Structure-based virtual screening,synthesis and SAR of novel inhibitors of hepatitis C virus NS5B polymerase

Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of therapeutic agents aimed at the treatment of HCV infections. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-based virtual screening, synthesis and structure–activity relationship (SAR) optimization approach. Virtual screening of 260,000 compounds from the ChemBridge database against the tetracyclic indole inhibitor binding pocket of NS5B (allosteric pocket-1, AP-1), sequentially down-sized the library by 4 orders of magnitude to yield 23 candidates. In vitro evaluation of the NS5B inhibitory activity of the in-silico selected compounds resulted in 17% hit rate, identifying two novel chemotypes. Of these, compound 3, bearing the rhodanine scaffold, proved amenable for productive SAR exploration and synthetic modification. As a result, 25 derivatives that exhibited IC₅₀ values ranging from 7.7 to 68.0 μM were developed. Docking analysis of lead compound 28 within the tetracyclic indole- and benzylidene-binding allosteric pockets (AP-1 and AP-3, respectively) of NS5B revealed topological similarities between these two pockets. Compound 28, a novel rhodanine analog with NS5B inhibitory potency in the low micromolar level range may be a promising lead for future development of more potent NS5B inhibitors.     

Discovery of novel potent HCV NS5B polymerase non-nucleoside inhibitors bearing a fused benzofuran scaffold

This letter describes the discovery of a fused benzofuran scaffold viable for preparing a series of novel potent HCV NS5B polymerase non-nucleoside inhibitors. Designed on the basis of the functionalized benzofuran derivative nesbuvir (HCV-796), these compounds presumably bind similarly to the allosteric binding site in the “palm” domain of HCV NS5B protein. SAR of each potential hydrogen-bonding interaction site of this novel scaffold is discussed along with some preliminary genotypic profile and PK data of several advanced compounds.     

Tri-substituted acylhydrazines as tertiary amide bioisosteres: HCV NS5B polymerase inhibitors

The use of a tri-substituted acylhydrazine as an isostere of a tertiary amide was explored in a series of HCV NS5B thumb site II inhibitors. Direct replacement generated an analog with similar conformational and physicochemical properties. The series was extended to produce compounds with potent binding affinities and encouraging levels of cellular potency.     

Discovery of a novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors

A novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors were derived from a fragment-based approach using information from X-ray crystallographic analysis of NS5B-inhibitor complexes and iterative rounds of parallel synthesis. Structure-based drug design strategies led to the discovery of potent sub-micromolar inhibitors 11a–c and 12a–c from a weak-binding fragment-like structure 1 as a starting point.     

Identification of novel thiadiazoloacrylamide analogues as inhibitors of dengue-2 virus NS2B/NS3 protease

Dengue virus is endemic throughout tropical and subtropical regions, and cause severe epidemic diseases. The NS2B/NS3 protease is a promising drug target for dengue virus. Herein, we report the discovery and modification of a novel class of thiadiazoloacrylamide derivatives with potent inhibitory activity against the NS2B/NS3 protease. Thiadiazolopyrimidinone 1 was firstly determined as a new chemical structure against NS2B/NS3 from a commercial compound library. Then, we sought to identify similar compounds with the thiadiazoloacrylamide core that would exhibit better activity. A series of analogues were synthesized and fourteen of them were identified with strong inhibitory activities, in which the nitrile group in the linker part was discovered as an essential group for the inhibitory activity. The best of these (8b) demonstrated an IC₅₀ at 2.24 μM based on in vitro DENV2 NS2B-NS3pro assays.     

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 2: Synthesis and structure–activity relationships of benzothiazine-substituted quinolinediones

A new series of benzothiazine-substituted quinolinediones were evaluated as inhibitors of HCV polymerase NS5B. SAR studies on this series revealed a methyl sulfonamide group as a high affinity feature. Analogues with this group showed submicromolar potencies in the HCV cell based replicon assay. Pharmacokinetic and toxicology studies were also performed on a selected compound (34) to evaluate in vivo properties of this new class of inhibitors of HCV NS5B polymerase.     

Design and synthesis of lactam–thiophene carboxylic acids as potent hepatitis C virus polymerase inhibitors

Herein we report the successful incorporation of a lactam as an amide replacement in the design of hepatitis C virus NS5B Site II thiophene carboxylic acid inhibitors. Optimizing potency in a replicon assay and minimizing potential risk for CYP3A4 induction led to the discovery of inhibitor 22a. This lead compound has a favorable pharmacokinetic profile in rats and dogs.     

Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase

Halosalicylamide derivatives were identified from high-throughput screening as potent inhibitors of HCV NS5B polymerase. The subsequent structure and activity relationship revealed the absolute requirement of the salicylamide moiety for optimum activity. Methylation of either the hydroxyl group or the amide group of the salicylamide moiety abolished the activity while the substitutions on both phenyl rings are acceptable. The halosalicylamide derivatives were shown to be non-competitive with respect to elongation nucleotide and demonstrated broad genotype activity against genotype 1-3 HCV NS5B polymerases. Inhibitor competition studies indicated an additive binding mode to the initiation pocket that is occupied by the thiadiazine class of compounds and an additive binding mode to the elongation pocket that is occupied by diketoacids, but a mutually exclusive binding mode with respect to the allosteric thumb pocket that is occupied by the benzimidazole class of inhibitors. Therefore, halosalicylamides represent a novel class of allosteric inhibitors of HCV NS5B polymerase.     

The discovery and structure–activity relationships of pyrano[3,4-b]indole based inhibitors of hepatitis C virus NS5B polymerase

We describe the structure–activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12.     

Non-nucleoside analogue inhibitors bind to an allosteric site on HCV NS5B polymerase. Crystal structures and mechanism of inhibition

X-ray crystal structures of two non-nucleoside analogue inhibitors bound to hepatitis C virus NS5B RNA-dependent RNA polymerase have been determined to 2.0 and 2.9 A resolution. These noncompetitive inhibitors bind to the same site on the protein, approximately 35 A from the active site. The common features of binding include a large hydrophobic region and two hydrogen bonds between both oxygen atoms of a carboxylate group on the inhibitor and two main chain amide nitrogen atoms of Ser(476) and Tyr(477) on NS5B. The inhibitor-binding site lies at the base of the thumb domain, near its interface with the C-terminal extension of NS5B. The location of this inhibitor-binding site suggests that the binding of these inhibitors interferes with a conformational change essential for the activity of the polymerase.     

Synthesis and SAR of piperazine amides as novel c-jun N-terminal kinase (JNK) inhibitors

A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.     

Synthesis and evaluation of non-dimeric HCV NS5A inhibitors

Based on the symmetrical bidentate structure of the NS5A inhibitor BMS-790052, a series of new monodentate molecules were designed. The synthesis of 36 new non-dimeric NS5A inhibitors is reported along with their ability to block HCV replication in an HCV 1b replicon system. Among them compound 5a showed picomolar range activity along with an excellent selectivity index (SI > 90,000).     

Discovery of pyrido[2,3-d]pyrimidine-based inhibitors of HCV NS5A

Efforts to improve the genotype 1a potency and pharmacokinetics of earlier naphthyridine-based HCV NS5A inhibitors resulted in the discovery of a novel series of pyrido[2,3-d]pyrimidine compounds, which displayed potent inhibition of HCV genotypes 1a and 1b in the replicon assay. SAR in this system revealed that the introduction of amides bearing an additional ‘E’ ring provided compounds with improved potency and pharmacokinetics. Introduction of a chiral center on the amide portion resulted in the observation of a stereochemical dependence for replicon potency and provided a site for the attachment of functional groups useful for improving the solubility of the series. Compound 21 was selected for administration in an HCV-infected chimpanzee. Observation of a robust viral load decline provided positive proof of concept for inhibition of HCV replication in vivo for the compound series.     

Inhibitors of hepatitis C virus polymerase: Synthesis and characterization of novel 2-oxy-6-fluoro-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-benzamides

SAR exploration from an initial hit, (S)-N-(2-cyclohexenylethyl)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)benzamide (1), identified using our proprietary automated ligand identification system (ALIS),¹ has led to a novel series of selective hepatitis C virus (HCV) NS5B polymerase inhibitors with improved in vitro potency as exemplified by (S)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)-N-isopentyl-N-methylbenzamidecarboxamide (41) (IC₅₀ = 0.5 μM). The crystal structure of an analogue (44) was solved and provided rationalization of the SAR of this series, which binds in a distinct manner in the palm domain of NS5B, consistent with biochemical analysis using enzyme mutant variants. These data warrant further lead optimization efforts on this novel series of non-nucleoside inhibitors targeting the HCV polymerase.     

Discovery of novel N-(5-(arylcarbonyl)thiazol-2-yl)amides and N-(5-(arylcarbonyl)thiophen-2-yl)amides as potent RORγt inhibitors

Novel series of N-(5-(arylcarbonyl)thiazol-2-yl)amides and N-(5-(arylcarbonyl)thiophen-2-yl)amides were discovered as potent retinoic acid receptor-related orphan receptor-gamma-t (RORγt) inhibitors. SAR studies of the RORγt HTS hit 6a led to identification of thiazole ketone amide 8h and thiophene ketone amide 9g with high binding affinity and inhibitory activity of Th17 cell differentiation. Compound 8h showed in vivo efficacy in both mouse experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis (CIA) models via oral administration.     

Design,synthesis and evaluation against Mycobacterium tuberculosis of azole piperazine derivatives as dicyclotyrosine (cYY) mimics

Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding the series A imidazole, whilst series C explores replacing the keto group of the piperidone ring of cYY with a CH₂-imidazole or CH₂-triazole moiety to enhance binding interaction with the heme of CYP121A1. The series displayed moderate to weak type II binding affinity for CYP121A1, with the exception of series B 10a, which displayed mixed type I binding. Of the three series, series C imidazole derivatives showed the best, although modest, inhibitory activity against M. tuberculosis (17d MIC?=?12.5?μg/mL, 17a 50?μg/mL). Crystal structures were determined for CYP121A1 bound to series A compounds 6a and 6b that show the imidazole groups positioned directly above the haem iron with binding between the haem iron and imidazole nitrogen of both compounds at a distance of 2.2?Å. A model generated from a 1.5?Å crystal structure of CYP121A1 in complex with compound 10a showed different binding modes in agreement with the heterogeneous binding observed. Although the crystal structures of 6a and 6b would indicate binding with CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1 as the target.     

Discovery of hit molecules targeting allosteric site of hepatitis C virus NS5B polymerase

Abstract

Nonstructural protein 5B (NS5B), the RNA-dependent RNA polymerase of Hepatitis C Virus (HCV), plays a key role in viral amplification and is an attractive and most explored target for discovery of new therapeutic agents for Hepatitis C. Though safe and effective, NS5B inhibitors were launched in 2013 (Sovaldi) and 2014 (Harvoni, Viekira Pak), the high price tags of these medications limit their use among poor people in developing countries. Hence, still there exists a need for cost-effective and short duration anti-HCV agents especially those targeting niche patient population who were non-respondent to earlier therapies or with comorbid conditions. The present study describes the discovery of novel non-nucleoside (NNI) inhibitors of NS5B using a series of rational drug design techniques such as virtual screening, scaffold matching and molecular docking. 2D and 3D structure based virtual screening technique identified 300 hit compounds. Top 20 hits were screened out from identified hits using molecular docking technique. Four molecules, that are representative of 20 hits were evaluated for binding affinity under in vitro conditions using surface plasmon resonance-based assay and the results emphasized that compound with CoCoCo ID: 412075 could exhibit good binding response toward NS5B and could be a potential candidate as NS5B inhibitor.

Communicated by Ramaswamy H. Sarma     

Discovery of benzophosphadiazine drug candidate IDX375: A novel hepatitis C allosteric NS5B RdRp inhibitor

Hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells, and as a consequence is an attractive target for selective inhibition. This paper describes the discovery of a novel family of HCV NS5B non-nucleoside inhibitors inspired by the bioisosterism between sulfonamide and phosphonamide. Systematic structural optimization in this new series led to the identification of IDX375, a potent non-nucleoside inhibitor that is selective for genotypes 1a and 1b. The structure and binding domain of IDX375 were confirmed by X-ray co-crystalisation study.     

Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B

Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin–proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A‐binding protein. Co‐immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh‐7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B‐binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A‐OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.     

An objective assessment of conformational variability in complexes of hepatitis C virus polymerase with non-nucleoside inhibitors

A major target for antiviral therapy against hepatitis C virus (HCV) is the HCV polymerase nonstructural protein 5B (NS5B). Huge efforts have been devoted to the development of nucleoside and non-nucleoside inhibitors (NNIs) of NS5B. An offshoot of these efforts has been the structural characterization of the interaction of NS5B with NNIs by X-ray crystallography. These works have shown that the conformation of recombinant NS5B is very similar across strains, constructs and complexes, making evaluation of the long-range conformational effects of NNIs nontrivial.Using procedures appropriate to the evaluation of such minor but potentially important differences, we objectively assessed the conformational diversity in the 78 available genotype 1b NS5B structures in the Protein Data Bank. We find that there are 20 significantly different NS5B conformations available, but all are geometrically close to a closed, RNA synthesis initiation-competent one. Within this fairly restricted range, differences can be mapped to movements of NS5B domains and subregions. Most of this information is actually defined by small but significant changes in complexes with NNIs. We thus establish rigorously the moving parts of the NS5B molecular machine and the previously unrecognized hinge points that come into play upon NNI binding.We propose that NNIs binding at three of the four distinct sites specifically inhibit the initiation step by the same mechanism: they prevent NS5B's “thumb” from quite reaching the proper initiation-competent position. Furthermore, we suggest that a small number of critical hinges in the NS5B structure may emerge as sites of resistance mutations during future antiviral treatment.