In response to the Funding Opportunity Announcement """"""""Development of Assays for High-Throughput Drug Screening,"""""""" we propose here to develop assays to identify and analyze inhibitors of helicases needed for RNA virus replication. All viruses require helicases to separate duplex DNA or RNA, yet helicases remain undeveloped antiviral drug targets. Although some helicase inhibitors that target DNA viruses are progressing in clinical trials, no antiviral agents targeting RNA viruses have entered the clinical arena. We therefore will target RNA viruses, focusing on pathogens with abnormally high global burdens of disease: hepatitis C virus (HCV), Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and human immunodeficiency virus (HIV). The assays to be developed are needed as part of an ongoing collaborative hypothesis-driven research project to develop HCV NS3 helicase inhibitors as chemical probes to facilitate basic science and for drug development. In that endeavor, we have initiated an uHTS at the MLPCN that uses our recently developed molecular beacon based helicase assay (MBHA) as a primary screen. To evaluate HCV helicase inhibitor specificity, MBHAs will be developed using helicases encoded by viruses related to HCV (DNV, WNV, YFV, and JEV) and human cellular helicases. The human proteins to be analyzed have all been linked to HIV replication and include the DEAD-box proteins DDX1, DDX3, DDX24, MDA-5, RNA helicase A (RHA), and Werner syndrome protein (WRN).
In aim 2, assays will be developed to analyze the cellular antiviral efficacy, and mechanism of action of HCV helicase inhibitors. Mechanistic assays include ATPase assays, ligand binding assays, protein oligomerization assays, and simultaneous protease/helicase assays. Most of these mechanistic assays could be used with either HCV helicase or one of the ten other helicases targeted here to screen for additional classes of helicase inhibitors.

Public Health Relevance

In collaboration with a NIH high throughput-screening center, my lab is searching a collection of hundreds of thousands of small molecules for compounds that might inhibit the helicase encoded by the hepatitis C virus (HCV). In this project, we will develop tests to rapidly search these HCV helicase inhibitors for compounds that could be used to treat hepatitis C. We will also develop similar assays to identify compounds that might inhibit viruses that cause AIDS, yellow fever, Dengue fever, West Nile and Japanese encephalitis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI088001-04
Application #
8211062
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Tseng, Christopher K
Project Start
2010-02-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2012
Total Cost
$365,558
Indirect Cost
$118,058
Name
University of Wisconsin Milwaukee
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
627906399
City
Milwaukee
State
WI
Country
United States
Zip Code
53201
Ndjomou, Jean; Corby, M Josie; Sweeney, Noreena L et al. (2015) Simultaneously Targeting the NS3 Protease and Helicase Activities for More Effective Hepatitis C Virus Therapy. ACS Chem Biol 10:1887-96
Provazzi, Paola J S; Mukherjee, Sourav; Hanson, Alicia M et al. (2015) Analysis of the Enzymatic Activity of an NS3 Helicase Genotype 3a Variant Sequence Obtained from a Relapse Patient. PLoS One 10:e0144638
Sweeney, Noreena L; Hanson, Alicia M; Mukherjee, Sourav et al. (2015) Benzothiazole and Pyrrolone Flavivirus Inhibitors Targeting the Viral Helicase. ACS Infect Dis 1:140-148
Mukherjee, Sourav; Weiner, Warren S; Schroeder, Chad E et al. (2014) Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication. ACS Chem Biol 9:2393-403
Shadrick, William R; Ndjomou, Jean; Kolli, Rajesh et al. (2013) Discovering new medicines targeting helicases: challenges and recent progress. J Biomol Screen 18:761-81
Shadrick, William R; Mukherjee, Sourav; Hanson, Alicia M et al. (2013) Aurintricarboxylic acid modulates the affinity of hepatitis C virus NS3 helicase for both nucleic acid and ATP. Biochemistry 52:6151-9
Aydin, Cihan; Mukherjee, Sourav; Hanson, Alicia M et al. (2013) The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities. Protein Sci 22:1786-98
Sweeney, Noreena L; Shadrick, William R; Mukherjee, Sourav et al. (2013) Primuline derivatives that mimic RNA to stimulate hepatitis C virus NS3 helicase-catalyzed ATP hydrolysis. J Biol Chem 288:19949-57
Mastrangelo, Eloise; Pezzullo, Margherita; De Burghgraeve, Tine et al. (2012) Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug. J Antimicrob Chemother 67:1884-94
Hanson, Alicia M; Hernandez, John J; Shadrick, William R et al. (2012) Identification and analysis of inhibitors targeting the hepatitis C virus NS3 helicase. Methods Enzymol 511:463-83

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