Discovery of Pyrano[3,4-<i>b</i>]indoles as Potent and Selective HCV NS5B Polymerase Inhibitors

Gopalsamy, Ariamala; Lim, Kitae; Ciszewski, Gregory; Park, Kaapjoo; Ellingboe, John W.; Bloom, Jonathan D.; Insaf, Shabana; Upeslacis, Janis; Mansour, Tarek S.; Krishnamurthy, Girija; Damarla, Murthy; Pyatski, Yelena; Ho, Douglas M.; Howe, Anita Y. M.; Orlowski, Mark; Feld, Boris; O’Connell, John F.

doi:10.1021/jm0401255

Cited by 100 publications

(59 citation statements)

References 16 publications

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“…The NNI-2 site is located in the thumb domain, next to NNI-1 (2,29,55). Chemotypes of NNI-2 binders include the thiophene (2, 7), phenylalanine (8), dihydropyranone (29), and pyranoindole analogs (17). The NNI-3 site is located adjacent to the active site.…”

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confidence: 99%

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

Pauwels¹,

Mostmans²,

Quirynen³

et al. 2007

J Virol

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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 ...

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confidence: 99%

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

Pauwels¹,

Mostmans²,

Quirynen³

et al. 2007

J Virol

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“…A number of candidate protease inhibitors (PIs) which have excellent potency in vitro have been developed (2,17,20); several of these compounds have also been evaluated in phase I/II trials, with encouraging results (15,16,29,36). Resistance to this class of inhibitors has been described, with some mutations conferring cross-resistance to several compounds (17,18,21,34,35).The NS5B RNA polymerase is also essential for viral replication, and a number of nucleoside inhibitors and nonnucleoside inhibitors (NNIs) of the HCV polymerase with potent activity in vitro and in early clinical trials have been described (5,12,13,27,30). Resistance to both nucleoside and nonnucleoside inhibitors in vitro has been described (22,24,26).…”

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“…The NS5B RNA polymerase is also essential for viral replication, and a number of nucleoside inhibitors and nonnucleoside inhibitors (NNIs) of the HCV polymerase with potent activity in vitro and in early clinical trials have been described (5,12,13,27,30). Resistance to both nucleoside and nonnucleoside inhibitors in vitro has been described (22,24,26).…”

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Synergy of Small Molecular Inhibitors of Hepatitis C Virus Replication Directed at Multiple Viral Targets

Wyles

Kaihara

Vaida

et al. 2007

J Virol

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Chronic hepatitis C virus (HCV) infection isChronic hepatitis C virus (HCV) infection is a major worldwide health problem; in the United States, an estimated 3 million persons are chronically infected (4). Estimates of the health care burden of chronic HCV infection predict a drastic increase in hospitalizations and medical costs related to complications such as cirrhosis and hepatocellular carcinoma over the next 1 to 2 decades (3). Effective and better-tolerated therapy for HCV could effectively stem this tide (7).Current interferon-based therapy for chronic HCV infection results in sustained responses in roughly 55% of patients and is accompanied by significant toxicity. Genotype 1 HCV, the most prevalent genotype in the United States, responds less well to therapy with pegylated interferon plus ribavirin, with response rates of 42 to 46% (11, 23). These limitations have spurred an intense drug discovery effort, resulting in a number of promising compounds (8).Hepatitis C virus replication takes place in the cytoplasm, with the replication complex being tightly associated with lipid membranes (1). Key components of the replication complex include several promising antiviral targets, including the NS3/4A protease and the NS5B RNA-dependent RNA polymerase. A number of candidate protease inhibitors (PIs) which have excellent potency in vitro have been developed (2,17,20); several of these compounds have also been evaluated in phase I/II trials, with encouraging results (15,16,29,36). Resistance to this class of inhibitors has been described, with some mutations conferring cross-resistance to several compounds (17,18,21,34,35).The NS5B RNA polymerase is also essential for viral replication, and a number of nucleoside inhibitors and nonnucleoside inhibitors (NNIs) of the HCV polymerase with potent activity in vitro and in early clinical trials have been described (5,12,13,27,30). Resistance to both nucleoside and nonnucleoside inhibitors in vitro has been described (22,24,26).We have assessed a number of combinations of HCV inhibitors with several molecular targets currently in development, using an HCV genotype 1 replicon-based luciferase reporter system.Replicon constructs. The BM4-5 replicon is a subgenomic HCV genotype 1b replicon which contains a deletion of a serine in NS5A and has been previously described (14). The firefly luciferase gene was inserted in the BM4-5 replicon, in a manner previously described (33), to create a luciferase/neomycin phosphotransferase fusion protein (FEO) and the replicon (BM4-5 FEO). Briefly, the Photinus pyralis luciferase gene was amplified using primers coding for the AscI restriction site. Following amplification, both the BM4-5 plasmid and luciferase PCR product were restriction digested with AscI. Ligation was then carried out to insert the luciferase gene in phase with the neomycin phosphotransferase gene, creating the desired BM4-5 FEO replicon. The sequence of the replicon was verified by DNA sequencing.Cell culture. Human hepatoma Huh-7.5.1 cells (a kind gift from Fra...

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“…HCV-570, a racemic mixture of pyranoindoles, has previously been shown to specifically inhibit HCV NS5B RdRp (17). The inhibitory activity of this racemate appears to reside in the R enantiomer (HCV-371) (20); no activity was found in the S enantiomer.…”

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confidence: 99%

Molecular Mechanism of a Thumb Domain Hepatitis C Virus Nonnucleoside RNA-Dependent RNA Polymerase Inhibitor

Howe¹,

Cheng²,

Thompson³

et al. 2006

Antimicrob Agents Chemother

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A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique ␤-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8-to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 M. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.A wealth of literature has documented that RNA viruses or viruses that use RNA replicative intermediates have high degrees of genetic variability (5, 15). The high mutation rates found in these organisms are largely due to the lack of proofreading mechanisms and/or a high recombination frequency during cellular DNA replication. A DNA-dependent DNA polymerase complex allows an error rate of 10 Ϫ9 mutations/ nucleotide, whereas the error rates for an RNA-dependent DNA polymerase (reverse transcriptase) and RNA-dependent RNA polymerase (RdRp), such as those in human immunodeficiency virus (HIV) and hepatitis C virus (HCV), respectively, are on the order of 10 Ϫ4 to 10 Ϫ5 mutations/nucleotide (35, 37). The robust viral replication that leads to a large population size during viral infection in these RNA viruses and retroviruses contributes to the generation of quasispecies in infected patients (7,12,13).During chemotherapy, the high rates of viral replication and the high frequency of mutation lead to the rapid generation of drug-resistant mutants. In the case of HIV, numerous mutations have been identified in patients treated with protease inhibitors as well as nucleoside and nonnucleoside reverse transcriptase inhibitors. The emergence of resistant viruses becomes one of the biggest challenges in developing effective antiviral therapies to eradicate these ...

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Discovery of Pyrano[3,4-b]indoles as Potent and Selective HCV NS5B Polymerase Inhibitors

Abstract: A novel series of HCV NS5B RNA-dependent RNA polymerase inhibitors containing a pyrano[3,4-b]indole scaffold is described leading to the discovery of compound 16, a highly potent and selective inhibitor that is active in the replicon system.

Cited by 100 publications

References 16 publications

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

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

Synergy of Small Molecular Inhibitors of Hepatitis C Virus Replication Directed at Multiple Viral Targets

Molecular Mechanism of a Thumb Domain Hepatitis C Virus Nonnucleoside RNA-Dependent RNA Polymerase Inhibitor

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