Identification and analysis of fitness of resistance mutations against the HCV protease inhibitor SCH 503034

Abstract

HCV NS3 protease variants resistant to the protease inhibitor SCH 503034 were selected. Three mutations, T54A, V170A and A156S mutations conferred low to moderate levels of resistance (<20-fold). Longer exposure (>10 passages) or selection with higher levels of compound led to the selection of a more resistant variant, A156T (>100-fold). [Lin, C., Lin, K., Luong, Y.P., Rao, B.G., Wei, Y.Y., Brennan, D.L., Fulghum, J.R., Hsiao, H.M., Ma, S., Maxwell, J.P., Cottrell, K.M., Perni, R.B., Gates, C.A., Kwong, A.D., 2004. In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms. J. Biol. Chem. 279(17), 17508–17514; Lu, L., Pilot-Matias, T.J., Stewart, K.D., Randolph, J.T., Pithawalla, R., He, W., Huang, P.P., Klein, L.L., Mo, H., Molla, A., 2004. Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro. Antimicrob. Agents Chemother. 48(6), 2260–2266.] Combination with IFN-α drastically reduced the number of emergent colonies. Resistant colonies showed no change in sensitivity to IFN-α.

Although the A156T mutation conferred the highest level of resistance to SCH 503034, it significantly reduced the colony formation efficiency (CFE) of the mutant replicon RNA, and rendered replicon cells less fit than those bearing wild-type replicons. Replicon cells bearing mutation A156S were less fit than wild-type in co-culture growth competition assays but showed no impact on CFE. The V170A mutation, on the other hand, did not affect replicon fitness in either assay, which was consistent with its emergence as the dominant mutant after 12 months of continuous selection. The reduced fitness of the most resistant variant suggests that it may be rare in naïve patients and that development of high-level resistance may be slow. Combination therapy with IFN-α should also greatly reduce the potential emergence of resistance.

Introduction

One of the major issues in development of antiviral therapy is the emergence of drug-resistant variants. In HIV patients treated with protease inhibitors, resistant viruses have been isolated and shown to correlate with relapse of viral replication (Eastman et al., 1998, Romano et al., 2002). Viral resistance can also be developed by selecting the virus with protease inhibitors in vitro, and in many cases the mutations that confer resistance in vitro are the same as those observed clinically (Carrillo et al., 1998, Condra et al., 1995, Hirsch et al., 1998, Markowitz et al., 1995, Molla et al., 1996, Tisdale et al., 1995). These results suggest that resistance studies in vitro could provide insight into what to expect in future clinical trials.

Like most RNA viruses, HCV has a high mutation rate and exists as a complex population of genetically distinct, but closely related, variants commonly referred to as a quasispecies (Cabot et al., 2001, Farci et al., 2002, Martell et al., 1992). Pre-existing minor viral species, resistant to the selecting drug, would gain a growth advantage over the existing wild-type viral population and rapidly become the dominant genotype (Harrigan et al., 2001, Hirsch et al., 1998, Lech et al., 1996, Paolucci et al., 2001).

In addition to reduced susceptibility to the selecting drug, another important factor affecting the outgrowth of the resistant population is the fitness of the mutant viruses. The replicative fitness of resistant viruses is also a critical parameter of viral resistance and has prognostic value with regard to achieving sustained virological response in the clinic. For example, the relative fitness of resistant HIV variants in response to treatment with various protease inhibitors can help guide which of the protease inhibitors should be used in combination therapy (Martinez-Picado et al., 2000). Understanding viral fitness is also essential to the predication of prevalence and transmission of resistant viruses in the population especially when the treatment is widely prescribed (Blower et al., 2001, Brenner et al., 2002).

Recently, several groups have reported selection of HCV resistant mutants against two protease inhibitors using the replicon system. Two major resistance loci were reported: mutations at 168 were found to confer resistance to BILN 2061 (a modified macrocyclic peptide, Fig. 1) and its analogues; whereas mutations at 156 impacted both BILN 2061 and VX-950 (a peptidic ketoamide, Fig. 1) (Lin et al., 2004, Lin et al., 2005, Lu et al., 2004, Trozzi et al., 2003). In this report, the selection and characterization of resistance mutations against a novel peptidic ketoamide inhibitor, SCH 503034 (Fig. 1) (Malcolm et al., in press), currently undergoing clinical evaluation, is described. In addition, the impact of the three major resistance mutations (V170A, A156S and A156T) on HCV replicon fitness was studied and the implications for future clinical utility are discussed.