High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication

Megan R. Edwards, Colette Pietzsch, Thibaut Vausselin, Megan L. Shaw, Alexander Bukreyev, Christopher F. Basler

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Ebola virus (EBOV), a member of the family Filoviridae, is a nonsegmented negative-sense RNA virus that causes severe, often lethal, disease in humans. EBOV RNA synthesis is carried out by a complex that includes several viral proteins. The function of this machinery is essential for viral gene expression and viral replication and is therefore a potential target for antivirals. We developed and optimized a high-throughput screening (HTS) assay based on an EBOV minigenome assay, which assesses the function of the polymerase complex. The assay is robust in 384-well format and displays a large signal to background ratio and high Z-factor values. We performed a pilot screen of 2080 bioactive compounds, identifying 31 hits (1.5% of the library) with >70% inhibition of EBOV minigenome activity. We further identified eight compounds with 50% inhibitory concentrations below their 50% cytotoxic concentrations, five of which had selectivity index (SI) values >10, suggesting specificity against the EBOV polymerase complex. These included an inhibitor of inosine monophosphate dehydrogenase, a target known to modulate the EBOV replication complex. They also included novel classes of inhibitors, including inhibitors of protein synthesis and hypoxia inducible factor-1. Five compounds were tested for their ability to inhibit replication of a recombinant EBOV that expresses GFP (EBOV-GFP), and four inhibited EBOV-GFP growth at sub-cytotoxic concentrations. These data demonstrate the utility of the HTS minigenome assay for drug discovery and suggest potential directions for antifiloviral drug development.

Original languageEnglish (US)
Pages (from-to)380-387
Number of pages8
JournalACS Infectious Diseases
Volume1
Issue number8
DOIs
StatePublished - Jan 8 2016

Fingerprint

Ebolavirus
Virus Replication
High-Throughput Screening Assays
Filoviridae
Hypoxia-Inducible Factor 1
Inosine Monophosphate
Protein Synthesis Inhibitors
Aptitude
Viral Genes
RNA Viruses
Essential Genes
Viral Proteins
Drug Discovery
Inhibitory Concentration 50
Libraries
Antiviral Agents
Oxidoreductases
RNA
Gene Expression

Keywords

  • antiviral
  • Ebola virus
  • filovirus
  • high-throughput screen
  • RNA polymerase
  • translation

ASJC Scopus subject areas

  • Infectious Diseases

Cite this

High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication. / Edwards, Megan R.; Pietzsch, Colette; Vausselin, Thibaut; Shaw, Megan L.; Bukreyev, Alexander; Basler, Christopher F.

In: ACS Infectious Diseases, Vol. 1, No. 8, 08.01.2016, p. 380-387.

Research output: Contribution to journalArticle

Edwards, Megan R. ; Pietzsch, Colette ; Vausselin, Thibaut ; Shaw, Megan L. ; Bukreyev, Alexander ; Basler, Christopher F. / High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication. In: ACS Infectious Diseases. 2016 ; Vol. 1, No. 8. pp. 380-387.
@article{aa6abbdaa1ab4aa1b9245c2e2e1ceef8,
title = "High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication",
abstract = "Ebola virus (EBOV), a member of the family Filoviridae, is a nonsegmented negative-sense RNA virus that causes severe, often lethal, disease in humans. EBOV RNA synthesis is carried out by a complex that includes several viral proteins. The function of this machinery is essential for viral gene expression and viral replication and is therefore a potential target for antivirals. We developed and optimized a high-throughput screening (HTS) assay based on an EBOV minigenome assay, which assesses the function of the polymerase complex. The assay is robust in 384-well format and displays a large signal to background ratio and high Z-factor values. We performed a pilot screen of 2080 bioactive compounds, identifying 31 hits (1.5{\%} of the library) with >70{\%} inhibition of EBOV minigenome activity. We further identified eight compounds with 50{\%} inhibitory concentrations below their 50{\%} cytotoxic concentrations, five of which had selectivity index (SI) values >10, suggesting specificity against the EBOV polymerase complex. These included an inhibitor of inosine monophosphate dehydrogenase, a target known to modulate the EBOV replication complex. They also included novel classes of inhibitors, including inhibitors of protein synthesis and hypoxia inducible factor-1. Five compounds were tested for their ability to inhibit replication of a recombinant EBOV that expresses GFP (EBOV-GFP), and four inhibited EBOV-GFP growth at sub-cytotoxic concentrations. These data demonstrate the utility of the HTS minigenome assay for drug discovery and suggest potential directions for antifiloviral drug development.",
keywords = "antiviral, Ebola virus, filovirus, high-throughput screen, RNA polymerase, translation",
author = "Edwards, {Megan R.} and Colette Pietzsch and Thibaut Vausselin and Shaw, {Megan L.} and Alexander Bukreyev and Basler, {Christopher F.}",
year = "2016",
month = "1",
day = "8",
doi = "10.1021/acsinfecdis.5b00053",
language = "English (US)",
volume = "1",
pages = "380--387",
journal = "ACS Infectious Diseases",
issn = "2373-8227",
publisher = "American Chemical Society",
number = "8",

}

TY - JOUR

T1 - High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication

AU - Edwards, Megan R.

AU - Pietzsch, Colette

AU - Vausselin, Thibaut

AU - Shaw, Megan L.

AU - Bukreyev, Alexander

AU - Basler, Christopher F.

PY - 2016/1/8

Y1 - 2016/1/8

N2 - Ebola virus (EBOV), a member of the family Filoviridae, is a nonsegmented negative-sense RNA virus that causes severe, often lethal, disease in humans. EBOV RNA synthesis is carried out by a complex that includes several viral proteins. The function of this machinery is essential for viral gene expression and viral replication and is therefore a potential target for antivirals. We developed and optimized a high-throughput screening (HTS) assay based on an EBOV minigenome assay, which assesses the function of the polymerase complex. The assay is robust in 384-well format and displays a large signal to background ratio and high Z-factor values. We performed a pilot screen of 2080 bioactive compounds, identifying 31 hits (1.5% of the library) with >70% inhibition of EBOV minigenome activity. We further identified eight compounds with 50% inhibitory concentrations below their 50% cytotoxic concentrations, five of which had selectivity index (SI) values >10, suggesting specificity against the EBOV polymerase complex. These included an inhibitor of inosine monophosphate dehydrogenase, a target known to modulate the EBOV replication complex. They also included novel classes of inhibitors, including inhibitors of protein synthesis and hypoxia inducible factor-1. Five compounds were tested for their ability to inhibit replication of a recombinant EBOV that expresses GFP (EBOV-GFP), and four inhibited EBOV-GFP growth at sub-cytotoxic concentrations. These data demonstrate the utility of the HTS minigenome assay for drug discovery and suggest potential directions for antifiloviral drug development.

AB - Ebola virus (EBOV), a member of the family Filoviridae, is a nonsegmented negative-sense RNA virus that causes severe, often lethal, disease in humans. EBOV RNA synthesis is carried out by a complex that includes several viral proteins. The function of this machinery is essential for viral gene expression and viral replication and is therefore a potential target for antivirals. We developed and optimized a high-throughput screening (HTS) assay based on an EBOV minigenome assay, which assesses the function of the polymerase complex. The assay is robust in 384-well format and displays a large signal to background ratio and high Z-factor values. We performed a pilot screen of 2080 bioactive compounds, identifying 31 hits (1.5% of the library) with >70% inhibition of EBOV minigenome activity. We further identified eight compounds with 50% inhibitory concentrations below their 50% cytotoxic concentrations, five of which had selectivity index (SI) values >10, suggesting specificity against the EBOV polymerase complex. These included an inhibitor of inosine monophosphate dehydrogenase, a target known to modulate the EBOV replication complex. They also included novel classes of inhibitors, including inhibitors of protein synthesis and hypoxia inducible factor-1. Five compounds were tested for their ability to inhibit replication of a recombinant EBOV that expresses GFP (EBOV-GFP), and four inhibited EBOV-GFP growth at sub-cytotoxic concentrations. These data demonstrate the utility of the HTS minigenome assay for drug discovery and suggest potential directions for antifiloviral drug development.

KW - antiviral

KW - Ebola virus

KW - filovirus

KW - high-throughput screen

KW - RNA polymerase

KW - translation

UR - http://www.scopus.com/inward/record.url?scp=84969131730&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84969131730&partnerID=8YFLogxK

U2 - 10.1021/acsinfecdis.5b00053

DO - 10.1021/acsinfecdis.5b00053

M3 - Article

VL - 1

SP - 380

EP - 387

JO - ACS Infectious Diseases

JF - ACS Infectious Diseases

SN - 2373-8227

IS - 8

ER -