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Antimicrobial Agents and Chemotherapy, June 2004, p. 2260-2266, Vol. 48, No. 6
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.6.2260-2266.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mutations Conferring Resistance to a Potent Hepatitis C Virus Serine Protease Inhibitor In Vitro

Liangjun Lu,1* Tami J. Pilot-Matias,1 Kent D. Stewart,2 John T. Randolph,1 Ron Pithawalla,1 Wenping He,1 Peggy P. Huang,1 Larry L. Klein,1 Hongmei Mo,1 and Akhteruzzaman Molla1

Antiviral Research,1 Structural Biology, Abbott Laboratories, Global Pharmaceutical Research and Development, Abbott Park, Illinois2

Received 17 November 2003/ Returned for modification 21 January 2004/ Accepted 3 February 2004

BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection. The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients. The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug. Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization. The 50% inhibitory concentrations of BILN 2061 for these colonies were 72- to 1,228-fold higher than that for the wild-type replicon. Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene. Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon. Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site. These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease.

* Corresponding author. Mailing address: Department R-47D, Building AP52-N, 200 Abbott Park Rd., Abbott Park, IL 60064-6217. Phone: (847) 935-8314. Fax: (847) 938-2756. E-mail: liangjun.lu{at}abbott.com.

Antimicrobial Agents and Chemotherapy, June 2004, p. 2260-2266, Vol. 48, No. 6
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.6.2260-2266.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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