Discovery of Proline Sulfonamides as Potent and Selective Hepatitis C Virus NS5b Polymerase Inhibitors. Evidence for a New NS5b Polymerase Binding Site

Modeling studies were performed on HCV NS5B Polymerase in an effort to design new inhibitors. The binding models of five different scaffold inhibitors were investigated and compared by using molecular dynamics simulations, free energy calculation and decomposition. Our results show Tyr448 plays the most critical role in the binding of most inhibitors. In addition, favorable contributions of residues Pro197, Arg200, Cys366, Met414 and Tyr448 in a deep hydrophobic pocket prove to be important for the selectivity of inhibitors. Furthermore, an optimized docking protocol was presented based on cross-docking the five inhibitors in the palm binding site of this enzyme using the Autodock program. This protocol was used later to virtually screen NCI and Maybridge diversity set libraries. The binding site was profiled via the statistics and analysis of the hydrogen bond networks formed between the receptor and the top-ranked diversity set compounds. Based on our detailed binding site analysis two useful rules were proposed to guide the selection of promising hits.Response to Reviewers: Question1: It is a pity that nothing in detail is discussed about found structures resulting from the virtual screening. Did the authors find new promising ligands with high (higher than the template ligands?) affinity and are they structurally diverse or similar to the used ones?Answer: We have found some promising compounds after the virtual screening. They are structurally diverse. Most of them form hydrogen bonds with the critical residues such as Tyr448 and have strong hydrophobic interaction with the residues in the deep hydrophobic pocket mentioned in our article. These compounds are under biological test.Question2: Additionally to figure 5 at least one figure should be added, showing the amino acid residues of the active site with the discussed most important interactions with one (the best?) ligand. This would considerably help to understand the discussion without looking in the original pdb-files. AbstractModeling studies were performed on HCV NS5B Polymerase in an effort to design new inhibitors. The binding models of five different scaffold inhibitors were investigated and compared by using molecular dynamics simulations, free energy calculation and decomposition. Our results show Tyr448 plays the most critical role in the binding of most inhibitors. In addition, favorable contributions of residues Pro197, Arg200, Cys366, Met414 and Tyr448 in a deep hydrophobic pocket prove to be important for the selectivity of inhibitors.Furthermore, an optimized docking protocol was presented based on cross-docking the five inhibitors in the palm binding site of this enzyme using the Autodock program. This protocol was used later to virtually screen NCI and Maybridge diversity set libraries. The binding site was profiled via the statistics and analysis of the hydrogen bond networks formed between the receptor and the top-ranked diversity set compounds. Based on our detailed binding site analysis two useful rules were proposed to ...

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“…and above the side chain of Met414 [14]. The cyclopropyl group of NNI-3 extends deeper into this lipophilic pocket [15].…”

Section: Binding Free Energy Calculation and Free Energy Decompositionmentioning

confidence: 99%

Insight into ligand selectivity in HCV NS5B polymerase: molecular dynamics simulations, free energy decomposition and docking

Froeyen

Herdewijn

2009

J Mol Model

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“…Stereo view of the ␤-flap and side chains that vary in genotypes 1b and 2a is illustrated in green for genotype 1b (PDB 1NB4) or in red for genotype 2a. The docking of acyl pyrrolidine 4 in genotype 1b was based on the structure of a proline sulfonamide analog (PDB 2GC8) (18). The represented ␤-flap wall is of genotype 2a.…”

Section: Addendummentioning

confidence: 99%

“…The NNI-3 site is located adjacent to the active site. Reported NNI-3 ligands include benzothiadiazine (11,47), proline sulfonamide (18), benzylidene (24,42), and acrylic acid (40,41) derivatives. In drug discovery, knowledge of the inhibitor site of action is crucial to guiding medicinal chemistry efforts.…”

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confidence: 99%

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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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“…Two other NNI sites have been characterized within the palm domain, distinct from but in close proximity to the RdRp active site. The palm I site (P1) has been targeted by proline (20), benzodiazepine (21), and benzothiadiazine (22) derivatives, such as RG7790 (setrobuvir), ABT-333, and ABT-072, whereas compounds that bind to the palm II site (P2) include benzofurans, such as HCV-796 (nesbuvir) (23). Imidazopyridines, including the NNI GS-9190 (tegobuvir), are another class of compounds which bind to the palm site of the polymerase; however, unlike other palm binders, this binding uniquely involves an interaction with the ␤-hairpin which extends from the thumb into the palm domain (site P-␤) (24,25).…”

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confidence: 99%

Cross-Genotypic Examination of Hepatitis C Virus Polymerase Inhibitors Reveals a Novel Mechanism of Action for Thumb Binders

Eltahla

Tay

Douglas

et al. 2014

Antimicrob Agents Chemother

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b Direct-acting antivirals (DAAs) targeting proteins encoded by the hepatitis C virus (HCV) genome have great potential for the treatment of HCV infections. However, the efficacy of DAAs designed to target genotype 1 (G1) HCV against non-G1 viruses has not been characterized fully. In this study, we investigated the inhibitory activities of nonnucleoside inhibitors (NNIs) against the HCV RNA-dependent RNA polymerase (RdRp). We examined the ability of six NNIs to inhibit G1b, G2a, and G3a subgenomic replicons in cell culture, as well as in vitro transcription by G1b and G3a recombinant RdRps. Of the six G1 NNIs, only the palm II binder nesbuvir demonstrated activity against G1, G2, and G3 HCV, in both replicon and recombinant enzyme models. The thumb I binder JTK-109 also inhibited G1b and G3a replicons and recombinant enzymes but was 41-fold less active against the G2a replicon. The four other NNIs, which included a palm I binder (setrobuvir), two thumb II binders (lomibuvir and filibuvir), and a palm ␤-hairpin binder (tegobuvir), all showed at least 40-fold decreases in potency against G2a and G3a replicons and the G3a enzyme. This antiviral resistance was largely conferred by naturally occurring amino acid residues in the G2a and G3a RdRps that are associated with G1 resistance. Lomibuvir and filibuvir (thumb II binders) inhibited primer-dependent but not de novo activity of the G1b polymerase. Surprisingly, these compounds instead specifically enhanced the de novo activity at concentrations of >100 nM. These findings highlight a potential differential mode of RdRp inhibition for HCV NNIs, depending on their prospective binding pockets, and also demonstrate a surprising enhancement of de novo activity for thumb RdRp binders. These results also provide a better understanding of the antiviral coverage for these polymerase inhibitors, which will likely be used in future combinational interferon-free therapies.

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Discovery of Proline Sulfonamides as Potent and Selective Hepatitis C Virus NS5b Polymerase Inhibitors. Evidence for a New NS5b Polymerase Binding Site

Cited by 55 publications

References 11 publications

Insight into ligand selectivity in HCV NS5B polymerase: molecular dynamics simulations, free energy decomposition and docking

Insight into ligand selectivity in HCV NS5B polymerase: molecular dynamics simulations, free energy decomposition and docking

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

Cross-Genotypic Examination of Hepatitis C Virus Polymerase Inhibitors Reveals a Novel Mechanism of Action for Thumb Binders

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