edward bayham, msbme, mba vice president, business development ibe 2007 march30,2007 st. louis,...

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.)


Serious Viral ThreatsSerious Viral Threats

Avian Influenza/SARS

Bioterrorism Threats»Hemorrhagic fever viruses»Venezuelan Equine Encephalitis


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)

http://www.uct.ac.za/depts/mmi/stannard/adeno.html

http://www.bocklabs.wisc.edu/movies/r14_ico.html

http://life.anu.edu.au/viruses/ICTVdB/em_pox.gif


What is the size of a virus?What is the size of a virus?

cellbacterium

virus

1 micron (one millionth of a meter)


Antiviral Drug Discovery Antiviral Drug Discovery ChallengesChallenges

Containment – Biosafety Level 4

Lack of reliable animal models Very specific host cell typesMeasuring efficacy in vitro


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)


Ebola outbreaksEbola outbreaks

1975

1967

2004/5198719801998-2000

422 cases (356 deaths; >80% case-fatality)

1996

2000

1996

1995

1994 1977

1975

1976

1979

2001

2005

Marburg outbreaksMarburg outbreaks


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 image Frederick A. Murphy, CDC

http://www.ncbi.nlm.nih.gov/ICTVdb/Images/Murphy/Ebola1.jpg


Trailer

-5’3’-Leader

3’ 5’Reporter gene

NP

L

NPN

NPNP

NPNP

NP VP35

VP35VP35

L

Replication and transcription(expression of reporter gene)

VP30

‘minigenome’

Replication

Transcriptionenhancer

EBOV subgenomic EBOV subgenomic replicationreplication

LVP30 VP24sGP/GPVP40VP35NP


Rationale for replicon-based Rationale for replicon-based screenscreen

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


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 gene

EBO-Rluc alone

110330

780

2220

0

5001000

15002000

2500

167 ng 250 ng 333 ng 417 ng

EBO-Rluc (ng)

Rlu

c (

LC

PS

)

Renilla lucpT7GS GE

leader trailer

0

50000

100000

150000

200000

250000

0 21 42 84

VP30 (ng)

Rlu

c (

LC

PS

)

EBO-Rluc: 167 ngNP: 208 ngVP35: 208 ngL: 208 ng

Signal: ca 150000 LCPSNoise: ca 100 LCPSS/N: 1500


EBO-Rluc screening setupEBO-Rluc screening setup

Transfect EBO-Rluc minigenomeNP, 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)


Ebola-GFP recombinant virusEbola-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)

LVP30 VP24GPVP40VP35NP GFP

BCL-4

Towner et al. Virology: 332(1):20-7; Feb. 5, 2005


Screening protocolScreening protocolPrimary screen (Minigenome)

25 M, n=1

EC50/CC50 (Minigenome)

EC50/CC50 (EBO virus)

Lead candidates

5 concentrationsn=4

EC50 <10 MSI >10

EC50 <10 MSI >10

> 80% inhibition


Novel 2Novel 200 Sulfonamide lead Sulfonamide lead candidatescandidates

AP80552

-20

0

20

40

60

80

100

120

0.1 1 10 100

Log M

% in

hib

itio

n

EBOV GFP EC50

VeroE6 CC50

EC50 3.4CC50: >75

AP80175

-40

-20

0

20

40

60

80

100

120

0.1 1 10 100

Log M

% in

hib

itio

n

EBOV GFP EC50

VeroE6 CC50

EC50 1.8CC50: >75


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

edward bayham, msbme, mba vice president, business development ibe 2007 march30,2007 st. louis,...

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