Binding-Site Identification and Genotypic Profiling of Hepatitis C Virus Polymerase Inhibitors

Abstract

The search for hepatitis C virus polymerase inhibitors has resulted in the identification of several nonnucleoside binding pockets. The shape and nature of these binding sites differ across and even within diverse hepatitis C virus genotypes. These differences confront antiviral drug discovery with the challenge of finding compounds that are capable of inhibition in variable binding pockets. To address this, we have established a hepatitis C virus mutant and genotypic recombinant polymerase panel as a means of guiding medicinal chemistry through the elucidation of the site of action of novel inhibitors and profiling against genotypes. Using a genotype 1b backbone, we demonstrate that the recombinant P495L, M423T, M414T, and S282T mutant enzymes can be used to identify the binding site of an acyl pyrrolidine analog. We assess the inhibitory activity of this analog and other nonnucleoside inhibitors with our panel of enzyme isolates generated from clinical sera representing genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a.Hepatitis C is estimated to affect 3% of the global population. In a number of individuals, it can lead to liver fibrosis, cirrhosis, and death. Although virus can be cleared by a combination of pegylated interferon and ribavirin, the treatment is successful in only around 50% of treated patients and has considerable liabilities. These weaknesses highlight the need for new drugs to treat hepatitis C virus (HCV) in patients who have failed current therapy, as well as in untreated patients (12,56).HCV is an enveloped virus with an RNA genome of ϳ9.6 kb. Its single-stranded RNA has a positive polarity and encodes a polyprotein of ϳ3,300 amino acids comprising 4 structural proteins (Core, E1, E2, and p7) and 6 nonstructural proteins (NS2, -3, -4A, -4B, -5A, and -5B) (43). These proteins, as well as the viral translation process using the internal ribosomal entry site and a range of host factors, are candidate targets for therapeutic intervention (3, 46). The remarkable clinical success of human immunodeficiency virus reverse transcriptase and protease inhibitors, as well as the availability of several crystal structures, has motivated HCV drug discovery efforts to focus mainly on the development of protease and polymerase inhibitors. HCV NS5B is an RNA-dependent RNA polymerase that is responsible for the replication of the viral genome, which is thought to occur through a primer-independent de novo mechanism (6, 31). Due to the lack of proofreading capacity, this replication process is subject to a high error rate (36). As a result, the virus has evolved into multiple variant strains, classified into six different genotypes (1 to 6) and several subtypes (a, b, c, etc.) (45). To add to this complexity, HCV-infected individuals also harbor different variants or quasispecies of the virus, together representing a pool of genomes on which selective pressure can act (16). It has been speculated that drug resistance will rapidly emerge upon administration of specific HCV antivirals and that together with viral ...

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

“…7). Recently, changes in the IC 50 value as a function of site mutation in NS5B were proposed to be useful for the early determination of NNI binding sites on NS5B (45). Thus, the use of a panel of site-specific NS5B mutant proteins in the binding assay like the one described here can be helpful for identifying binding pockets of allosteric inhibitors before co-crystal structures are available.…”

1,3-Dipolar cycloadditions: applications to the synthesis of antiviral agents

“…Nucleosidic molecules 5-7 and 9, and the non-nucleosidic structures 8 and 10 are examples of triazole-containing entities able to inhibit HIV proliferation. 70 One of the first contributions in this area was published in 1989, but nucleoside 5 showed moderate activity in HIV-1 infected cells. 17 The same author used hydrazoic acid as 1,3-dipole and a carbodiimide as dipolarophile, generated from AZT, for the elaboration of compound 6, which also exhibited 75 moderate activity against HIV-1.…”

“…7 70 The reaction between 1,3-dipoles and alkenes or alkynes is very versatile allowing the presence of many functional groups in both components, namely 1,3-dipole I and dipolarophile II (Scheme 1). Yields of heterocycles III are elevated with a few and easily removable impurities.…”

Functional Characterization of Fingers Subdomain-specific Monoclonal Antibodies Inhibiting the Hepatitis C Virus RNA-dependent RNA Polymerase

“…The NNI class of compounds represents allosteric inhibitors that interfere with initiation of RNA synthesis. At least four binding sites for NNI on the HCV RdRp have been reported (23,24). Surprisingly, all these binding sites are located exclusively in palm and thumb subdomains of HCV polymerase.…”