Targeting NS5B RNA-dependent RNA polymerase for anti-HCV chemotherapy

The global prevalence of persistent hepatitis C virus (HCV) infection and the lack of a highly effective and well-tolerated antiviral therapy have spurred intensive efforts to discover and develop novel anti-HCV therapy in the pharmaceutical industry. HCV NS5B RNA-dependent RNA polymerase (RdRp), the centerpiece for viral replication, constitutes a valid target for drug discovery. Compared to the host RNA and DNA polymerases, NS5B RdRp has distinct subcellular localization at the interface of the endoplasmic reticulum (ER) membrane and cytoplasm, a novel catalytic mechanism and many unique structural features, all of which make it an attractive target for developing effective anti-HCV therapeutics. High genetic variation among the major HCV genotypes commands that any efficacious NS5B inhibitors have to be broadly active against NS5Bs from various genotypes. Rapid viral replication and its inherent genetic diversity will certainly culminate drug resistance to any NS5B inhibitors. Therefore, iterative drug design and combination therapies of drugs that intervene with different steps in the HCV replicative cycle are needed to combat the viral infection. Many classes of nucleoside and non-nucleoside inhibitors of NS5B RdRp have been identified and appeared in literatures and patent applications. These progresses hold a considerable promise to the development of novel, specific and highly effective therapeutics to achieve sustained response and ultimately the eradication of HCV infection.     

丙型肝炎病毒NS5B RNA聚合酶抑制剂研究进展

丙型肝炎病毒（hepatitis C virus,HCV）是引起慢性肝炎并进而发展为肝硬化和肝细胞癌的主要病原体之一。目前,临床上采用α-干扰素（IFN-α）和利巴韦林（RBV）联合用药治疗丙型肝炎,但有效率仅为40%~50%。寻找HCV特定靶向抗病毒治疗药物是抗HCV研究的重要方向,相应靶点包括NS2和NS3蛋白酶,NS4A、NS4B、NS5A和NS5B,其中以NS5B RNA依赖性RNA聚合酶（NS5B RdRp）为靶标的抗HCV药物研究近年来颇受关注。本文在介绍NS5B及NS5B RdRp结构和功能的基础上,总结归纳以NS5B RdRp为靶点的HCV特定靶向抗病毒治疗药物研究的主要策略,以及近年来相关NS5BRdRp抑制剂的研究进展。     

Allosteric inhibition of the hepatitis C virus NS5B RNA dependent RNA polymerase

The human and monetary costs of chronic hepatitis C and the complications arising from this disease emphasize the urgency to find a treatment for Hepatitis C Virus (HCV) infected patients. The current standard of treatment for patients chronically infected with HCV is combination therapy with pegylated interferon plus ribavirin. Recently, viral enzymes have become the target of efforts to develop small molecule inhibitors interfering with the essential steps in the life cycle of the virus. Amongst these enzymes the HCV-encoded NS5B RNA-dependent RNA polymerase (NS5B RdRp) is essential for viral replication and has been recognized as a prime target for therapeutic intervention. Several distinct classes of inhibitors of NS5B RdRp have been disclosed in the literature, including active site inhibitors such as nucleosides and pyrophosphate mimetics, as well as non-nucleoside inhibitors. The latter, based on the success of allosteric inhibitors in the treatment of HIV infection, have been developed into compounds which show activity in the subgenomic cell-culture assay of HCV replication. This review provides an account of the recent developments in this field.     

Benzimidazole derivatives bearing substituted biphenyls as hepatitis C virus NS5B RNA-dependent RNA polymerase inhibitors: structure-activity relationship studies and identification of a potent and highly selective inhibitor JTK-109

Following the discovery of a new series of benzimidazole derivatives bearing a diarylmethyl group as inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase (HCV NS5B RdRp),1,2 we extended the structure-activity relationship (SAR) study to analogues bearing a substituted biphenyl group and succeeded in a significant advancement of activity. Starting from compound 1, optimization of the A, B, and C rings afforded potent inhibitors with low nanomolar potency against genotype 1b NS5B. The compounds, which have a substituent with a carbonyl function at the 4-position of the B-ring, efficiently blocked subgenomic viral RNA replication in the replicon cell assay at low submicromolar concentrations. Among the new compounds, compound 10n (JTK-109) exhibited favorable pharmacokinetic profiles, high selectivity for NS5B, and good safety profiles, suggesting the potential for a clinical candidate in the treatment of hepatitis C.     

Recent progress in the development of inhibitors of the hepatitis C virus RNA-dependent RNA polymerase

The global prevalence of hepatitis C virus (HCV) infection and the serious consequences associated with the chronic state of the disease have become a worldwide health problem. A combination therapy comprising Interferon-alpha and Ribavirin represents the current standard treatment for chronic HCV infection, although it has demonstrated limited success and causes serious side effects. Promising alternative approaches toward the control of HCV infection include the development of small molecule inhibitors of viral enzymes interfering with the essential steps in the life cycle of the virus. In this review we will focus on inhibitors of the HCV-encoded NS5B RNA-dependent RNA polymerase (NS5B RdRp) which is essential for viral replication and has been recognized as a prime target for therapeutic intervention.     

Ligand and structure based virtual screening strategies for hit-finding and optimization of hepatitis C virus (HCV) inhibitors

Virtual Screening (VS) has experienced increased attention into the recent years due to the large datasets made available, the development of advanced VS techniques and the encouraging fact that VS has contributed to the discovery of several compounds that have either reached the market or entered clinical trials. Hepatitis C Virus (HCV) nonstructural protein 5B (NS5B) has become an attractive target for the development of antiviral drugs and many small molecules have been explored as possible HCV NS5B inhibitors. In parallel with experimental practices, VS can serve as a valuable tool in the identification of novel effective inhibitors. Different techniques and workflows have been reported in literature with the goal to prioritize possible potent hits. In this context, different virtual screening strategies have been deployed for the identification of novel Hepatitis C Virus (HCV) inhibitors. This work reviews recent applications of virtual screening in an effort to identify novel potent HCV inhibitors.     

Efficient in silico assay of inhibitors of hepatitis C Virus RNA-dependent RNA polymerase by structure-based virtual screening and in vitro evaluation

To identify a new protective or therapeutic intervention for hepatitis C virus (HCV) infection, we performed efficient structure-based virtual screening to identify novel inhibitory agents for HCV. To this end, we selected NS5B, an RNA-dependent RNA polymerase (RdRp), as the target for the treatment of HCV infection. To decipher the dockable nature of various RdRp X-ray crystals, we docked the crystal ligand (inhibitor) to the crystal receptor (enzyme). The accuracy of regeneration of the crystal pose indicates the amenability of the RdRp binding pocket for structure-based virtual screening. We also utilized a consensus scoring scheme to reduce false positives, thereby ensuring efficient virtual screening. In this study, each molecule that ranked in the top 1% among all screening molecules gained 1 consensus point in a scoring function. Thus, after virtual screening of 57,177 chemicals from the Maybridge Screening collection, 14 molecules gained 8 points across 11 scoring functions. One of them, an isoxazole, showed significant dose-dependent inhibition of HCV RdRp activity and replication. In this study, we have developed a structure-based virtual screening method using HCV RdRp for efficient identification of novel inhibitors.     

Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase

We have identified naturally occurring 2-benzylidenebenzofuran-3-ones (aurones) as new templates for non-nucleoside hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) inhibitors. The aurone target site, identified by site-directed mutagenesis, is located in thumb pocket I of HCV RdRp. The RdRp inhibitory activity of 42 aurones was rationally explored in an enzyme assay. Molecular docking studies were used to determine how aurones bind to HCV RdRp and to predict their range of inhibitory activity. Seven aurone derivatives were found to have potent inhibitory effects on HCV RdRp, with IC(50) below 5 μM and excellent selectivity index (inhibition activity versus cellular cytotoxicity). The most active aurone analogue was (Z)-2-((1-butyl-1H-indol-3-yl)methylene)-4,6-dihydroxybenzofuran-3(2H)-one (compound 51), with an IC(50) of 2.2 μM. Their potent RdRp inhibitory activity and their low toxicity make these molecules attractive candidates as direct-acting anti-HCV agents.     

Identification of anthranilic acid derivatives as a novel class of allosteric inhibitors of hepatitis C NS5B polymerase

A series of potent anthranilic acid-based inhibitors of the hepatitis C NS5B polymerase has been identified. The inhibitors bind to a site on NS5B between the thumb and palm regions adjacent to the active site as determined by X-ray crystallography of the enzyme-inhibitor complex. Guided by both molecular modeling and traditional SAR, the enzyme activity of the initial hit was improved by approximately 100-fold, yielding a series of potent and selective NS5B inhibitors with IC50 values as low as 10 nM. These compounds were also inhibitors of the HCV replicon in cultured HUH7 cells.     

Structure modeling and docking study of HCV NS5B-3a RNA polymerase for the identification of potent inhibitors

Homology modeling in combination with molecular docking studies has been applied to predict the binding modes of the Hepatitis C virus (HCV) NS5B-3a inhibitors within the pocket of NS5B polymerase of HCV genotype 3a. Structure modeling and docking using Genetic Optimization for Ligand Docking (GOLD) were carried out to understand the ligand binding properties of NS5B-3a and to identify a potent inhibitor against it. The three-dimensional homology model of Pakistani strain is not available and was built based on the HCV-J4 NS5B polymerase structure. Compound 1 possessing high GOLD fitness score of 62.17 with IC50 value of 0.04 μM was selected as the potent inhibitor. The docking analysis depicted that hydrogen bonding, ionic and hydrophobic interactions contributed significantly for its ligand binding and the amino acid residues Arg442 and His403 seemed to be the anchor residues for receptor recognition. For developing a connection between the experimental bioactivities and GOLD fitness scores, a squared correlation coefficient was calculated and found as acceptable with the value of r ² = 0.67. These findings may act as potent lead in designing effective NS5B-3a inhibitors.     

Potent inhibitors of subgenomic hepatitis C virus RNA replication through optimization of indole-N-acetamide allosteric inhibitors of the viral NS5B polymerase

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off-target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC(50) = 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.     

Inhibitors of hepatitis C--a review of the recent patent literature

A review of the patent literature for inhibitors of hepatitis C virus (HCV) is presented for the period of January 2001 to December 2001. This review focuses on inhibitors of virally encoded enzymes/proteins, especially HCV NS3 serine protease and NS5B RNA-dependent RNA polymerase (RDRP), both of which have emerged as primary anti-HCV targets as a result of detailed understanding of their biological functions and the high quality structural information available. A number of potent small molecule inhibitors for these enzymes have been disclosed during this period of time. Inhibitors of other virally encoded enzymes have also been reported, but in lower numbers. With recent advances in obtaining a stable HCV cell-based assay to facilitate screening and selection of inhibitors, it is highly likely that effective small molecule antiviral therapies for HCV infection should soon emerge.     

Current perspective of HCV NS5B inhibitors: a review

Hepatitis C virus (HCV) infection has emerged as one of the most significant disease to affect humans. Despite its large medical and economical impact, there are no vaccines or efficient therapies without major side effects. The HCV non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase responsible for the complete copy of the RNA viral genome and is a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric inhibitors of NS5B, very few are active in clinical applications. Developments in the field have prompted us to review the research work on HCV NS5B polymerase inhibitors, especially their structure activity relationships and molecular modeling studies. This review will focus on the journey of drug discovery of HCV NS5B inhibitors covering both nucleoside and non-nucleosides.     

SAR and mode of action of novel non-nucleoside inhibitors of hepatitis C NS5b RNA polymerase

Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochemical potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases. The structures of the complexes formed between several of these inhibitors and HCV NS5b were determined by X-ray crystallography, and the inhibitors were found to bind in an allosteric binding site separate from the active site. Structure-activity relationships and structural studies have identified the mechanism of action for compounds in this series, several of which possess drug-like properties, as unique, reversible, covalent inhibitors of HCV NS5b.     

Novel nucleoside analogues targeting HCV replication through an NS5A‐dependent inhibition mechanism

A series of new tricyclic nucleosides were synthesized and evaluated as hepatitis C virus (HCV) replication inhibitors. Initial screening in a HCV replicon system, derived from a genotype 1b isolate, identified 9‐benzylamino‐3‐(β‐D‐ribofuranosyl)‐3H‐imidazo[4′,5′:5,6]pyrido[2,3‐b]pyrazine ( 15d ) as the most potent analogue. Comparative assessment of 15d activity against HCV full‐length viruses or subgenomic replicons derived from genotypes 1 to 4 revealed a specificity of the compound for genotypes 1 and 3. Surprisingly, resistance mutations selected against 15d were mapped to domains II and III of the non‐structural protein 5A (NS5A), but not to the RNA‐dependent RNA polymerase residing in NS5B. These results argue that compound 15d might represent a lead for the development of a novel class of NS5A inhibitors.     

Structure-activity relationship of purine ribonucleosides for inhibition of hepatitis C virus RNA-dependent RNA polymerase

As part of a continued effort to identify inhibitors of hepatitis C viral (HCV) replication, we report here the synthesis and evaluation of a series of nucleoside analogues and their corresponding triphosphates. Nucleosides were evaluated for their ability to inhibit HCV RNA replication in a cell-based, subgenomic replicon system, while nucleoside triphosphates were evaluated for their ability to inhibit in vitro RNA synthesis mediated by the HCV RNA-dependent RNA polymerase, NS5B. 2'-C-Methyladenosine and 2'-C-methylguanosine were identified as potent inhibitors of HCV RNA replication, and the corresponding triphosphates were found to be potent inhibitors of HCV NS5B-mediated RNA synthesis. The data generated in the cell-based assay demonstrated a fairly stringent structure-activity relationship around the active nucleosides. Increase in steric bulk beyond methyl on C2, change in the stereo- or regiochemistry of the methyl substituent, or change of identity of the heterobase beyond that of the endogenous adenine or guanine was found to lead to loss of inhibitory activity. The results highlight the importance of the ribo configuration 2'- and 3'-hydroxy pharmacophores for inhibition of HCV RNA replication in the cell-based assay and demonstrate that inclusion of the 2'-C-methylribonucleoside pharmacophore leads to increased resistance to adenosine deaminase and purine nucleoside phosphorylase mediated metabolism.     

2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid with inbuilt β-N-hydroxy-γ-keto-acid pharmacophore as HCV NS5B polymerase inhibitors

The inbuilt 2-N-hydroxy-1-oxo-3-carboxylic acid of isoquinolone was designed as pyrophosphate mimic for hepatitis C NS5B polymerase. Various 2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid derivatives 11a-p were synthesized and evaluated as HCV NS5B polymerase inhibitors. Compound 11c exhibited moderate inhibitory potency based on the inorganic pyrophosphate generation (IC?? = 9.5 μM) and based on NTP incorporation by NS5B enzyme (IC?? = 5.9 μM). Compound 11c demonstrated antiviral activity (EC?? = 15.7 μM) and good selectivity in HCV genotype 1b replicon Ava.5 cells. Compound 11c reduced the interaction of NTP to NS5B polymerase. Docking model showed that 11c situated in similar orientation to the bound uridine triphosphate in the active site of NS5B polymerase. As a result, 2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid was disclosed as a novel inbuilt β-N-Hydroxy-γ-keto-acid pharmacophore for HCV NS5B polymerase inhibitors.     

Development and characterization of a replicon-based phenotypic assay for assessing HCV NS4B from clinical isolates

The hepatitis C virus (HCV) NS4B inhibitors have shown potent inhibition of HCV replication in vitro. To assess the effect of viral diversity on the susceptibility to NS4B inhibitors, genotype (GT)-specific GT1a and GT1b replicon shuttle vectors were designed and created for cloning HCV NS4B genes from clinical isolates. For the GT1b NS4B shuttle vector, the S2204I adaptive mutation was introduced in NS5A to improve replication due to the replacement of the K1846T adaptive mutation in NS4B with NS4B from the clinical isolates. In addition to the adaptive mutations, a newly identified Huh-7 cell line, Huh-7-1C, which is highly permissive for both GT1a and GT1b replication, was used to further enhance the replication levels. HCV NS4B gene from clinical isolates was amplified and inserted into the corresponding GT1a and GT1b modified lab strain chimeric replicons. GT1a and GT1b chimeric replicons expressing diverse NS4B genes from corresponding subtypes of clinical isolates replicated at highly efficient levels for phenotypic analysis. Due to natural variation in their amino acid residues in NS4B, these isolates displayed varying drug susceptibilities to an NS4B inhibitor. In mixed populations with wild-type, the sensitivity of resistance detection of NS4B resistant mutants H94R and V105M was between 20% and 80%. The chimeric shuttle vectors can be used to characterize the activity of antiviral drugs targeting NS4B from diverse natural clinical isolates and aid in the development of novel compounds against HCV NS4B.     

Discovery and development of VX-950, a novel, covalent, and reversible inhibitor of hepatitis C virus NS3.4A serine protease

The hepatitis C virus (HCV) NS3.4A protease, which is essential for viral replication, is considered one of the most attractive targets for developing novel anti-HCV therapies. However, discovery of potent and selective small-molecule inhibitors of HCV NS3.4A protease as oral drug candidates has been hampered by the shallow substrate-binding groove of the protease. Serine trap warheads have been used to covalently anchor inhibitor scaffolds and to increase their affinity to the protease. This review will examine the evolution of covalent inhibitors of the HCV NS3.4A protease from early aldehyde molecules to alpha-ketoamide inhibitors. Kinetic and structural studies of alpha-ketoacid and alpha-ketoamide inhibitors revealed an unusual mechanism of binding in the catalytic site. Optimization of alpha-ketoamide scaffolds by scientists at Vertex and Eli Lilly led to the discovery of VX-950, a novel, potent, selective inhibitor of HCV NS3.4A protease. VX-950 possesses excellent antiviral activity in both HCV replicon cells and human fetal hepatocytes infected with HCV-positive patient sera. In addition, VX-950 exhibits a favorable pharmacokinetic profile in several animal species and demonstrates potent inhibition of the HCV NS3.4A protease activity in a mouse model. In a recent phase 1b clinical trial, VX-950 was able to rapidly reduce the plasma viral load of patients chronically infected with genotype 1 HCV by a mean approximately 3 log(10) in 2 days. The median viral load reduction was 4.4 log(10) for the best dose group after 14 days of dosing. The pre-clinical profile and early clinical data of VX-950 will be discussed in this review.