Binding kinetics, potency, and selectivity of the hepatitis C virus NS3 protease inhibitors GS-9256 and vedroprevir.

BACKGROUND: GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays. METHODS: Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods. RESULTS: GS-9256 and vedroprevir had measured Ki values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; Ki values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was >10,000-fold against all tested off-target proteases. Association rate constants of 4×10(5)M(-1)s(-1) and 1×10(6)M(-1)s(-1), respectively, were measured, and dissociation rate constants of 4.8×10(-5)s(-1) and 2.6×10(-4)s(-1) were determined. CONCLUSIONS: GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics. GENERAL SIGNIFICANCE: The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.

Preclinical characterization of the novel hepatitis C virus NS3 protease inhibitor GS-9451.

GS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, including in vitro antiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50 = 316 nM). Additive to synergistic in vitro antiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ∼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results of in vitro cross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.

No resistance to tenofovir disoproxil fumarate through 96 weeks of treatment in patients with lamivudine-resistant chronic hepatitis B.

BACKGROUND & AIMS: A recent study compared the efficacy of tenofovir disoproxil fumarate (TDF) vs the combination of emtricitabine and TDF (FTC/TDF) in patients with lamivudine-resistant chronic hepatitis B who were treated for as long as 96 weeks. We report findings from resistance analyses conducted for this study. METHODS: Two hundred eighty patients with chronic hepatitis B virus (HBV) infection and lamivudine resistance (confirmed by INNO-LiPA Multi-DR) were randomly assigned (1:1) to groups treated with TDF or FTC/TDF. The HBV reverse transcriptase domain from the polymerase gene from all patients was sequenced at baseline and from 18 viremic patients at week 96 or early discontinuation. RESULTS: At screening for the efficacy study, 99% of patients were found to have lamivudine resistance. Prior exposure to entecavir or entecavir resistance was observed in 12% of patients, and 22% of patients had been previously exposed to adefovir; 1.8% were resistant to adefovir. Only 18 patients (6.4%) qualified for sequence analysis, including 1 patient who experienced virologic breakthrough and 17 with persistent viremia. Six of these patients did not have any sequence changes from baseline in HBV reverse transcriptase (33%), and sequence analysis could not be performed for 5 patients (28%). In 2 patients who qualified for phenotypic analysis (1 given TDF and 1 given FTC/TDF), no resistance to TDF was observed. Neither previous treatment exposure nor resistance to entecavir or adefovir affected viral kinetics. However, the mean baseline level of HBV DNA was significantly higher in viremic patients than in patients with viral suppression by week 96 (7.28 log10 IU/mL vs 5.62 log10 IU/mL; P = .0003). CONCLUSIONS: No resistance to TDF was detected through 96 weeks of treatment in patients with lamivudine-resistant chronic hepatitis B. Prior treatment or resistance to entecavir or adefovir did not affect viral kinetics through 96 weeks. No additional benefit was observed with the addition of emtricitabine vs TDF monotherapy. ClinicalTrial.gov number: NCT00737568.

Novel, sulfonamide linked inhibitors of the hepatitis C virus NS3 protease.

A sulfonamide replacement of the P2-P3 amide bond in the context of macrocyclic HCV NS3 protease inhibitors was investigated. These analogs displayed good inhibitory potency in the absence of any P3 capping group. The synthesis and preliminary SAR are described.

Randomized, placebo-controlled trial of tenofovir disoproxil fumarate in adolescents with chronic hepatitis B.

UNLABELLED: Tenofovir disoproxil fumarate (DF) is highly effective for the suppression of hepatitis B virus (HBV) in chronically infected adults. This study evaluated the safety and efficacy of tenofovir DF in adolescents with chronic hepatitis B (CHB). In this double-blind, placebo-controlled trial, adolescents 12 to <18 years of age with CHB were randomized to tenofovir DF 300 mg (n = 52) or placebo (n = 54) once daily for 72 weeks. The primary endpoint was virologic response (HBV DNA <400 copies/mL) at week 72. One hundred six patients were enrolled; 101 patients completed 72 weeks of treatment. At baseline, 91% of patients were hepatitis B e antigen-positive and 85% had prior exposure to HBV therapy. A virologic response was observed in 89% (46/52) of patients who received tenofovir DF and 0% (0/54) of patients who received placebo (P < 0.001). Treatment response was not affected by prior HBV treatment. Furthermore, no resistance to tenofovir DF developed through week 72. Among patients with an alanine aminotransferase (ALT) level greater than the upper limit of normal at baseline, normalization of ALT occurred in 74% of patients receiving tenofovir DF and 31% of patients receiving placebo (P < 0.001). The rate of grade 3/4 adverse events was higher among patients treated with placebo (24%) than patients treated with tenofovir DF (10%). No patients met the safety endpoint of a 6% decrease in spine bone mineral density at week 72. CONCLUSION: Tenofovir DF therapy in HBV-infected adolescents was well tolerated and highly effective at suppressing HBV DNA and normalizing ALT values in both treatment-naïve adolescents and those with prior exposure to HBV therapy.

Localization of a class III myosin to filopodia tips in transfected HeLa cells requires an actin-binding site in its tail domain.

Bass Myo3A, a class III myosin, was expressed in HeLa cells as a GFP fusion in order to study its cellular localization. GFP-Myo3A localized to the cytoplasm and to the tips of F-actin bundles in filopodia, a localization that is consistent with the observed concentration toward the distal ends of F-actin bundles in photoreceptor cells. A mutation in the motor active site resulted in a loss of filopodia localization, suggesting that Myo3A motor activity is required for filopodial tip localization. Deletion analyses showed that the NH2-terminal kinase domain is not required but the CO2H-terminal 22 amino acids of the Myo3A tail are required for filopodial localization. Expression of this tail fragment alone produced fluorescence associated with F-actin throughout the cytoplasm and filopodia and a recombinant tail fragment bound to F-actin in vitro. An actin-binding motif was identified within this tail fragment, and a mutation within this motif abolished both filopodia localization by Myo3A and F-actin binding by the tail fragment alone. Calmodulin localized to filopodial tips when coexpressed with Myo3A but not in the absence of Myo3A, an observation consistent with the previous proposal that class III myosins bind calmodulin and thereby localize it in certain cell types.

In vitro efficacy of approved and experimental antivirals against novel genotype 3 hepatitis C virus subgenomic replicons.

Infection with genotype 3 hepatitis C virus (HCV) is common throughout the world, however no direct-acting antiviral (DAA) has been approved to treat this genotype. We therefore attempted to develop novel genotype 3 replicons to facilitate the discovery and development of new HCV therapies. A novel Huh-7-derived cell line 1C but not Lunet cells enabled the selection of a few stable colonies of a genotype 3a subgenomic replicon (strain S52). Genotypic analysis revealed a mutation of P89L in the viral NS3 protease domain, which was confirmed to enhance genotype 3a RNA replication and enable the establishment of highly replicating luciferase-encoding replicons. Secondary adaptive mutations that further enhanced RNA replication were identified in the viral NS3 and NS4A proteins. In addition, cell lines that were cured of genotype 3a replicons demonstrated higher permissiveness specifically to genotype 3a HCV replication. These novel replicons and cell lines were then used to study the activity of approved and experimental HCV inhibitors. NS3 protease and non-nucleoside NS5B polymerase inhibitors often demonstrated substantially less antiviral activity against genotype 3a compared to genotype 1b. In contrast, nucleoside analog NS5B inhibitors and host-targeting HCV inhibitors showed comparable antiviral activity between genotypes 3a and 1b. Overall, the establishment of this novel genotype 3a replicon system, in conjunction with those derived from other genotypes, will aid the development of treatment regimens for all genotypes of HCV.

Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus.

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.

The kinase domain alters the kinetic properties of the myosin IIIA motor.

Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells.