Binding site characterization and resistance to a class of non-nucleoside inhibitors of the hepatitis C virus NS5B polymerase

The virally encoded NS5B RNA-dependent RNA polymerase has emerged as a prime target in the search for specific HCV antivirals. A series of benzimidazole 5-carboxamide compounds inhibit the cellular RNA replication of a HCV subgenomic replicon and we have advanced our understanding of this class of inhibitors through a combination of complementary approaches that include biochemical cross-linking experiments with a photoreactive analogue followed by mass spectrometry analysis of the enzyme. A novel binding site has been localized for these inhibitors at the junction of the thumb domain and the N-terminal finger loop. Furthermore, the isolation and characterization of resistant replicon mutants that co-localize to this region distinguished this class of compounds from other non-nucleoside NS5B inhibitors that bind to distinct allosteric sites. Resistant mutations that emerged with the benzimidazole 5-carboxamide and related compounds were found at three amino acid positions in the thumb domain: Pro(495) with substitutions to Ser, Leu, Ala, or Thr; Pro(496) substitutions to Ser or Ala; and a V499A substitution. Mutations at each of these positions conferred different levels of resistance to this drug class: the Pro(495) changes provided the greatest shifts in compound potency, followed by moderate changes in potency with the Pro(496) substitutions, and finally only minor shifts in potency with V499A. Combinations that include the benzimidazole 5-carboxamide polymerase inhibitors and compounds that bind other sites or other HCV targets, including HCV protease inhibitors, are complementary in cell culture models of HCV RNA replication at suppressing the emergence of resistant variants. This novel class of compounds and unique binding site expand the diversity of HCV antivirals currently under development and offer the potential to improve the treatment of chronic HCV infection.     

Non-nucleoside inhibitors binding to hepatitis C virus NS5B polymerase reveal a novel mechanism of inhibition

The RNA-dependent RNA polymerase (NS5B) from hepatitis C virus (HCV) is a key enzyme in HCV replication. NS5B is a major target for the development of antiviral compounds directed against HCV. Here we present the structures of three thiophene-based non-nucleoside inhibitors (NNIs) bound non-covalently to NS5B. Each of the inhibitors binds to NS5B non-competitively to a common binding site in the "thumb" domain that is approximately 35 Angstroms from the polymerase active site located in the "palm" domain. The three compounds exhibit IC(50) values in the range of 270 nM to 307 nM and have common binding features that result in relatively large conformational changes of residues that interact directly with the inhibitors as well as for other residues adjacent to the binding site. Detailed comparisons of the unbound NS5B structure with those having the bound inhibitors present show that residues Pro495 to Arg505 (the N terminus of the "T" helix) exhibit some of the largest changes. It has been reported that Pro495, Pro496, Val499 and Arg503 are part of the guanosine triphosphate (GTP) specific allosteric binding site located in close proximity to our binding site. It has also been reported that the introduction of mutations to key residues in this region (i.e. Val499Gly) ablate in vivo sub-genomic HCV RNA replication. The details of NS5B polymerase/inhibitor binding interactions coupled with the observed induced conformational changes provide new insights into the design of novel NNIs of HCV.     

Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.     

Aminothiazole inhibitors of HCV RNA polymerase

Aminothiazole-based inhibitors designed for HCV polymerase display low micromolar potencies in biochemical assays. These compounds show a stringent preference for a cyclohexyl hydrophobe at the 2-amino position. The composition of these compounds suggests that they may be interacting at a recently discovered allosteric site on the polymerase.     

Identification and structure-based optimization of novel dihydropyrones as potent HCV RNA polymerase inhibitors

A novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal structure revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor binding. Herein we report the structure-activity relationships (SARs) of this novel class of dihydropyrone-containing compounds that show potent inhibitory activities against the HCV RNA polymerase in biochemical assays.     

Interdomain communication in hepatitis C virus polymerase abolished by small molecule inhibitors bound to a novel allosteric site

The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have determined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3- and 2.4-Angstroms resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small alpha-helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen.     

Mechanistic study of HCV polymerase inhibitors at individual steps of the polymerization reaction

Little is known about the mechanism of HCV polymerase-catalyzed nucleotide incorporation and the individual steps employed by this enzyme during a catalytic cycle. In this paper, we applied various biochemical tools and examined the mechanism of polymerase catalysis. We found that formation of a productive RNA-enzyme complex is the slowest step followed by RNA dissociation and initiation of primer strand synthesis. Various groups have reported several classes of small molecule inhibitors of hepatitis C virus NS5B polymerase; however, the mechanism of inhibition for many of these inhibitors is not clear. We undertook a series of detailed mechanistic studies to characterize the mechanisms of inhibition for these HCV polymerase inhibitors. We found that the diketoacid derivatives competitively bind to the elongation NTP pocket in the active site and inhibit both the initiation and elongation steps of polymerization. While both benzimidazoles and benzothiadiazines are noncompetitive with respect to the active site elongation NTP pocket, benzothiadiazine compounds competitively bind to the initiation pocket in the active site and inhibit only the initiation step of de novo RNA polymerization. The benzimidazoles bind to the thumb allosteric pocket and inhibit the conformational changes during RNA synthesis. We also observed a cross interaction between the thumb allosteric pocket and the initiation pocket using inhibitor-inhibitor cross competition studies. This information will be very important in designing combination therapies using two small molecule drugs to treat hepatitis C virus.     

Improved replicon cellular activity of non-nucleoside allosteric inhibitors of HCV NS5B polymerase: from benzimidazole to indole scaffolds

Benzimidazole-based allosteric inhibitors of the hepatitis C virus (HCV) NS5B polymerase were diversified to a variety of topologically related scaffolds. Replacement of the polar benzimidazole core by lipophilic indoles led to inhibitors with improved potency in the cell-based subgenomic HCV replicon system. Transposing the indole scaffold into a previously described series of benzimidazole-tryptophan amides generated the most potent inhibitors of HCV RNA replication in cell culture reported to date in this series (EC(50) approximately 50 nM).     

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.     

Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.     

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.     

Structure based virtual screening of inhibitors for binding at the allosteric site of HCV RNA dependent RNA polymerase

Hepatitis C Virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at a risk of developing significant morbidity and mortality. There is no effective treatment or prevention till date for HCV infection. We describe the computed binding of 10 compounds to the allosteric binding site of RNA dependent RNA polymerase enzyme. These compounds were identified from the ZINC database through virtual screening followed by ADMET evaluation.     

Thumb inhibitor binding eliminates functionally important dynamics in the hepatitis C virus RNA polymerase

Hepatitis C virus (HCV) has infected almost 200 million people worldwide, typically causing chronic liver damage and severe complications such as liver failure. Currently, there are few approved treatments for viral infection. Thus, the HCV RNA‐dependent RNA polymerase (gene product NS5B) has emerged as an important target for small molecule therapeutics. Potential therapeutic agents include allosteric inhibitors that bind distal to the enzyme active site. While their mechanism of action is not conclusively known, it has been suggested that certain inhibitors prevent a conformational change in NS5B that is crucial for RNA replication. To gain insight into the molecular origin of long‐range allosteric inhibition of NS5B, we employed molecular dynamics simulations of the enzyme with and without an inhibitor bound to the thumb domain. These studies indicate that the presence of an inhibitor in the thumb domain alters both the structure and internal motions of NS5B. Principal components analysis identified motions that are severely attenuated by inhibitor binding. These motions may have functional relevance by facilitating interactions between NS5B and RNA template or nascent RNA duplex, with presence of the ligand leading to enzyme conformations with narrower and thus less accessible RNA binding channels. This study provides the first evidence for a mechanistic basis of allosteric inhibition in NS5B. Moreover, we present evidence that allosteric inhibition of NS5B results from intrinsic features of the enzyme free energy landscape, suggesting a common mechanism for the action of diverse allosteric ligands. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Specific inhibitors of HCV polymerase identified using an NS5B with lower affinity for template/primer substrate

The interaction of the hepatitis C virus (HCV) RNA-dependent RNA polymerase with RNA substrate is incompletely defined. We have characterized the activities of the HCV NS5B polymerase, modified by different deletions and affinity tags, with a routinely used homopolymeric substrate, and established apparent affinities of the various NS5B constructs both for the NTP and the template/primer substrates. We identified a uniquely tagged HCV NS5B RNA polymerase construct with a lower affinity (higher K(m)) than mature HCV NS5B for template/ primer substrate and highlighted the use of such a polymerase for the identification of inhibitors of NS5B activity, particularly inhibitors of productive RNA binding. The characterization of specific benzimidazole-5-carboxamide-based inhibitors, identified in a screening campaign, revealed that this class of compounds was non-competitive with regard to NTP incorporation and had no effect on processive elongation, but inhibited an initiation phase of the HCV polymerase activity. The potency of these compounds versus a panel of different NS5B polymerase constructs was inversely proportional to the enzymes' affinities for template/primer substrate. The benzimidazole-5-carboxamide compounds also inhibited the full-length, untagged NS5B de novo initiation reaction using HCV 3'-UTR substrate RNA and expand the diversifying pool of potential HCV replication inhibitors.     

Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors

The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.     

Non-nucleoside inhibitors of the hepatitis C virus NS5B polymerase: discovery of benzimidazole 5-carboxylic amide derivatives with low-nanomolar potency

Optimization of benzimidazole 5-carboxamide derivatives previously identified as specific inhibitors of the NS5B polymerase of the hepatitis C virus (HCV) has led to the discovery of potent analogues that inhibit the enzyme at low-nanomolar concentrations. Greater than 800-fold improvement in potency from the original lead structure was achieved through the combined effects of conformational rigidification, molecular size extension and the identification of previously unexploited interactions. Furthermore, these inhibitors retain specificity for HCV polymerase relative to other viral and mammalian RNA polymerases.     

Identification of novel inhibitors of HCV RNA-dependent RNA polymerase by pharmacophore-based virtual screening and in vitro evaluation

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B hepatitis where no effective treatment is available. The HCV NS5B with RNA-dependent RNA polymerase (RdRp) activity is a key target for the treatment of HCV infection. Here we report novel NS5B polymerase inhibitors identified by virtual screening and in vitro evaluation of their inhibitory activities. On the basis of a newly identified binding pocket of NS5B, distinct from the nucleotide binding site but highly conserved among various HCV isolates, we performed virtual screening of compounds that fit this binding pocket from the available chemical database of 3.5 million compounds. The inhibitory activities of the in silico selected 119 compounds were estimated with in vitro RdRp assay. Three compounds with IC50 values of about 20 μM were identified, and their kinetic analyses suggest that these compounds are noncompetitive inhibitors with respect to the ribonucleotide substrate. Furthermore, the single-point mutations of the conserved residues in the binding pocket of NS5B resulted in the significant decrease of the RdRp activity, indicating that the binding pocket presented here might be important for the therapeutic intervention of HCV. These novel inhibitors would be useful for the development of effective anti-HCV agents.     

2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry site

2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic benzoxazole system as a replacement for the core scaffold in previously discovered benzimidazole viral translation inhibitors. Structure–activity relationships in the target binding of substituted benzoxazole ligands were investigated.     

Thiazolone-acylsulfonamides as novel HCV NS5B polymerase allosteric inhibitors: convergence of structure-based drug design and X-ray crystallographic study

A novel series of thiazolone-acylsulfonamides were designed as HCV NS5B polymerase allosteric inhibitors. The structure based drug designs (SBDD) were guided by docking results that revealed the potential to explore an additional pocket in the allosteric site. In particular, the designed molecules contain moieties of previously described thiazolone and a newly designed acylsulfonamide linker that is in turn connected with a substituted aromatic ring. The selected compounds were synthesized and demonstrated low muM activity. The X-ray complex structure was determined at a 2.2A resolution and converged with the SBDD principle.     

Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors

Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.