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IBE 2007
March30,2007
St. Louis, Missouri
Automated Antiviral Drug Screening
Using Engineered Replication Systems
Edward Bayham, MSBME, MBA
Vice President, Business Development
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.)
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Serious Viral Threats
Avian Influenza/SARS
Bioterrorism Threats
»Hemorrhagic fever viruses
»Venezuelan Equine Encephalitis
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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?
bacterium
cell
1 micron (one millionth of a meter)
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virus
Antiviral Drug Discovery Challenges
Containment – Biosafety Level 4
Lack of reliable animal models
Very specific host cell types
Measuring efficacy in vitro
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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)
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Ebola outbreaks
Marburg outbreaks
1975
1979
2000
1994
1996
2001 1976
2005 1995
1977
1967
1998-2000
2004/5
1996
1980
1987
1975
422 cases (356 deaths; >80% case-fatality)
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Ebola virus
•
•
•
family: Filoviridae (filo (Latin): ‘threadlike’)
enveloped
genome: negative-sense, single-stranded RNA, 19 kb
NP
VP35
VP40
sGP/GP
VP30
VP24
L
EBOV
Viral Proteins:
L = polymerase
VP35 = polymerase cofactor
NP = nucleoprotein
VP30= transcription factor
GP = glycoprotein
VP40 = matrix protein
VP24 = matrix protein
EM image
Frederick A. Murphy, CDC
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EBOV subgenomic replication
NP
VP35
VP40
sGP/GP
VP30
VP24
L
Trailer
Leader
3’-
-5’
L
NP
VP35
Reporter gene
3’
VP35
VP35
NP NP NP
NP N NP NP
Transcription
enhancer
5’
L
‘minigenome’
Replication
VP30
Replication and transcription
(expression of reporter gene)
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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
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Reporter gene expression is
dependent on viral proteins
Minigenome with reporter gene
pT7
GS
Renilla luc
leader
GE
trailer
Rluc (LCPS)
EBO-Rluc alone
2220
2500
2000
1500
1000
500
0
780
330
110
167 ng
250 ng
333 ng
417 ng
EBO-Rluc (ng)
250000
Rluc (LCPS)
T7 pol expression vectors for:
• NP
• VP35
• L
• VP30
200000
150000
100000
50000
0
0
Signal: ca 150000 LCPS
Noise: ca
100 LCPS
S/N:
1500
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21
42
84
VP30 (ng)
EBO-Rluc:
NP:
VP35:
L:
167 ng
208 ng
208 ng
208 ng
EBO-Rluc screening setup
Transfect EBO-Rluc minigenome
NP, VP35, L, VP30
Trypsinize and freeze in aliquots
Thaw and plate into 96 well plate
4 hours
Add compounds (1% DMSO)
24 hours
PBS wash/20 ul lysis buffer
Measure luciferase
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Ebola-GFP recombinant virus
NP
GFP
VP35
VP40
GP
VP30
VP24
L
• 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)
BCL-4
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)
Towner et al. Virology: 332(1):20-7; Feb. 5, 2005
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Screening protocol
Primary screen
(Minigenome)
25 mM, n=1
> 80% inhibition
EC50/CC50
(Minigenome)
EC50 <10 mM
SI >10
EC50/CC50
(EBO virus)
EC50 <10 mM
SI >10
Lead candidates
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5 concentrations
n=4
Novel 20 Sulfonamide lead candidates
AP80552
AP80175
120
100
100
80
80
60
EBOV GFP EC50
VeroE6 CC50
40
20
% inhibition
% inhibition
120
60
EBOV GFP EC50
VeroE6 CC50
40
20
0
-20
0
-40
-20
0.1
1
10
Log m M
EC50 3.4
CC50: >75
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100
0.1
1
10
Log mM
EC50 1.8
CC50: >75
100
Summary
Subgenomic replication represents a useful cellbased 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
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