Discovery and Development of SARS-CoV 3CL Protease Inhibitors

Transcription

1 Discovery and Development of SARS-CoV 3CL Protease Inhibitors Amy K. Patick, Ph.D. Group Director, Head of Virology Pfizer Global Research and Development La Jolla Laboratories

2 SARS: verview First known case of atypical pneumonia Nov in Guandong Province, China; first official outbreak reported to WH Feb. 2003

3 SARS: verview (2) Disease characterized by fever, malaise, dry cough, dyspnea, hypoxaemia, GI symptoms, lymphopeania, elevated aminotransferase levels. Death due to progressive respiratory failure. As of August 2003: 8422 cases; 916 deaths (11% CFR). Virus isolated from nasopharyngeal aspirates, BAL, sputum, kidney, urine, feces, plasma.

4 SARS: Coronavirus (CoV) Identified 1 CPE in cell culture and EM: RT-PCR analysis confirms identity Demonstration of seroconversion Interstitial pneumonia in monkeys 1. C. Drosten, et al. N. Engl. J. Med. 2003, 348:20, 1967; T.Ksiazek, et al. N. Engl. J. Med. 2003, 348:20, 1953; J. Peiris, et al. Lancet, 2003, 361, 1319; R. Fouchier, et al. Nature, 2003, 423, 240

5 SARS: A New CoV 1. T.Ksiazek, et al. N. Engl. J. Med. 2003, 348:20, 1953

6 CoV: verview Large, enveloped (+) stranded RNA viruses Respiratory and enteric disease in man and animals 2 human CoVs cause 15-30% of colds with seasonal incidence Fatal systemic and epizootic disease in animals: FIPV, HEV, IBV, TGEV Narrow host range and cell/tissue specificity

7 CoV Life Cycle 1. J. Ziebuhr, et al. J. Gen. Virol. 2000, 81, J. Ziebuhr, et al. J. Gen. Virol. 2000, 81, 853

8 SARS CoV 3CL Protease CoV known to encode critical enzyme (3CL) homologous with picornavirus 3C protease 1 Substrate specificity (Q/S,G) Cysteine as active site nucleophile 1. J. Ziebuhr, et al. J. Gen. Virol. 2000, 81, 853

9 CoV 3CL Protease Structural similarities also noted Purple: Human rhinovirus (HRV- 2) 3C protease 1 Green: porcine transmissible gastroenteritis coronavirus (TGEV) 3CL protease 2 1. D.A. Matthews, et al. Proc. Natl. Acad. Sci. USA 1999, 96, K. Anand, et al. EMB J. 2002, 21, 3213.

10 HRV 3C Protease Inhibitors Project Summary X-Ray crystal structure of HRV 3CP solved in 1994 Peptidomimetic irreversible protease inhibitor incorporating ester as Michael acceptor developed AG7088 (Ruprintrivir) selected and formulated for intranasal delivery. Potent in vitro antiviral activity against diverse rhinovirus and other picornaviruses (mean EC90 = 82 nm) Clinical studies: Phase I: Safe and well tolerated Phase II challenge: moderated illness severity when initiated either before or one day after rhinovirus infection Phase II (natural infection): no antiviral activity or reduction in symptom severity D.A. Matthews, et al. Cell 1994, 77, 761; P. Dragovich, et al. J. Med. Chem. 1999, 42, 1213; A. Patick, et al. Antimicrob Agents and Chemother. 1999, 43, 2444

11 HRV Relationship Homology modeling suggests Michael acceptorcontaining HRV-3CP inhibitors may also interact with SARS 3CL 1,2 Pink: HRV-2 3C protease 3 Cyan: SARS-CoV 3CL protease homology model 2 1. K. Anand et al. Science 2003, 300, D.A. Matthews et al. Bioorg. Med. Chem. Lett. 2003, submitted. 3. D.A. Matthews, et al. Proc. Natl. Acad. Sci. USA 1999, 96,

12 SARS CoV: In vitro antiviral assay (USAMRIID, SRI) SARS-CoV infection of Vero 76 cells Cell viability assay using neutral red or MTS as endpoint Cell Ctrl Virus Ctrl

13 AG-7088 vs SARS? AG-7088 not optimized for SARS 3CL (multiple hypotheses why) 1,2 Poor fit to SARS 3CL 2 NH N N H N H C 2 Et F Easily accommodated by SARS 3CL 1 para-f too large for SARS 3CL but H may fit 1 1. K. Anand et al. Science, 2003, 300, D.A. Matthews et al. Bioorg. Med. Chem. Lett. 2003, submitted.

14 AG-7088 vs SARS? AG-7088 does not inhibit SARS-CoV in vitro NH N N H N H C 2 Et EC 50 >167 µg/ml CC 50 >167 µg/ml (Strain ) F D.A. Matthews et al. Bioorg. Med. Chem. Lett. 2003, submitted.

15 ther Michael Acceptors Truncated molecule (AG-7122) exhibits in vitro anti-sars-cov activity NH N H C 2 Et EC 50 = 14.1 µg/ml CC 50 >100 µg/ml (Strain ) Circles = SARS-infected cells (AV) Squares = uninfected cells (cytotox.) D.A. Matthews et al. Bioorg. Med. Chem. Lett. 2003, submitted.

16 Next Steps Evaluate physiochemical properties of active SARS-CoV 3CL protease inhibitors Utilize structure-based design and combinatorial synthesis strategies to design novel peptidomimetic 3C protease inhibitors

17 Key Learnings Rapid identification of etiological agent Rapid access to key scientific publications Existence of significant knowledge base, technical expertises and compounds (3C protease inhibitors) Efficient external networking and deep sense of collaboration Antiviral assay in development

18 Key Learnings Streamline processes Establish infrastructure and identify single points of contacts Early and frequent communication

19 Acknowledgements Virology (La Jolla) George Smith Mary Brothers Chemistry Ted Johnson Siegfried Reich Pete Dragovich Crystallography Dave Matthews Comp. Chemistry Peter Rose Paul Rejto NIAID/NIH Catherine Laughlin Christopher Tseng SRI Jack Secrist Jeff Hogan Tom Voss USAMRIID John Huggins Robert Baker Chris Hartmann Eric Mucker Susan Zwiers