The HCV RNA-dependent RNA polymerase has emerged as one of the key targets for novel anti-HCV therapy development. Herein, we report the optimization of the dihydropyrone series inhibitors to improve compound aqueous solubility and reduce CYP2D6 inhibition, which led to the discovery of compound 24 (PF-00868554). Compound 24 is a potent and selective HCV polymerase inhibitor with a favorable pharmacokinetic profile and has recently entered a phase II clinical evaluation in patients with genotype 1 HCV.

PF-00868554 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, which exerts its inhibitory effect by binding to the thumb base domain of the protein. It is a potent and selective inhibitor, with a mean 50% inhibitory concentration of 0.019 M against genotype 1 polymerases and a mean 50% effective concentration (EC 50 ) of 0.075 M against the genotype 1b-Con1 replicon. To determine the in vitro antiviral activity of PF-00868554 against various HCV strains, a panel of chimeric replicons was generated, in which polymerase sequences derived from genotype 1a and 1b clinical isolates were cloned into the 1b-Con1 subgenomic reporter replicon. Our results indicate that PF-00868554 has potent in vitro antiviral activity against a majority (95.8%) of genotype 1a and 1b replicons, with an overall mean EC 50 of 0.059 M. PF-00868554 showed no cytotoxic effect in several human cell lines, up to the highest concentration evaluated (320 M). Furthermore, the antiviral activity of PF-00868554 was retained in the presence of human serum proteins. An in vitro resistance study of PF-00868554 identified M423T as the predominant resistance mutation, resulting in a 761-fold reduction in susceptibility to PF-00868554 but no change in susceptibility to alpha interferon and a polymerase inhibitor that binds to a different region. PF-00868554 also showed good pharmacokinetic properties in preclinical animal species. Our results demonstrate that PF-00868554 has potent and broadspectrum antiviral activity against genotype 1 HCV strains, supporting its use as an oral antiviral agent in HCV-infected patients.

To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The NS5B region was amplified by nested PCR and introduced into the corresponding region of the GT1b (Con-1) subgenomic reporter replicon by Splicing by Overlap Extension (SOEing) PCR. Stable cell lines were generated with replication-competent chimeras for in vitro antiviral activity determination of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Compounds that bind to the NNI2 (thiophene carboxylic acid) or NNI3 (benzothiadiazine) allosteric sites showed 8-to >1,280-fold reductions in antiviral activity against non-GT1 NS5B chimeric replicons compared to that against the GT1b subgenomic replicon. Smaller reductions in susceptibility, ranging from 0.2-to 33-fold, were observed for the inhibitor binding to the NNI1 (benzimidazole) site. The inhibitor binding to the NNI4 (benzofuran) site showed broad-spectrum antiviral activity against all chimeric replicons evaluated in this study. In conclusion, evaluation of HCV NNIs against intergenotypic chimeric replicons showed differences in activity spectrum for inhibitors that target different regions of the enzyme, some of which could be associated with specific residues that differ between GT1 and non-GT1 polymerases. Our study demonstrates the utility of chimeric replicons for broad-spectrum activity determination of HCV inhibitors.

A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 Å away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 M. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.Hepatitis C virus (HCV) has emerged as one of most important etiological factors for blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (34, 38). The infection becomes persistent in about 85% of infected individuals, despite the presence of a strong humoral and cellular immune response (3). Currently, about 4.5 million individuals in the United States and more than 170 million worldwide are infected with HCV, which represents an important public health problem. A combination of pegylated forms of alpha interferon (IFN-␣) and ribavirin is the only therapy available against HCV, but the success rate observed in individuals infected with genotype 1, which is the most prevalent genotype in the United States and worldwide, is only about 40% to 50% (7,8,25). In addition, IFN-␣ therapy is associated with significant side effects including fatigue, headache, myalgia, fever, nausea, and insomnia in more than 30% of patients. Ribavirin also causes hemolytic anemia in 10% to 20% of patients (22,36). Consequently, there remains a significant unmet medical need for more effective and safer HCV therapy.The HCV genome is a single-stranded, positive-sense RNA of approximately 9.6 kb (5). The genom...

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neo r marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. Approximately 170 million people globally test positive for hepatitis C virus (HCV) (9, 11). Infection by HCV results in a high degree of chronic hepatitis. In addition to inducing liver damage, a significant proportion of these infections also result in hepatocellular carcinoma. Although current treatments for hepatitis caused by HCV include interferon in combination with ribavirin (18), approximately 50 to 60% of individuals still are not able to resolve infection (15). Therefore, there is an unmet medical need to develop more effective therapies to treat HCV infection. Until 1999, all cell-based screening efforts for HCV drug discovery relied on surrogate viral systems, such as bovine viral diarrhea virus, and the potential development of assays where activities of specific viral targets could be monitored. In 1999, a significant breakthrough in studying HCV RNA replication occurred when the Bartenschlager laboratory developed the HCV replicon system, a tissue culture system that faithfully mimicked all of the RNA replication events of the HCV life cycle (21). This initiated a phase of intensified research into the mechanisms of HCV RNA translation, replication, and protein processing. It also ushered in a new era for HCV drug discovery, since it was now possible to test the effects of inhibitors of traditional targets, such as NS3 protease, helicase, and NS5B polymerase, in an authentic, in vitro HCV RNA replication system (1).The original replicon system (21) was constructed by replacement of genes from the HCV genome that are not essential for HCV RNA replication, e.g., the structural genes, p7 and NS2, with a genetic cassette carrying an antibiotic resistance gene and the internal ribosomal entry site (IRES) from encephalomyocarditis virus (EMCV). This resulted in the formation of a dicistronic, selectable, subgenomic HCV replicon (2-4, 21) whose replication requires RNA elements in both nontranslated regions as well as the nonstructural proteins, including NS3 protease, helicase, and polymerase. Therefore, the HCV replicon system can be used for identifying inhibitors against all of these components (1).Cell-based screening efforts in a high-throughput format to identify novel inhibitors and viral or host targets have recently been described (6,23,34). The first ...

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