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.

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.

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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 biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit poor systemic exposure in rat pharmacokinetic studies after oral dosing. The structure-activity relationship investigations that improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification of the highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was the realization that the arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50 values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38-108% in preclinical species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has recently been sought in Japan.

BackgroundThe purpose of this study was to investigate the potential of antibody-directed immunotherapy targeting the aminophospholipid phosphatidylserine, which promotes immunosuppression when exposed in the tumor microenvironment, alone and in combination with antibody treatment towards the T-cell checkpoint inhibitor PD-1 in breast carcinomas, including triple-negative breast cancers.MethodsImmune-competent mice bearing syngeneic EMT-6 or E0771 tumors were subjected to treatments comprising of a phosphatidylserine-targeting and an anti-PD-1 antibody either as single or combinational treatments. Anti-tumor effects were determined by tumor growth inhibition and changes in overall survival accompanying each treatment. The generation of a tumor-specific immune response in animals undergoing complete tumor regression was assessed by secondary tumor cell challenge and splenocyte-produced IFNγ in the presence or absence of irradiated tumor cells. Changes in the presence of tumor-infiltrating lymphocytes were assessed by flow cytometry, while mRNA-based immune profiling was determined using NanoString PanCancer Immune Profiling Panel analysis.ResultsTreatment by a phosphatidylserine-targeting antibody inhibits in-vivo growth and significantly enhances the anti-tumor activity of antibody-mediated PD-1 therapy, including providing a distinct survival advantage over treatment by either single agent. Animals in which complete tumor regression occurred with combination treatments were resistant to secondary tumor challenge and presented heightened expression levels of splenocyte-produced IFNγ. Combinational treatment by a phosphatidylserine-targeting antibody with anti-PD-1 therapy increased the number of tumor-infiltrating lymphocytes more than that observed with single-arm therapies. Finally, immunoprofiling analysis revealed that the combination of anti-phosphatidylserine targeting antibody and anti-PD-1 therapy enhanced tumor-infiltrating lymphocytes, and increased expression of pro-immunosurveillance-associated cytokines while significantly decreasing expression of pro-tumorigenic cytokines that were induced by single anti-PD-1 therapy.ConclusionsOur data suggest that antibody therapy targeting phosphatidylserine-associated immunosuppression, which has activity as a single agent, can significantly enhance immunotherapies targeting the PD-1 pathway in murine breast neoplasms, including triple-negative breast cancers.

A series of 2-substituted benzofuran hydroxyamic acids were synthesized as rigid analogs of simple (benzyloxy)phenyl hydroxamates, evaluated for their in vitro and in vivo 5-lipoxygenase activity and found to be potent inhibitors of the enzyme. Substituents which enhanced lipophilicity near the 2-position of the benzofuran nucleus increased inhibitor potency but reduced oral activity. Incorporation of small polar substituents such as methoxymethylene, hydroxymethylene, and amino (urea) on the acyl group led to more consistent oral activity. The most potent inhibitors of this series in vitro were N-hydroxy-N-[1-(2-phenyl-5-benzofuranyl)-ethyl]furancarboxamide (12) and methyl 5-[N-hydroxy-N-[1-(2-(3,4,5-trimethoxyphenyl)-5-benzofuranyl]ethyl]-5- oxopentanoate (17), both with IC50 values of 40 nM, and in vivo the most potent compound was N-hydroxy-N-[1-(2-phenyl-5-benzofuranyl)ethyl]urea, 20, with an ED50 = 10.3 mg/kg.

A medicinal chemistry campaign that was conducted to address a potential genotoxic liability associated with an aniline-derived scaffold in a series of HCV NS5A inhibitors with dual GT-1a/-1b inhibitory activity is described. Anilides 3b and 3c were used as vehicles to explore structural modifications that retained antiviral potency while removing the potential for metabolism-based unmasking of the embedded aniline. This effort resulted in the discovery of a highly potent biarylimidazole chemotype that established a potency benchmark in replicon assays, particularly toward HCV GT-1a, a strain with significant clinical importance. Securing potent GT-1a activity in a chemotype class lacking overt structural liabilities was a critical milestone in the effort to realize the full clinical potential of targeting the HCV NS5A protein.