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Iterative structure–activity analyses in a class of highly functionalized furo[2,3-b]pyridines led to the identification of the second generation pan-genotypic hepatitis C virus NS5B polymerase primer grip inhibitor BMT-052 (14), a potential clinical candidate. The key challenge of poor metabolic stability was overcome by strategic incorporation of deuterium at potential metabolic soft spots. The preclinical profile and status of BMT-052 (14) is described.

[Image: see text] In solving the P-gp and BCRP transporter-mediated efflux issue in a series of benzofuran-derived pan-genotypic palm site inhibitors of the hepatitis C virus NS5B replicase, it was found that close attention to physicochemical properties was essential. In these compounds, where both molecular weight (MW >579) and TPSA (>110 Å(2)) were high, attenuation of polar surface area together with weakening of hydrogen bond acceptor strength of the molecule provided a higher intrinsic membrane permeability and more desirable Caco-2 parameters, as demonstrated by trifluoroacetamide 11 and the benchmark N-ethylamino analog 12. In addition, the tendency of these inhibitors to form intramolecular hydrogen bonds potentially contributes favorably to the improved membrane permeability and absorption. The functional group minimization that resolved the efflux problem simultaneously maintained potent inhibitory activity toward a gt-2 HCV replicon due to a switching of the role of substituents in interacting with the Gln414 binding pocket, as observed in gt-2a NS5B/inhibitor complex cocrystal structures, thus increasing the efficiency of the optimization. Noteworthy, a novel intermolecular S=O···C=O n → π* type interaction between the ligand sulfonamide oxygen atom and the carbonyl moiety of the side chain of Gln414 was observed. The insights from these structure–property studies and crystallography information provided a direction for optimization in a campaign to identify second generation pan-genotypic NS5B inhibitors.

The drug daclatasvir (DCV), which inhibits the hepatitis C virus (HCV) non-structural protein 5A (NS5A), can successfully reduce viral load in patients; here, a combination of DCV and an NS5A analogue is shown to enhance DCV potency on multiple genotypes and overcome resistance in vitro and in a mouse model. The recently approved drug daclatasivir can successfully reduce viral load in patients infected with hepatitis C virus (HCV) by inhibiting the virus's non-structural protein 5A (NS5A) replication complex, although HCV NS5A inhibitor resistance mutations arise in some cases. Min Gao and colleagues now show that a combination of daclatasivir and an NS5A analogue can enhance daclatasivir potency, and the combination also overcomes resistance in vitro and in a mouse model. The authors develop a model to explain this effect, whereby inhibitor binding to one resistant NS5A causes a conformational change that is transmitted to adjacent NS5A molecules, restoring drug sensitivity to the resistant molecules. It is estimated that more than 170 million people are infected with hepatitis C virus (HCV) worldwide1,2. Clinical trials have demonstrated that, for the first time in human history, the potential exists to eradicate a chronic viral disease using combination therapies that contain only direct-acting antiviral agents3. HCV non-structural protein 5A (NS5A) is a multifunctional protein required for several stages of the virus replication cycle4. NS5A replication complex inhibitors, exemplified by daclatasvir (DCV; also known as BMS-790052 and Daklinza), belong to the most potent class of direct-acting anti-HCV agents described so far, with in vitro activity in the picomolar (pM) to low nanomolar (nM) range. The potency observed in vitro has translated into clinical efficacy, with HCV RNA declining by ~3–4 log10 in infected patients after administration of single oral doses of DCV. Understanding the exceptional potency of DCV was a key objective of this study. Here we show that although DCV and an NS5A inhibitor analogue (Syn-395) are inactive against certain NS5A resistance variants, combinations of the pair enhance DCV potency by >1,000-fold, restoring activity to the pM range. This synergistic effect was validated in vivo using an HCV-infected chimaeric mouse model. The cooperative interaction of a pair of compounds suggests that NS5A protein molecules communicate with each other: one inhibitor binds to resistant NS5A, causing a conformational change that is transmitted to adjacent NS5As, resensitizing resistant NS5A so that the second inhibitor can act to restore inhibition. This unprecedented synergistic anti-HCV activity also enhances the resistance barrier of DCV, providing additional options for HCV combination therapy and new insight into the role of NS5A in the HCV replication cycle.

BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC 50 ) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC 50 s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC 50 , 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC 50 s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC 50 . Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures in vivo in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC 50 s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.

The clinical efficacy of a pegylated form of human lambda 1 interferon (IFN-λ1; also referred to herein as lambda) has been demonstrated in patients chronically infected with hepatitis C virus (HCV) representing genotypes 1 through 4. In these proof-of-concept studies, lambda showed an improved safety profile compared to the pegylated form of alfa interferon (referred to herein as alfa). In the study described in this report, an assessment of the in vitro antiviral activity of type III IFNs toward different HCV replicons revealed that the unpegylated recombinant form of IFN-λ1 (rIFN-λ1) exerted the most robust effect, while rIFN-λ3 exhibited greater activity than rIFN-λ2. More importantly, cross-resistance to rIFN-λ1 was not observed in replicon cell lines known to have reduced susceptibility to investigational direct-acting antiviral (DAA) agents targeting the essential HCV nonstructural protein NS3, NS5A, or NS5B. When combined with either rIFN-α, the NS3 protease inhibitor (NS3 PI) asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFN-λ1 displayed a mixture of additive and synergistic effects. In three-drug combination studies, inclusion of lambda with ASV and DCV also yielded additive to synergistic effects. In line with these observations, it was demonstrated that a regimen that used a combination of rIFN-λ1 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant sequences. Overall, these data support further clinical development of lambda as part of alternative combination treatments with DAAs for patients chronically infected with HCV.

The development of a series of novel 7-azabenzofurans exhibiting pan-genotype inhibition of HCV NS5B polymerase via binding to the primer grip site is presented. Many challenges, including poor oral bioavailability, high clearance, bioactivation, high human serum shift, and metabolic stability were encountered and overcome through SAR studies. This work culminated in the selection of BMS-986139 (43) as a preclinical candidate.

Three hepatitis C virus (HCV) inhibitors, asunaprevir (ASV; BMS-650032), daclatasvir (DCV; BMS-790052), and BMS-791325, each targeting a different nonstructural protein of the virus (NS3, NS5A, and NS5B, respectively), have independently demonstrated encouraging preclinical profiles and are currently undergoing clinical evaluation. Since drug-resistant variants have rapidly developed in response to monotherapy with almost all direct-acting antiviral agents (DAAs) for HCV, the need for combination therapies to effectively eradicate the virus from infected patients is clear. These studies demonstrated the additive-synergistic effects on replicon inhibition and clearance of combining NS3 protease or NS5B RNA polymerase inhibitors with the first-in-class, NS5A replication complex inhibitor daclatasvir (DCV) and reveal new resistance pathways for combinations of two small-molecule inhibitors that differ from those that develop during monotherapy. The results suggest that under a specific selective pressure, a balance must be reached in the fitness costs of substitutions in one target gene when substitutions are also present in another target gene. Further synergies and additional novel resistance substitutions were observed during triple-combination treatment relative to dual-drug therapy, indicating that, in combination, HCV inhibitors can exert cross-target influences on resistance development. Enhanced synergies in replicon inhibition and a reduced frequency of resistance together lend strong support to the utility of combinations of DAAs for the treatment of HCV, and the identification of altered resistance profiles during combination treatment provides useful information for monitoring resistance in the clinic.

The iminothiazolidinone BMS-858 (2) was identified as a specific inhibitor of HCV replication in a genotype 1b replicon assay via a high-throughput screening campaign. A more potent analogue, BMS-824 (18), was used in resistance mapping studies, which revealed that inhibitory activity was related to disrupting the function of the HCV nonstructural protein 5A. Despite the development of coherent and interpretable SAR, it was subsequently discovered that in DMSO 18 underwent an oxidation and structural rearrangement to afford the thiohydantoin 47, a compound with reduced HCV inhibitory activity. However, HPLC bioassay fractionation studies performed after incubation of 18 in assay media led to the identification of fractions containing a dimeric species 48 that exhibited potent antiviral activity. Excision of the key elements hypothesized to be responsible for antiviral activity based on SAR observations reduced 48 to a simplified, symmetrical, pharmacophore realized most effectively with the stilbene 55, a compound that demonstrated potent inhibition of HCV in a genotype 1b replicon with an EC50 = 86 pM.

The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC(50)) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log(10) reduction in mean viral load measured 24 h post-dose that was sustained for an additional 120 h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144 h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.

Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ∼5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.

Objective: To utilize chimeric hepatitis C virus (HCV) replicons to select adaptive mutation(s) that allow replication of a genotype 1a replicon. Methods: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural genes of a 1b replicon with genotype 1a sequences. Results: A chimeric replicon containing a genotype 1a NS5A protein did not replicate in a transient assay, but could be used to establish stable cell lines using G418 selection. The cell lines contained a K1846T mutation in NS4B which functioned as an adaptive mutation that now allowed the chimera to replicate at levels similar to wild-type replicons. Similarly, replication of a 1a NS5A5B chimera was only observed after establishment of stable cell lines, even in the presence of the K1846T mutation. Sequence analysis of this cell line revealed an additional adaptive mutation of M1496L in NS3. Lastly, by including the K1846T mutation in a replicon that was entirely genotype 1a sequence, stable 1a cell lines could be established. Conclusion: These studies identify an NS4B adaptive mutation, K1846T, which allows establishment of a replication-competent 1a replicon and demonstrate the utility of this chimeric approach for establishing replicons for various HCV genotypes.

BMS-791325 is a hepatitis C virus (HCV) inhibitor binding to the thumb domain of the NS5B RNA-dependent RNA polymerase. BMS-791325 is well characterized in genotype 1 (GT1) and exhibits good inhibitory activity (50% effective concentration [EC 50 ], http://dx.doi.org/10.1128/AAC.02495-13 ). BMS-791325 potency against GT6a hybrid replicons is more variable, with two of three hybrid clones having EC 50 s similar to that for GT1 while a third patient clone was ∼10 times less susceptible to BMS-791325. To characterize the resistance profile of BMS-791325 beyond GT1, curing studies were performed across GT1a and -3a to -6a and demonstrated that GT1a has the highest resistance barrier versus BMS-791325 while GT6a has the lowest. Selection of GT3 to -6 NS5B chimeric replicon cells at different concentrations of BMS-791325 revealed substitutions in the thumb domain of NS5B at residues 494 and 495 that conferred different levels of resistance to BMS-791325 but remained susceptible to NS5A or NS3 protease inhibitors. In addition, we demonstrate that the reduced potency of BMS-791325 against one GT6a patient is due to an A494 polymorphism present in ∼21% of sequences in the European HCV database. The results from this report suggest that BMS-791325 is a candidate for combination treatment of HCV GT3 to -6 chronic infections, and the resistance profiles identified will provide useful information for future clinical development.

BMS-791325 is a nonnucleoside inhibitor of hepatitis C virus (HCV) NS5B polymerase with low-nanomolar potency against genotypes 1a (50% effective concentration [EC 50 ], 3 nM) and 1b (EC 50 , 7 nM) in vitro . BMS-791325 safety, pharmacokinetics, and antiviral activity were evaluated in a double-blind, placebo-controlled, single-ascending-dose study in 24 patients (interferon naive and experienced) with chronic HCV genotype 1 infection, randomized (5:1) to receive a single dose of BMS-791325 (100, 300, 600, or 900 mg) or placebo. The prevalence and phenotype of HCV variants at baseline and specific posttreatment time points were assessed. Antiviral activity was observed in all cohorts, with a mean HCV RNA decline of ≈2.5 log 10 copies/ml observed 24 h after a single 300-mg dose. Mean plasma half-life among cohorts was 7 to 9 h; individual 24-hour levels exceeded the protein-adjusted EC 90 for genotype 1 at all doses. BMS-791325 was generally well tolerated, with no serious adverse events or discontinuations. Enrichment for resistance variants was not observed at 100 to 600 mg. At 900 mg, variants (P495L/S) associated with BMS-791325 resistance in vitro were transiently observed in one patient, concurrent with an observed HCV RNA decline of 3.4 log 10 IU/ml, but were replaced with wild type by 48 h. Single doses of BMS-791325 were well tolerated; demonstrated rapid, substantial, and exposure-related antiviral activity; displayed dose-related increases in exposure; and showed viral kinetic and pharmacokinetic profiles supportive of once- or twice-daily dosing. These results support its further development in combination with other direct-acting antivirals for HCV genotype 1 infection. (This trial has been registered at ClinicalTrials.gov under registration no. NCT00664625.)

The encephalitic alphaviruses are useful models for understanding virus-neuron interactions. A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and inducing apoptosis of these nonrenewable cells. Antibodies to the surface glycoproteins suppress virus replication, but other recovery-promoting components of the immune response have not been recognized. We assessed the effect on the outcome of NSV-induced encephalomyelitis of immunization of mice with nonstructural proteins (nsPs) by using recombinant vaccinia viruses. Mice immunized with vaccinia virus expressing nsPs and challenged with NSV initially developed paralysis similar to unimmunized mice but then recovered neurologic function. Mice preimmunized with vaccinia virus expressing structural proteins were completely protected from paralysis. Mice immunized with vaccinia virus alone showed paralysis with little evidence of recovery. Vaccinia virus expressing only nsP2 was as effective as vaccinia virus expressing all the nsPs. Protection provided by immunity to nsPs was not associated with a reduction in virus replication or with improved antibody responses to structural proteins. Protection could not be passively transferred with nsP immune serum. The depletion of T cells at the time of NSV infection decreased protection. The data show that antiviral immune responses can improve the ability of neurons to survive infection and to recover function without altering virus replication.

The exceptional in vitro potency of the hepatitis C virus (HCV) NS5A inhibitor BMS-790052 has translated into an in vivo effect in proof-of-concept clinical trials. Although the 50% effective concentration (EC 50 ) of the initial lead, the thiazolidinone BMS-824, was ∼10 nM in the replicon assay, it underwent transformation to other inhibitory species after incubation in cell culture medium. The biological profile of BMS-824, including the EC 50 , the drug concentration required to reduce cell growth by 50% (CC 50 ), and the resistance profile, however, remained unchanged, triggering an investigation to identify the biologically active species. High-performance liquid chromatography (HPLC) biogram fractionation of a sample of BMS-824 incubated in medium revealed that the most active fractions could readily be separated from the parental compound and retained the biological profile of BMS-824. From mass spectral and nuclear magnetic resonance data, the active species was determined to be a dimer of BMS-824 derived from an intermolecular radical-mediated reaction of the parent compound. Based upon an analysis of the structural elements of the dimer deemed necessary for anti-HCV activity, the stilbene derivative BMS-346 was synthesized. This compound exhibited excellent anti-HCV activity and showed a resistance profile similar to that of BMS-824, with changes in compound sensitivity mapped to the N terminus of NS5A. The N terminus of NS5A has been crystallized as a dimer, complementing the symmetry of BMS-346 and allowing a potential mode of inhibition of NS5A to be discussed. Identification of the stable, active pharmacophore associated with these NS5A inhibitors provided the foundation for the design of more potent inhibitors with broad genotype inhibition. This culminated in the identification of BMS-790052, a compound that preserves the symmetry discovered with BMS-346.

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.

The hepatitis C virus (HCV) replicon is a unique system for the development of a high-throughput screen (HTS), since the analysis of inhibitors requires the quantification of a decrease in a steady-state level of HCV RNA. HCV replicon replication is dependent on host cell factors, and any toxic effects may have a significant impact on HCV replicon replication. Therefore, determining the antiviral specificity of compounds presents a challenge for the identification of specific HCV inhibitors. Here we report the development of an HCV/bovine viral diarrhea virus (BVDV) dual replicon assay suitable for HTS to address these issues. The HCV reporter enzyme is the endogenous NS3 protease contained within the HCV genome, while the BVDV reporter enzyme is a luciferase enzyme engineered into the BVDV genome. The HTS uses a mixture of HCV and BVDV replicon cell lines placed in the same well of a 96-well plate and isolated in the same cell backgrounds (Huh-7). The format consists of three separate but compatible assays: the first quantitates the amount of cytotoxicity based upon the conversion of Alamar blue dye via cellular enzymes, while the second indirectly quantitates HCV replicon replication through measurement of the amount of NS3 protease activity present. The final assay measures the amount of luciferase activity present from the BVDV replicon cells, as an indicator of the specificity of the test compounds. This HCV/BVDV dual replicon assay provides a reliable format to determine the potency and specificity of HCV replicon inhibitors.

Compound-1453 was identified and characterized as a specific inhibitor of bovine viral diarrhea virus (BVDV). The concentration of compound-1453 which results in 50% protection from virus-induced cytopathic effect is ∼2.2 μM, with a therapeutic index of 60, and it is not active against a panel of RNA and DNA viruses. A time-of-addition experiment suggested that compound-1453 targets a stage of the viral life cycle after viral entry. To determine the target of compound-1453, resistant virus was generated. Resistant variants grew efficiently in the presence or absence of 33 μM compound-1453 and exhibited replication efficiency in the presence of compound-1453 approximately 1,000-fold higher than that of the wild-type (wt) virus. Functional mapping and sequence analysis of resistant cDNAs revealed a single amino acid substitution (Glu to Gly) at residue 291 in the NS5B polymerase in all eight independently generated cDNA clones. Recombinant virus containing this single mutation retained the resistance phenotype and a replication efficiency similar to that of the original isolated resistant virus. Since compound-1453 did not inhibit BVDV polymerase activity in vitro (50% inhibitory concentration > 300 μM), we developed a membrane-based assay that consisted of a BVDV RNA replicase complex isolated from virus-infected cells. Compound-1453 inhibited the activity of the wt, but not the drug-resistant, replicase in the membrane assay at concentrations similar to those observed in the viral infection assay. This work presents a novel inhibitor of a viral RNA-dependent RNA replicase.

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn 2+ than in the presence of Mg 2+ . When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

The hepatitis C virus NS5A protein is an established and clinically validated target for antiviral intervention by small molecules. Characterizations are presented of compounds identified as potent inhibitors of HCV replication to provide insight into structural elements that interact with the NS5A protein. UV-activated cross linking and affinity isolation was performed with one series to probe the physical interaction between the inhibitors and the NS5A protein expressed in HCV replicon cells. Resistance mapping with the second series was used to determine the functional impact of specific inhibitor subdomains on the interaction with NS5A. The data provide evidence for a direct high-affinity interaction between these inhibitors and the NS5A protein, with the interaction dependent on inhibitor stereochemistry. The functional data supports a model of inhibition that implicates inhibitor binding by covalently combining distinct pharmacophores across an NS5A dimer interface to achieve maximal inhibition of HCV replication.