In vitro studies of cross-resistance mutations against two hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.     

The design of a potent inhibitor of the hepatitis C virus NS3 protease: BILN 2061--from the NMR tube to the clinic

The virally encoded serine protease NS3/NS4A is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. Until very recently, the design of inhibitors for the HCV NS3 protease was limited to large peptidomimetic compounds with poor pharmacokinetic properties, making drug discovery an extremely challenging endeavor. In our quest for the discovery of a small-molecule lead that could block replication of the hepatitis C virus by binding to the HCV NS3 protease, the critical protein-polypeptide interactions between the virally encoded NS3 serine protease and its polyprotein substrate were investigated. Lead optimization of a substrate-based hexapeptide, guided by structural data, led to the understanding of the molecular dynamics and electronic effects that modulate the affinity of peptidomimetic ligands for the active site of this enzyme. Macrocyclic beta-strand scaffolds were designed that allowed the discovery of potent, highly selective, and orally bioavailable compounds. These molecules were the first HCV NS3 protease inhibitors reported that inhibit replication of HCV subgenomic RNA in a cell-based replicon assay at low nanomolar concentrations. Optimization of their biopharmaceutical properties led to the discovery of the clinical candidate BILN 2061. Oral administration of BILN 2061 to patients infected with the hepatitis C genotype 1 virus resulted in an impressive reduction of viral RNA levels, establishing proof-of-concept for HCV NS3 protease inhibitors as therapeutic agents in humans.     

Molecular basis of telaprevir resistance due to V36 and T54 mutations in the NS3-4A protease of the hepatitis C virus

Background

The inhibitor telaprevir (VX-950) of the hepatitis C virus (HCV) protease NS3-4A has been tested in a recent phase 1b clinical trial in patients infected with HCV genotype 1. This trial revealed residue mutations that confer varying degrees of drug resistance. In particular, two protease positions with the mutations V36A/G/L/M and T54A/S were associated with low to medium levels of drug resistance during viral breakthrough, together with only an intermediate reduction of viral replication fitness. These mutations are located in the protein interior and far away from the ligand binding pocket.

Results

Based on the available experimental structures of NS3-4A, we analyze the binding mode of different ligands. We also investigate the binding mode of VX-950 by protein-ligand docking. A network of non-covalent interactions between amino acids of the protease structure and the interacting ligands is analyzed to discover possible mechanisms of drug resistance. We describe the potential impact of V36 and T54 mutants on the side chain and backbone conformations and on the non-covalent residue interactions. We propose possible explanations for their effects on the antiviral efficacy of drugs and viral fitness. Molecular dynamics simulations of T54A/S mutants and rotamer analysis of V36A/G/L/M side chains support our interpretations. Experimental data using an HCV V36G replicon assay corroborate our findings.

Conclusion

T54 mutants are expected to interfere with the catalytic triad and with the ligand binding site of the protease. Thus, the T54 mutants are assumed to affect the viral replication efficacy to a larger degree than V36 mutants. Mutations at V36 and/or T54 result in impaired interaction of the protease residues with the VX-950 cyclopropyl group, which explains the development of viral breakthrough variants.     

Sequence analysis of NS3 protease gene in clinical strains of hepatitis C virus

The amino terminal region of the non structural gene 3 (NS3) of hepatitis C virus (HCV) is a chymotripsinlike serine-protease responsible for cleavage of the non structural proteins of Hepatitis C virus (HCV). In order to investigate the genetic variation of this region, we developed a nested PCR to obtain NS3 protease sequences from 54 patients chronically infected with HCV genotypes 1a, 1b and 3, respectively. Comparison of nucleotide and amino acids sequences of NS3 protease domain with consensus sequence obtained within the same genotype, showed 3.73% nucleotide divergence and 1.64% amino acid divergence in isolates of genotype 3a, whereas isolates 1a exhibited 4.45% nucleotide and 4% amino acid change, respectively. Finally, NS3 sequence from 1b isolates revealed 6.47% nucleotide and 3.5 % aa changes. Comparison of consensus amino acid sequences derived from isolates 1a, 1b and 3, with the HCV prototypes showed a low amino acid sequence diversity. However, the consensus sequence of HCV genotype 3 isolates showed an amino acid changed from the prototype, that was located within a region important for enzyme structure and activity. These results indicated that the NS3 protease gene is highly conserved within the same HCV genotype. The domains involved in enzyme function were highly conserved in 1a and 1b strains, whereas consensus sequence of isolates 3a showed that the majority of these strains were not perfectly conserved in one of such regions. These findings altogether suggested that the NS3 protease enzyme of HCV may constitute an important target for antiviral therapy, but the NS3 protease variability of isolates 3 within a region that is a potential target for antiviral therapy could pose a problem for structure based drug development.     

Resistance outside the substrate envelope: hepatitis C NS3/4A protease inhibitors

Direct acting antivirals have dramatically increased the efficacy and tolerability of hepatitis C treatment, but drug resistance has emerged with some of these inhibitors, including nonstructural protein 3/4?A protease inhibitors (PIs). Although many co-crystal structures of PIs with the NS3/4A protease have been reported, a systematic review of these crystal structures in the context of the rapidly emerging drug resistance especially for early PIs has not been performed. To provide a framework for designing better inhibitors with higher barriers to resistance, we performed a quantitative structural analysis using co-crystal structures and models of HCV NS3/4A protease in complex with natural substrates and inhibitors. By comparing substrate structural motifs and active site interactions with inhibitor recognition, we observed that the selection of drug resistance mutations correlates with how inhibitors deviate from viral substrates in molecular recognition. Based on this observation, we conclude that guiding the design process with native substrate recognition features is likely to lead to more robust small molecule inhibitors with decreased susceptibility to resistance.     

An NS3 serine protease inhibitor abrogates replication of subgenomic hepatitis C virus RNA

The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.     

Consideration of Viral Resistance for Optimization of Direct Antiviral Therapy of Hepatitis C Virus Genotype 1-Infected Patients

Different highly effective interferon-free treatment options for chronic hepatitis C virus (HCV) infection are currently available. Pre-existence of resistance associated variants (RAVs) to direct antiviral agents (DAAs) reduces sustained virologic response (SVR) rates by 3–53% in hepatitis C virus (HCV) genotype 1 infected patients depending on different predictors and the DAA regimen used. Frequencies of single and combined resistance to NS3, NS5A and NS5B inhibitors and consequences for the applicability of different treatment regimens are unknown. Parallel population based sequencing of HCV NS3, NS5A and NS5B genes in 312 treatment-naïve Caucasian HCV genotype 1 infected patients showed the presence of major resistant variants in 20.5% (NS3), 11.9% (NS5A), and 22.1% (NS5B) with important differences for HCV subtypes. In NS3, Q80K was observed in 34.7% and 2.1% of subtype 1a and 1b patients, respectively while other RAVs to second generation protease inhibitors were detected rarely (1.4%). Within NS5A RAVs were observed in 7.1% of subtype 1a and 17.6% in subtype 1b infected patients. RAVs to non-nucleoside NS5B inhibitors were observed in 3.5% and 44.4% of subtype 1a and 1b patients, respectively. Considering all three DAA targets all subtype 1a and 98.6% of subtype 1b infected patients were wildtype for at least one interferon free DAA regimen currently available. In conclusion, baseline resistance testing allows the selection of at least one RAVs-free treatment option for nearly all patients enabling a potentially cost- and efficacy-optimized treatment of chronic hepatitis C.     

Hepatitis C Virus Variants with Decreased Sensitivity to Direct-Acting Antivirals (DAAs) Were Rarely Observed in DAA-Naive Patients prior to Treatment

The prevalence of naturally occurring hepatitis C virus (HCV) variants that are less sensitive to direct-acting antiviral (DAA) inhibitors has not been fully characterized. We used population sequence analysis to assess the frequency of such variants in plasma samples from 3,447 DAA-naive patients with genotype 1 HCV. In general, HCV variants with lower-level resistance (3- to 25-fold increased 50% inhibitor concentration [IC₅₀]) to telaprevir were observed as the dominant species in 0 to 3% of patients, depending on the specific variant, whereas higher-level resistant variants (>25-fold-increased IC₅₀) were not observed. Specific variants resistant to NS5A inhibitors were predominant in up to 6% of patients. Most variants resistant to nucleo(s/t)ide active-site NS5B polymerase inhibitors were not observed, whereas variants resistant to non-nucleoside allosteric inhibitors were observed in up to 18% of patients. The presence of DAA-resistant variants in NS5A, NS5B, or NS3 (including telaprevir-resistant variants), in baseline samples of treatment-naive patients receiving a telaprevir-based regimen in phase 3 studies did not affect the sustained viral response (SVR). Treatment-naive patients with viral populations containing the telaprevir-resistant variants NS3 V36M, T54S, or R155K at baseline achieved a 74% SVR rate, whereas patients with no resistant variants detected prior to treatment achieved a 76% SVR rate. The effect of specific resistant variant frequency on response to various DAA treatments in different patient populations, including interferon nonresponders, should be further studied.     

Compensatory substitutions in the HCV NS3/4A protease cleavage sites are not observed in patients treated unsuccessfully with telaprevir combination treatment

ABSTRACT: BACKGROUND: Development of compensatory mutations within the HIV p7/p1 and p1/p6 protease cleavage site region has been observed in HIV-infected patients treated with protease inhibitors. Mechanisms of fitness compensation may occur in HCV populations upon treatment of HCV protease inhibitors as well. FINDINGS: In this study, we investigated whether substitutions in protease cleavage site regions of HCV occur in response to a treatment regimen containing the NS3/4A protease inhibitor telaprevir (TVR). Evaluation of viral populations from 569 patients prior to treatment showed that the four NS3/4A cleavage sites were well conserved. Few changes in the cleavage site regions were observed in the 159 patients who failed TVR combination treatment, and no residues displayed evidence of directional selection after the acquisition of TVR-resistance. CONCLUSIONS: Cleavage site mutations did not occur after treatment with the HCV protease inhibitor telaprevir.     

Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells

The hepatitis C virus (HCV) contains a positive-sense RNA genome that encodes a unique polyprotein precursor, which must be processed by proteases to enable viral maturation. Virally encoded NS3/4A protease has thus become an attractive target for the development of antiviral drugs. To establish an assay system for monitoring NS3/4A protease activity in mammalian cells, this study describes a substrate vector, pEG(Delta4AB)SEAP, in which enhanced green fluorescent protein (EGFP) was fused to secreted alkaline phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, Delta4AB, which spans the NS4A and NS4B junction region. Secretion of SEAP into the culture medium was demonstrated to depend on the cleavage of Delta4AB by HCV NS3/4A protease. We demonstrated that the accumulation of SEAP activity in the culture medium depends on time up to 60h with the coexpression of active NS3/4A protease. The amount of SEAP in the culture medium was around 10 times greater than that of cells with coexpression of inactive NS3/4A mutant protease. This strategy has made it possible to monitor NS3/4A activity inside mammalian cells. Moreover, by using cells containing the HCV subgenomic replicon, the EG(Delta4AB)SEAP reporter can be used to detect the anti-HCV activity of interferon-alpha (IFN-alpha). Consequently, this EG(Delta4AB)SEAP reporter can be used to screen for NS3/4A protease inhibitors in the cellular environment and for anti-HCV drugs in replicon cells.     

Functional and therapeutic analysis of hepatitis C virus NS3.4A protease control of antiviral immune defense

Chronic hepatitis C virus (HCV) infection is a major global public health problem. HCV infection is supported by viral strategies to evade the innate antiviral response wherein the viral NS3.4A protease complex targets and cleaves the interferon promoter stimulator-1 (IPS-1) adaptor protein to ablate signaling of interferon alpha/beta immune defenses. Here we examined the structural requirements of NS3.4A and the therapeutic potential of NS3.4A inhibitors to control the innate immune response against virus infection. The structural composition of NS3 includes an amino-terminal serine protease domain and a carboxyl-terminal RNA helicase domain. NS3 mutants lacking the helicase domain retained the ability to control virus signaling initiated by retinoic acid-inducible gene-I (RIG-I) or melanoma differentiation antigen 5 and suppressed the downstream activation of interferon regulatory factor-3 (IRF-3) and nuclear factor kappaB (NF-kappaB) through the targeted proteolysis of IPS-1. This regulation was abrogated by truncation of the NS3 protease domain or by point mutations that ablated protease activity. NS3.4A protease control of antiviral immune signaling was due to targeted proteolysis of IPS-1 by the NS3 protease domain and minimal NS4A cofactor. Treatment of HCV-infected cells with an NS3 protease inhibitor prevented IPS-1 proteolysis by the HCV protease and restored RIG-I immune defense signaling during infection. Thus, the NS3.4A protease domain can target IPS-1 for cleavage and is essential for blocking RIG-I signaling to IRF-3 and NF-kappaB, whereas the helicase domain is dispensable for this action. Our results indicate that NS3.4A protease inhibitors have immunomodulatory potential to restore innate immune defenses to HCV infection.     

Phenotypic and structural analyses of hepatitis C virus NS3 protease Arg155 variants: sensitivity to telaprevir (VX-950) and interferon alpha

Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg(155) of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg(155) was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg(155) with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5A. The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys(155) side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon alpha or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells.     

Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells

Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.     

Similar Prevalence of Low-Abundance Drug-Resistant Variants in Treatment-Naive Patients with Genotype 1a and 1b Hepatitis C Virus Infections as Determined by Ultradeep Pyrosequencing

Background and Objectives

Hepatitis C virus (HCV) variants that confer resistance to direct-acting-antiviral agents (DAA) have been detected by standard sequencing technology in genotype (G) 1 viruses from DAA-naive patients. It has recently been shown that virological response rates are higher and breakthrough rates are lower in G1b infected patients than in G1a infected patients treated with certain classes of HCV DAAs. It is not known whether this corresponds to a difference in the composition of G1a and G1b HCV quasispecies in regards to the proportion of naturally occurring DAA-resistant variants before treatment.

Methods

We used ultradeep pyrosequencing to determine the prevalence of low-abundance (<25% of the sequence reads) DAA-resistant variants in 191 NS3 and 116 NS5B isolates from 208 DAA-naive G1-infected patients.

Results

A total of 3.5 million high-quality reads of ≥200 nucleotides were generated. The median coverage depth was 4150x and 4470x per NS3 and NS5B amplicon, respectively. Both G1a and G1b populations showed Shannon entropy distributions, with no difference between G1a and G1b in NS3 or NS5B region at the nucleotide level. A higher number of substitutions that confer resistance to protease inhibitors were observed in G1a isolates (mainly at amino acid 80 of the NS3 region). The prevalence of amino acid substitutions that confer resistance to NS5B non-nucleoside inhibitors was similar in G1a and G1b isolates. The NS5B S282T variant, which confers resistance to the polymerase inhibitors mericitabine and sofosbuvir, was not detected in any sample.

Conclusion

The quasispecies genetic diversity and prevalence of DAA-resistant variants was similar in G1a and G1b isolates and in both NS3 and NS5B regions, suggesting that this is not a determinant for the higher level of DAA resistance observed across G1a HCV infected patients upon treatment.     

Evaluation of persistence of resistant variants with ultra-deep pyrosequencing in chronic hepatitis C patients treated with telaprevir

Background & Aims

Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment.

Methods

Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103) were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline.

Results

Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing.

Conclusion

In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that long-term persistence of resistant variants is rare.     

Discovery of velpatasvir (GS-5816): A potent pan-genotypic HCV NS5A inhibitor in the single-tablet regimens Vosevi® and Epclusa®

Direct-acting antiviral inhibitors have revolutionized the treatment of hepatitis C virus (HCV) infected patients. Herein is described the discovery of velpatasvir (VEL, GS-5816), a potent pan-genotypic HCV NS5A inhibitor that is a component of the only approved pan-genotypic single-tablet regimens (STRs) for the cure of HCV infection. VEL combined with sofosbuvir (SOF) is Epclusa^®, an STR with 98% cure-rates for genotype 1–6 HCV infected patients. Addition of the pan-genotypic HCV NS3/4A protease inhibitor voxilaprevir to SOF/VEL is the STR Vosevi^®, which affords 97% cure-rates for genotype 1–6 HCV patients who have previously failed another treatment regimen.     

HCV NS3/4A蛋白酶抑制剂细胞筛选模型

丙型肝炎病毒(hepatitis C virus,HCV)持续感染可导致慢性丙型肝炎,并可发展为肝硬化和肝细胞癌。HCV NS3/4A蛋白酶在HCV复制和致病机制中起着非常关键的作用,已成为HCV研究热点。由于候选药物分子必须具有易进入细胞、稳定性好等特征,建立一种细胞水平上酶活性的测定系统对于筛选抗NS3/4A药物无疑有着重要意义。目前已有多种NS3/4A蛋白酶筛选系统开发出来,本文将对此作一综述。     

Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors

The mechanism and kinetics of the interactions between ligands and immobilized full‐length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3–NS4A interaction consisted of a high‐affinity (K_D = 50 nM) and a low‐affinity (K_D = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism‐based inhibitor VX 950 exhibited a time‐dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN‐191 showed no signs of time‐dependent interactions, but ITMN‐191 had the highest affinity of the tested compounds, with both the slowest dissociation (k_off) and fastest association rate, closely followed by BILN 2061. The k_off for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti‐HCV lead discovery and optimization. Copyright © 2010 John Wiley & Sons, Ltd.     

Thermodynamics of zinc binding to hepatitis C virus NS3 protease: A folding by binding event

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease is responsible for the processing of the non‐structural region of the viral precursor polyprotein in infected hepatic cells. HCV NS3 is a zinc‐dependent serine protease. The zinc ion, which is bound far away from the active site and considered to have a structural role, is essential for the structural integrity of the protein; furthermore, the ion is required for the hydrolytic activity. Consequently, the NS3 zinc binding site has been considered for a long time as a possible target for drug discovery. As a first step towards this goal, the energetics of the NS3‐zinc interaction and its effect on the NS3 conformation must be established and discussed. The thermodynamic characterization of zinc binding to NS3 protease by isothermal titration calorimetry and spectroscopy is presented here. Spectroscopic and calorimetric results suggest that a considerable conformational change in the protein is coupled to zinc binding. The energetics of the conformational change is comparable to that of the folding of a protein of similar size. Therefore, zinc binding to NS3 protease can be considered as a “folding by binding” event. Proteins 2009. © 2009 Wiley‐Liss, Inc.