo, Jim Wells(UCSF), Medicinal Chemistry: Adam Renslo, Jim Wells(UCSF),

Bill Roush(Scripps)Bill Roush(Scripps) ADME: SRI International(Jon Mirsalis), Les ADME: SRI International(Jon Mirsalis), Les

Benet(UCSF), iOWH, NIAIDBenet(UCSF), iOWH, NIAID Process Chemistry: iOWH, now NIAID and Process Chemistry: iOWH, now NIAID and

Pharmastart(SRI)Pharmastart(SRI) GMP synthesis, IND enabling: Phil Coyne, John Rogers GMP synthesis, IND enabling: Phil Coyne, John Rogers

and Beth Spinelli(NIAID)with Pharmastartand Beth Spinelli(NIAID)with Pharmastart

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

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:electrostaticsvan der Waalssolvation energy

Brian Shoichet

Rafaela Ferreiro

Virtual ligand librariesVirtual ligand librariesZINK (ZINK (http://blaster.docking.org/zinc/http://blaster.docking.org/zinc/))

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

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 hitsExperimental hits Computational hitsComputational hitsCOMPARISON

docking poses

IC50 = 11 uM, Rank 113

O

NH

O

O

O

RN

O

OO

O

HN

O

MeO

Cl

IC50 = 1uM

IC50 = 260 nMNow in hit to lead chemistry

Two general approachesTwo general approaches

Diversity or phenotypic screensDiversity or phenotypic screens: new uses : new uses for existing drugs and development of high for existing drugs and development of high throughput screen for drugs targeting throughput screen for drugs targeting amastigotes in muscle cells.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/721,2 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-X10/73 cloned stock from the strain Silvio-X10 isolated from an infected insect and the Y strain4 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

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

DirectorMcKerrow

Co-DirectorsDeRisi, Rosenthal

Animal Models/CultureScreensAbdulla, Doyle, Engel, Rosenthal

Technical Staff2.5

DrugMetabolismBenet, Mirsalis (SRI)

Technical Staff1.0

MedChem/SMDCRenslo

Technical Staff2.5

HTSArkin

Technical Staff1.5

StructuralBiology/CrystallographyBrinen

Technical Staff1.0

Bioinformatics /ComputationBabbitt, Jacobson, Sali, Shoichet

Technical Staff1.5

Biochemistry/ ProteinProductionCaffrey, Craik, Sakanari

Technical Staff4.0

Genomics/ProteomicsBurlingame, DeRisi

Technical Staff1.25

MicroscopyandImagingEngel

Technical Staff1.0

Scienti cAdvisoryBoardColley, Litterst, Roush

ExecutiveBoardDeRisi, McKerrow, Rosenthal,

Sali, Wang, Wells

Industrial andGovernmentCollaborations

SandlerCenterOrganization

Chart

ProjectManager(Robertson)

QB3RegulatoryAffairsandIndustryLiaison(Saxton)

Communications Coordinator(Soto)

Staff number only those supported by philantropy

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