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Antimicrobial Agents and Chemotherapy, November 2003, p. 3525-3530, Vol. 47, No. 11
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.11.3525-3530.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Resistance Profile of a Hepatitis C Virus RNA-Dependent RNA Polymerase Benzothiadiazine Inhibitor

Tammy T. Nguyen,1 Adam T. Gates,1 Lester L. Gutshall,1 Victor K. Johnston,1 Baohua Gu,1 Kevin J. Duffy,2 and Robert T. Sarisky1*

Departments of Virology,1 Medicinal Chemistry, The Metabolic and Viral Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 194262

Received 16 April 2003/ Returned for modification 30 June 2003/ Accepted 20 August 2003

Recently, a benzo-1,2,4-thiadiazine antiviral agent (C21H21N3O4S; compound 4) was shown to be a potent, highly specific inhibitor of the primary catalytic enzyme of the hepatitis C virus (HCV) replicase complex. In this study, we selected for resistance to confirm the mechanism of action for compound 4 in HCV replicon cells. As expected, spontaneous mutations or fluidity in the HCV polymerase (NS5B) coding sequence occurred upon routine passage of the HCV replicon cells in the absence of compound 4. After 1 month of culture in the presence of 10 µM compound 4, or 20 times the 50% inhibitory concentration of the replicon, replicon cells were almost 20-fold less susceptible to compound 4. Twenty-one NS5B cDNA clones were generated from the resistant replicon cells. Five mutations in the 21 NS5B clones were present at frequencies higher than that of control replicon cells, and no clone contained more than a single mutation within the polymerase gene. RNA-dependent RNA polymerase studies using purified recombinant NS5B containing these single point mutations allowed the identification of residue 414 as sufficient for biochemical resistance to compound 4. Further, the contribution of this residue to confer cell-based resistance to compound 4 was validated using a stable recombinant mutant replicon cell line which harbors a methionine-to-threonine change at residue 414. The potential for additional mutations in other nonstructural genes of HCV to contribute to the resistance profile of compound 4 is discussed.

* Corresponding author. Present address: Infectious Diseases Research, Centocor, Inc., 200 Great Valley Pkwy., R-4-1, Malvern, PA 19355. Phone: (610) 240-8207. Fax: (610) 240-4064. E-mail: Rsarisky{at}cntus.jnj.com.

Antimicrobial Agents and Chemotherapy, November 2003, p. 3525-3530, Vol. 47, No. 11
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.11.3525-3530.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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