Identification of <i>N,N</i>-Disubstituted Phenylalanines as a Novel Class of Inhibitors of Hepatitis C NS5B Polymerase

Chan, Laval; Reddy, T. Jagadeeswar; Proulx, Melanie; Das, Sanjoy K.; Pereira, Oswy Z.; Wang, Wuyi; Siddiqui, A. M.; Yannopoulos, Constantin G.; Poisson, Carl; Turcotte, Nathalie; Drouin, Alexandre; Alaoui-Ismaili, Moulay Hicham; Bethell, Richard C.; Hamel, Martine; L’Heureux, Lucille; Bilimoria, Darius; Nguyen-Ba, Nghe

doi:10.1021/jm0340400

J. Med. Chem.

2003

DOI: 10.1021/jm0340400

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Identification of N,N-Disubstituted Phenylalanines as a Novel Class of Inhibitors of Hepatitis C NS5B Polymerase

Laval Chan¹,

T. Jagadeeswar Reddy²,

Melanie Proulx³

et al.

Abstract: The HCV NS5B RNA dependent RNA polymerase plays an essential role in viral replication. The discovery of a novel class of inhibitors based on an N,N-disubstituted phenylalanine scaffold and structure-activity relationships studies to improve potency are described.

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Cited by 72 publications

(49 citation statements)

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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.…”

mentioning

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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“…The majority of the compounds evaluated in vitro and in early clinical trials have belonged to one of three classes of HCV inhibitors: NS3 protease inhibitors (PI) (11,17,29), NS5B nucleoside polymerase inhibitors (21,22,24), and NS5B nonnucleoside (NNI) polymerase inhibitors (2,7,8,12). Importantly, resistance to each of these compound classes has been described with some resistance mutations conveying cross-resistance to several inhibitors within a given class (e.g., A156T in HCV protease) (15,18,20,28).…”

mentioning

confidence: 99%

Synergy of a Hepatitis C Virus (HCV) NS4A Antagonist in Combination with HCV Protease and Polymerase Inhibitors

Wyles

Kaihara

Schooley

2008

Antimicrob Agents Chemother

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Rapid emergence of resistance to monotherapy with virus-specific inhibitors necessitates combination therapy. ACH-806 is a hepatitis C virus NS4A inhibitor with a novel mechanism of action and resistance pathway. This compound was synergistic with NS3 protease inhibitors and NS5B nucleoside and nonnucleoside polymerase inhibitors.Significant progress has been made in the discovery and testing of novel inhibitors of hepatitis C virus (HCV) replication (4). The majority of the compounds evaluated in vitro and in early clinical trials have belonged to one of three classes of HCV inhibitors: NS3 protease inhibitors (PI) (11,17,29), NS5B nucleoside polymerase inhibitors (21,22,24), and NS5B nonnucleoside (NNI) polymerase inhibitors (2,7,8,12). Importantly, resistance to each of these compound classes has been described with some resistance mutations conveying cross-resistance to several inhibitors within a given class (e.g., A156T in HCV protease) (15,18,20,28). Analogous to the experience with human immunodeficiency virus type 1 therapy, combinations of several classes of viral inhibitors with unique mechanisms of action and resistance pathways will be integral to the success of small-molecule-based antiviral therapy for chronic HCV infection.ACH-806 [1-(4-pentyloxy-3-trifluoromethylphenyl)-3-(pyridine-3-carbonyl)thiourea] is a novel acylthiourea compound with a 50% effective concentration (EC 50 ) of 14 nM in the genotype 1b replicon system and 30 nM in a genotype 1a replicon system (13). A phase 1b proof-of-concept study showed significant antiviral activity at the lowest dose tested (23). ACH-806 possesses a unique mechanism of action. It selectively binds to the NS4A protein, resulting in altered protein composition and inactivation of the replicase complex (13). Given its unique mechanism of action, we sought to evaluate ACH-806 in combination with other small-molecule inhibitors of HCV replication, as well as alpha interferon, in a genotype 1b luciferase reporter replicon system.(A portion of the results described here was presented as an abstract at the XVI International HIV Drug Resistance Workshop, Hilton Barbados, Bridgetown, Barbados, 12 to 16 June 2007.)Replicon constructs. The BM4-5 replicon is a subgenomic HCV genotype 1b replicon which contains a deletion of a serine in NS5A (10). The firefly luciferase gene was inserted into the BM4-5 replicon, in a manner we and others have previously described (26,27), to generate the BM4-5 FEO replicon. The sequence of the replicon was verified by DNA sequencing.Cell culture and luciferase compound assays. Cell culture and luciferase compound assays were performed as previously described in detail (9, 27). Briefly, 10,000 BM4-5 FEO cells/ well were seeded into 96-well plates and incubated for 4 h. Medium was then aspirated and replaced with 100 l of complete medium containing a single compound or combinations at the desired concentration(s). Plates with compounds were incubated for 48 h and then assayed for luciferase expression (Bright-Glo; Promega). All conditio...

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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).…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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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Identification of N,N-Disubstituted Phenylalanines as a Novel Class of Inhibitors of Hepatitis C NS5B Polymerase

Abstract: The HCV NS5B RNA dependent RNA polymerase plays an essential role in viral replication. The discovery of a novel class of inhibitors based on an N,N-disubstituted phenylalanine scaffold and structure-activity relationships studies to improve potency are described.

Cited by 72 publications

References 13 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 a Hepatitis C Virus (HCV) NS4A Antagonist in Combination with HCV Protease and Polymerase Inhibitors

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

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