Edward Bayham, MSBME, MBA Vice President, Business Development IBE 2007 March30,2007 St. Louis, Missouri Automated Antiviral Drug Screening Using Engineered Replication Systems
Edward Bayham, MSBME, MBAVice President, Business Development
IBE 2007March30,2007
St. Louis, Missouri
Automated Antiviral Drug Screening Using Engineered Replication Systems
Apath Copyright 2007
Antiviral Market OpportunitiesAntiviral Market OpportunitiesBig 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.)
Apath Copyright 2007
Serious Viral ThreatsSerious Viral Threats
Avian Influenza/SARS
Bioterrorism Threats»Hemorrhagic fever viruses»Venezuelan Equine Encephalitis
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What is a Virus?What is a Virus?Virus: poison (Latin)Submicroscopic entity that exists on the edge between biology and chemistry (between life forms and inanimate matter)
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What is the size of a virus?What is the size of a virus?
cellbacterium
virus
1 micron (one millionth of a meter)
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Antiviral Drug Discovery ChallengesAntiviral Drug Discovery Challenges
Containment – Biosafety Level 4
Lack of reliable animal models Very specific host cell typesMeasuring efficacy in vitro
Apath Copyright 2007
ApathApath
Apath is an early stage drug discovery company focused on antiviral drug discovery
Drug discovery platform is well suited to bioterrorism agentsScreening platform based on subgenomic (replicons) and full length replication systems • 10 viruses (including 4 biodefense pathogens)
Apath Copyright 2007
Ebola outbreaksEbola outbreaks
1975
1967
2004/5198719801998-2000
422 cases (356 deaths; >80% case-fatality)
1996
2000
1996
1995
1994 1977
1975
1976
1979
20012005
Marburg outbreaksMarburg outbreaks
Apath Copyright 2007
Ebola virusEbola virus• family: Filoviridae (filo (Latin): ‘threadlike’)• enveloped• genome: negative-sense, single-stranded RNA, 19 kb
Viral Proteins:L = polymeraseVP35 = polymerase cofactorNP = nucleoproteinVP30= transcription factorGP = glycoproteinVP40 = matrix proteinVP24 = matrix protein
LVP30 VP24sGP/GPVP40VP35NP EBOV
EM imageFrederick A. Murphy, CDC
Apath Copyright 2007
Trailer-5’3’-
Leader
3’ 5’Reporter gene
NP
L
NPN
NPNP
NPNP
NP VP35
VP35VP35L
Replication and transcription(expression of reporter gene)
VP30
‘minigenome’
Replication
Transcriptionenhancer
EBOV subgenomic replicationEBOV subgenomic replication
LVP30 VP24sGP/GPVP40VP35NP
Apath Copyright 2007
Rationale for repliconRationale for replicon--based screenbased screen
Focus on viral RNA replication, transcription and translation of viral proteinsCell-based assay that can be carried out at BSL-1/2Automated/High-throughput screening capability
Apath Copyright 2007
T7 pol expression vectors for:• NP• VP35• L• VP30
Reporter gene expression is Reporter gene expression is dependent on viral proteinsdependent on viral proteins
Minigenome with reporter geneEBO-Rluc alone
110 330780
2220
0500
1000150020002500
167 ng 250 ng 333 ng 417 ng
EBO-Rluc (ng)
Rlu
c (L
CPS
)
Renilla lucpT7GS GE
leader trailer
050000
100000150000200000250000
0 21 42 84
VP30 (ng)
Rlu
c (L
CPS
)
EBO-Rluc: 167 ngNP: 208 ngVP35: 208 ngL: 208 ng
Signal: ca 150000 LCPSNoise: ca 100 LCPSS/N: 1500
Apath Copyright 2007
EBOEBO--Rluc screening setupRluc screening setupTransfect EBO-Rluc minigenome
NP, VP35, L, VP30
Trypsinize and freeze in aliquots
24 hours
PBS wash/20 ul lysis bufferMeasure luciferase
Thaw and plate into 96 well plate
4 hours
Add compounds (1% DMSO)
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EbolaEbola--GFP recombinant virusGFP 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)
LVP30 VP24GPVP40VP35NP GFP
BCL-4
Towner et al. Virology: 332(1):20-7; Feb. 5, 2005
Apath Copyright 2007
Screening protocolScreening protocolPrimary screen (Minigenome)
25 μM, n=1
EC50/CC50 (Minigenome)
EC50/CC50 (EBO virus)
Lead candidates
5 concentrationsn=4
EC50 <10 μMSI >10
EC50 <10 μMSI >10
> 80% inhibition
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Novel 2Novel 200 Sulfonamide lead candidatesSulfonamide lead candidates
AP80552
-20
0
20
40
60
80
100
120
0.1 1 10 100
Log μM
% in
hibi
tion
EBOV GFP EC50VeroE6 CC50
EC50 3.4CC50: >75
AP80175
-40
-20
0
20
40
60
80
100
120
0.1 1 10 100
Log μM
% in
hibi
tion
EBOV GFP EC50VeroE6 CC50
EC50 1.8CC50: >75
Apath Copyright 2007
SummarySummary
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