The Safety and Antiviral Activity of BZF961 with or without Ritonavir in Patients Infected with Hepatitis C Virus: A Randomized, Multicenter Trial.

PURPOSE: Infection with hepatitis C virus is the leading cause of infectious disease mortality in the United States. BZF961 is a novel small molecule inhibitor of the hepatitis C virus NS3-4A protease. Here we present the results of a randomized, double-blinded, placebo-controlled, multicentered study in treatment-naïve patients with chronic hepatitis C virus genotype-1 infection. METHODS: Patients were enrolled sequentially in 2 parts and treated for 3days. BZF961 was administered as monotherapy (500mg BID for 3 days) or in combination with the cytochrome P450 3A4 inhibitor ritonavir to boost its exposure (BZF961 10, 20, or 50mg QD or BID). FINDINGS: BZF961 was safe and well tolerated in the patients studied with no serious adverse events. There were no appreciable differences in adverse events among patients who received BZF961, BZF961 with ritonavir, or placebo. There was a significant, clinically meaningful reduction in viral load from baseline in patients treated either with BZF961 500mg every 12hours alone or BZF961 50mg every 12hours in combination with ritonavir. Activity against the hepatitis C virus of the lower-dose regimens was apparent but more modest. There were no relevant changes from baseline viral loads in placebo-treated patients. IMPLICATIONS: Coadministration of ritonavir with BZF961 boosted BZF961 exposure (including Cmin, which is the clinically relevant parameter associated with antiviral activity) in a therapeutic range with less variability compared with BZF961 alone. For strategic reasons, BZF961 is no longer under development.

Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor.

EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.).

Alisporivir plus ribavirin, interferon free or in combination with pegylated interferon, for hepatitis C virus genotype 2 or 3 infection.

UNLABELLED: Alisporivir is a cyclophilin inhibitor with pan-genotypic anti-hepatitis C virus (HCV) activity and a high barrier to viral resistance. The VITAL-1 study assessed alisporivir as interferon (IFN)-free therapy in treatment-naïve patients infected with HCV genotype 2 or 3. Three hundred forty patients without cirrhosis were randomized to: arm 1, alisporivir (ALV) 1,000 mg once-daily (QD); arm 2, ALV 600 mg QD and ribavirin (RBV); arm 3, ALV 800 mg QD and RBV; arm 4, ALV 600 mg QD and pegylated IFN (Peg-IFN); or arm 5, Peg-IFN and RBV. Patients receiving IFN-free ALV regimens who achieved rapid virological response (RVR) continued the same treatment throughout, whereas those with detectable HCV RNA at week 4 received ALV, RBV, and Peg-IFN from weeks 6 to 24. Overall, 300 patients received ALV-based regimens. In arm 1 to arm 4, the intent-to-treat rates of sustained virological response (SVR) 24 weeks after treatment (SVR24) were from 80% to 85%, compared with 58% (n = 23 of 40) with Peg-IFN/RBV. Per-protocol analysis showed higher SVR24 rates in patients who received ALV/RBV, IFN-free after RVR (92%; n = 56 of 61) than with ALV alone after RVR (72%; n = 13 of 18) or with Peg-IFN/RBV (70%; n = 23 of 33). Both RVRs and SVRs to ALV IFN-free regimens were numerically higher in genotype 3- than in genotype 2-infected patients. Viral breakthrough was infrequent (3%; n = 7 of 258). IFN-free ALV treatment showed markedly better safety/tolerability than IFN-containing regimens. CONCLUSIONS: ALV plus RBV represents an effective IFN-free option for a proportion of patients with HCV genotype 2 or 3 infections, with high SVR rates for patients with early viral clearance. Further investigations of ALV in IFN-free combination regimens with direct-acting antiviral drugs deserve exploration in future trials.

The combination of alisporivir plus an NS5A inhibitor provides additive to synergistic anti-hepatitis C virus activity without detectable cross-resistance.

Alisporivir (ALV), a cyclophilin inhibitor, is a host-targeting antiviral (HTA) with multigenotypic anti-hepatitis C virus (HCV) activity and a high barrier to resistance. Recent advances have supported the concept of interferon (IFN)-free regimens to treat chronic hepatitis C. As the most advanced oral HTA, ALV with direct-acting antivirals (DAAs) represents an attractive drug combination for IFN-free therapy. In this study, we investigated whether particular DAAs exhibit additive, synergistic, or antagonistic effects when combined with ALV. Drug combinations of ALV with NS3 protease, NS5B polymerase, and NS5A inhibitors were investigated in HCV replicons from genotypes 1a, 1b, 2a, 3, and 4a (GT1a to -4a). Combinations of ALV with DAAs exerted an additive effect on GT1 and -4. A significant and specific synergistic effect was observed with ALV-NS5A inhibitor combination on GT2 and -3. Furthermore, ALV was fully active against DAA-resistant variants, and ALV-resistant variants were fully susceptible to DAAs. ALV blocks the contact between cyclophilin A and domain II of NS5A, and NS5A inhibitors target domain I of NS5A; our data suggest a molecular basis for the use of these two classes of inhibitors acting on two distinct domains of NS5A. These results provide in vitro evidence that ALV with NS5A inhibitor combination represents an attractive strategy and a potentially effective IFN-free regimen for treatment of patients with chronic hepatitis C. Due to its high barrier and lack of cross-resistance, ALV could be a cornerstone drug partner for DAAs.

Inhibition of hepatitis C virus replication by GS-6620, a potent C-nucleoside monophosphate prodrug.

As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 µM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 µM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

Characterization of Hepatitis C virus resistance from a multiple-dose clinical trial of the novel NS5A inhibitor GS-5885.

GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log10 HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at ≥3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30- and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.).

Characterization of resistance to the protease inhibitor GS-9451 in hepatitis C virus-infected patients.

GS-9451, a novel hepatitis C virus (HCV) nonstructural 3/4a (NS3/4a) protease inhibitor, is highly active in patients infected with HCV genotype 1 (GT 1). The aim of this study is to characterize the clinical resistance profile of GS-9451 in GT 1 HCV-infected patients in a phase 1, 3-day monotherapy study. The full-length NS3/4A gene was population sequenced at baseline, on the final treatment day, and at follow-up time points. NS3 protease domains from patient isolates with emerging mutations were cloned into an NS3 shuttle vector, and their susceptibilities to GS-9451 and other HCV inhibitors were determined using a transient replication assay. No resistance mutations at NS3 position 155, 156, or 168 were detected in any of the baseline samples or in viruses from patients treated with 60 mg of GS-9451 once daily. Among patients who received 200 mg and 400 mg of GS-9451, viruses with mutations at position D168 (D168E/G/V) and R155 (R155K), which confer high-level resistance to GS-9451, were detected in those with GT 1b and GT 1a virus, respectively. Viruses with D168 mutations were no longer detected in any GT 1b patient at day 14 and subsequent time points. In GT 1a patients, R155K mutants were replaced by the wild type in 57% of patients at week 24. These NS3 clinical mutants were sensitive to NS5B and NS5A inhibitors, as well as alpha interferon (IFN-α) and ribavirin. The lack of cross-resistance between GS-9451 and other classes of HCV inhibitors supports the utility of combination therapy.

Tegobuvir (GS-9190) potency against HCV chimeric replicons derived from consensus NS5B sequences from genotypes 2b, 3a, 4a, 5a, and 6a.

With the exception of nucleoside analogs, few direct acting antivirals in clinical development are active across the six major hepatitis C virus genotypes. We report novel consensus sequence chimeras for genotypes 2b, 3a, 4a, 5a, and 6a NS5B and show variable susceptibilities over a panel of NS5B inhibitors. Tegobuvir (GS-9190) had EC(50)s of <16 nM against genotype 1 and >100 nM for other genotypes tested here. An NS5B F445C mutation engineered into the GT3a, 4a, and 6a chimeric replicons lowered the tegobuvir EC(50) to levels comparable to those for genotype 1a, but did not considerably alter the EC(50) of site 2 or nucleoside analog inhibitors. These data support the use of HCV chimeras in profiling direct acting antivirals across genotypes and specifically determines the impact of the C445F NS5B polymorphism on tegobuvir potency against genotypes 3a, 4a, and 6a.

A phase 1, randomized, placebo-controlled, 3-day, dose-ranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C.

BACKGROUND & AIMS: GS-5885 is an inhibitor of the hepatitis C virus (HCV) NS5A protein and exhibits potent suppression of genotype 1 HCV replicons. The safety, tolerability, pharmacokinetics, antiviral activity, and resistance profile of once-daily GS-5885 doses of 1-90 mg were evaluated in patients with chronic genotype 1 HCV. METHODS: Genotype 1 HCV-infected patients were randomized to 3 days of once-daily (QD) dosing with placebo (n=12) or GS-5885 1 mg (n=10), 3 mg (n=10), 10 mg (n=20), 30 mg (n=10), or 90 mg (n=10). Plasma samples for pharmacokinetics, HCV RNA, and NS5A sequencing were collected through day 14. RESULTS: GS-5885 was well tolerated and resulted in median maximal reductions in HCV RNA ranging from 2.3 log(10) IU/ml (1 mg QD) to 3.3 log(10) IU/ml (10 mg QD in genotype 1b and 30 mg QD). E(max) modeling indicated GS-5885 30 mg was associated with>95% of maximal antiviral response to HCV genotype 1a. HCV RNA reductions were generally more sustained among patients with genotype 1b vs. 1a. Three of 60 patients had a reduced response and harbored NS5A-resistant virus at baseline. NS5A sequencing identified residues 30 and 31 in genotype 1a, and 93 in genotype 1b as the predominant sites of mutation following GS-5885 dosing. Plasma pharmacokinetics was consistent with QD dosing. CONCLUSIONS: During 3 days of monotherapy, low doses of GS-5885 demonstrated significant antiviral activity in genotype 1a and 1b HCV-infected patients. GS-5885 is currently being evaluated in combination with direct antiviral regimens with and without peginterferon.

Susceptibility of treatment-naive hepatitis C virus (HCV) clinical isolates to HCV protease inhibitors.

In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two α-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the α-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the α-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo, particularly at lower doses.

Control of specificity and magnitude of NF-kappa B and STAT1-mediated gene activation through PIASy and PIAS1 cooperation.

NF-kappaB and STATs regulate multiple cellular processes through the transcriptional activation of genes with diversified functions. Although the molecular mechanisms that can turn on/off the overall NF-kappaB/STAT signaling have been extensively studied, how NF-kappaB/STAT-target genes can be differentially regulated is poorly understood. Here we report that PIASy, a member of the PIAS (for protein inhibitor of activated STAT) protein family, is a physiologically important transcriptional repressor of NF-kappaB and STAT1. Piasy deletion in dendritic cells resulted in enhanced expression of a subset of NF-kappaB and STAT1-dependent genes in response to LPS or IFN-gamma treatment, respectively. Consistently, Piasy null mice are hypersensitive to the LPS-induced endotoxic shock. Furthermore, PIASy and PIAS1 display specific as well as redundant effects on the regulation of NF-kappaB/STAT1 signaling. Pias1-/-Piasy-/- embryos died before day 11.5. The disruption of one allele of Pias1 in the Piasy-/- background significantly enhanced the effect of Piasy deletion on the transcriptional induction of NF-kappaB/STAT1-dependent genes, and vice versa. Our results demonstrate that PIASy cooperates with PIAS1 to regulate the specificity and magnitude of NF-kappaB/STAT1-mediated gene activation.

A revised model for AMP-activated protein kinase structure: The alpha-subunit binds to both the beta- and gamma-subunits although there is no direct binding between the beta- and gamma-subunits.

The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.

Negative regulation of NF-kappaB signaling by PIAS1.

The NF-kappaB family of transcription factors is activated by a wide variety of signals to regulate a spectrum of cellular processes. The proper regulation of NF-kappaB activity is critical, since abnormal NF-kappaB signaling is associated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here that PIAS1 (protein inhibitor of activated STAT1) is an important negative regulator of NF-kappaB. Upon cytokine stimulation, the p65 subunit of NF-kappaB translocates into the nucleus, where it interacts with PIAS1. The binding of PIAS1 to p65 inhibits cytokine-induced NF-kappaB-dependent gene activation. PIAS1 blocks the DNA binding activity of p65 both in vitro and in vivo. Consistently, chromatin immunoprecipitation assays indicate that the binding of p65 to the promoters of NF-kappaB-regulated genes is significantly enhanced in Pias1-/- cells. Microarray analysis indicates that the removal of PIAS1 results in an increased expression of a subset of NF-kappaB-mediated genes in response to tumor necrosis factor alpha and lipopolysaccharide. Consistently, Pias1 null mice showed elevated proinflammatory cytokines. Our results identify PIAS1 as a novel negative regulator of NF-kappaB.

PIAS1 selectively inhibits interferon-inducible genes and is important in innate immunity.

Interferon (IFN) activates the signal transducer and activator of transcription (STAT) pathway to regulate immune responses. The protein inhibitor of activated STAT (PIAS) family has been suggested to negatively regulate STAT signaling. To understand the physiological function of PIAS1, we generated Pias1(-/-) mice. Using PIAS1-deficient cells, we show that PIAS1 selectively regulates a subset of IFN-gamma- or IFN-beta-inducible genes by interfering with the recruitment of STAT1 to the gene promoter. The antiviral activity of IFN-gamma or IFN-beta was consistently enhanced by Pias1 disruption. Pias1(-/-) mice showed increased protection against pathogenic infection. Our data indicate that PIAS1 is a physiologically important negative regulator of STAT1 and suggest that PIAS1 is critical for the IFN-gamma- or IFN-beta-mediated innate immune responses.

Protein inhibitor of activated STAT Y (PIASy) and a splice variant lacking exon 6 enhance sumoylation but are not essential for embryogenesis and adult life.

Protein inhibitor of activated STAT Y (PIASy) is the shortest member of the PIAS family and has been reported to modulate the transcriptional activities of STAT1, lymphoid enhancer factor 1 (LEF-1), and the androgen receptor. PIAS proteins have also been identified as E3 ligases for the small ubiquitin-like modifier (SUMO) proteins. PIASy in particular has been reported to mediate SUMO-2/3 modification of LEF-1, sequestering it into nuclear bodies, and SUMO-1 ligation to c-Myb, modulating its transcriptional activation properties. We have cloned murine Piasy and a splice variant which omits exon 6, containing the nuclear retention PINIT motif. Cell culture studies indicate that both the full length and the splice variant are localized in the nucleus but differentially enhance SUMO ligation. To further understand the functions of PIASy, we have generated PIASy-deficient mice. Surprisingly, Piasy(-/-) mice appear phenotypically normal. Activation of STAT1 is not significantly perturbed in Piasy(-/-) cells, and sumoylation patterns for SUMO-1 or SUMO-3 modification are similar when comparing tissues and embryonic fibroblasts from wild-type and knockout mice. Our study demonstrates that at steady state, PIASy is either dispensable or compensated for by other PIAS family members or by other mechanisms when deleted.