1. Edward Bayham, MSBME, MBA Vice President, Business Development IBE 2007 March30,2007 St. Louis, Missouri Automated Antiviral Drug Screening Using Engineered Replication Systems

2. Apath Copyright 2007 Antiviral Market Opportunities Big Markets (Chronic/high prevalence/high incidence) Human immunodeficiency virus (HIV) Hepatitis C Virus (HCV) Herpes viruses (HSV, VZV, EBV, CMV) Modest Markets (acute/high incidence) Influenza (A & B) Respiratory syncytial virus (RSV) Niche Markets (acute/low incidence) Respiratory viruses (SARS, PIV1-3, hMPV) Enteric viruses (rotavirus, enteroviruses, caliciviruses) Encephalitis viruses (VEE, JE, TBE) Hepatitis viruses (hepatitis A, hepatitis B, hepatitis E) Hemorrhagic fever viruses (Ebola, Marburg, Lassa fever, etc.)

3. Apath Copyright 2007 Serious Viral Threats Avian Influenza/SARS Bioterrorism Threats »Hemorrhagic fever viruses »Venezuelan Equine Encephalitis

4. Apath Copyright 2007 What is a Virus? Virus: poison (Latin) Submicroscopic entity that exists on the edge between biology and chemistry (between life forms and inanimate matter)

5. Apath Copyright 2007 What is the size of a virus? cell bacterium virus 1 micron (one millionth of a meter)

6. Apath Copyright 2007 Antiviral Drug Discovery Challenges Containment – Biosafety Level 4 Lack of reliable animal models Very specific host cell types Measuring efficacy in vitro

7. Apath Copyright 2007 Apath Apath is an early stage drug discovery company focused on antiviral drug discovery Drug discovery platform is well suited to bioterrorism agents Screening platform based on subgenomic (replicons) and full length replication systems 10 viruses (including 4 biodefense pathogens)

8. Apath Copyright 2007 Ebola outbreaks 1975 1967 2004/5 1987 1980 1998-2000 422 cases (356 deaths; >80% case-fatality) 1996 2000 1996 1995 1994 1977 1975 1976 1979 2001 2005 Marburg outbreaks

9. Apath Copyright 2007 Ebola virus family: Filoviridae (filo (Latin): ‘threadlike’) enveloped genome: negative-sense, single-stranded RNA, 19 kb Viral Proteins: L = polymerase VP35 = polymerase cofactor NP = nucleoprotein VP30= transcription factor GP = glycoprotein VP40 = matrix protein VP24 = matrix protein L VP30 VP24 sGP/GPVP40VP35NP EBOV EM image Frederick A. Murphy, CDC

10. Apath Copyright 2007 Trailer -5’3’- Leader 3’5’ Reporter gene NP L N VP35 L Replication and transcription (expression of reporter gene) VP30 ‘minigenome’ Replication Transcription enhancer EBOV subgenomic replication L VP30 VP24 sGP/GPVP40VP35NP

11. Apath Copyright 2007 Rationale for replicon-based screen Focus on viral RNA replication, transcription and translation of viral proteins Cell-based assay that can be carried out at BSL-1/2 Automated/High-throughput screening capability

12. Apath Copyright 2007 T7 pol expression vectors for: NP VP35 L VP30 Reporter gene expression is dependent on viral proteins Minigenome with reporter gene Renilla luc pT7 GSGE leadertrailer EBO-Rluc:167 ng NP:208 ng VP35:208 ng L:208 ng Signal: ca 150000 LCPS Noise: ca 100 LCPS S/N:1500

13. Apath Copyright 2007 EBO-Rluc screening setup Transfect EBO-Rluc minigenome NP, VP35, L, VP30 Trypsinize and freeze in aliquots 24 hours PBS wash/20 ul lysis buffer Measure luciferase Thaw and plate into 96 well plate 4 hours Add compounds (1% DMSO)

14. Apath Copyright 2007 Ebola-GFP recombinant virus Zaire strain of Ebola virus High level of GFP expression Not cytolytic Ebola-GFP infection assay: Vero E6 cells in 96 plates Infection (MOI = 0.1) IFN a control (IC90) 48h incubation Formalin fixation Wash out formalin with PBS and soak in PBS (1h) GFP detection:Spectrofluorometer (bottom read) Signal to noise: S/N = >12 Cytotoxicity: crystal violet staining (CC50 @ Apath by ATP-content) L VP30 VP24GPVP40VP35NP GFP BCL-4 Towner et al. Virology: 332(1):20-7; Feb. 5, 2005

15. Apath Copyright 2007 Screening protocol Primary screen (Minigenome) 25  M, n=1 EC50/CC50 (Minigenome) EC50/CC50 (EBO virus) Lead candidates 5 concentrations n=4 EC 50 <10  M SI >10 EC 50 <10  M SI >10 > 80% inhibition

16. Apath Copyright 2007 Novel 2 0 Sulfonamide lead candidates EC50 3.4 CC50: >75 EC50 1.8 CC50: >75

17. Apath Copyright 2007 Summary Subgenomic replication represents a useful cell- based screening tool for identifying inhibitors of viruses (particularly BSL3 and 4 viruses). A number of lead candidates have been identified. Novel sulfonamide lead compounds have been identified Mouse efficacy studies have been initiated at USAMRIID under Project Bioshield