Origène Nyanguile

Binding-Site Identification and Genotypic Profiling of Hepatitis C Virus Polymerase Inhibitors

ABSTRACT The search for hepatitis C virus polymerase inhibitors has resulted in the identification of several nonnucleoside binding pockets. The shape and nature of these binding sites differ across and even within diverse hepatitis C virus genotypes. These differences confront antiviral drug discovery with the challenge of finding compounds that are capable of inhibition in variable binding pockets. To address this, we have established a hepatitis C virus mutant and genotypic recombinant polymerase panel as a means of guiding medicinal chemistry through the elucidation of the site of action of novel inhibitors and profiling against genotypes. Using a genotype 1b backbone, we demonstrate that the recombinant P495L, M423T, M414T, and S282T mutant enzymes can be used to identify the binding site of an acyl pyrrolidine analog. We assess the inhibitory activity of this analog and other nonnucleoside inhibitors with our panel of enzyme isolates generated from clinical sera representing genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a.

Antiviral Suppression vs Restoration of RIG-I Signaling by Hepatitis C Protease and Polymerase Inhibitors

BACKGROUND & AIMS Expression of the nonstructural protein (NS)3/4A protease in cells infected with hepatitis C virus (HCV) results in cleavage of the mitochondrial antiviral-signaling protein (MAVS) and disruption of signaling pathways that lead to viral activation of interferon regulatory factor 3 (IRF-3) and synthesis of type 1 interferons (IFN-alpha/beta). High concentrations of inhibitors of NS3/4A reverse this signaling defect, but quantitative analyses of this potential therapeutic effect are lacking. This study quantitatively assessed the rescue of IRF-3 signaling by NS3/4A inhibitors, compared with in vitro antiviral activity. METHODS Antiviral activities of 2 NS3/4A protease inhibitors (TMC435350 and an analog, TMC380765) and a nonnucleoside polymerase inhibitor (Tib-3) were determined in HCV replicon cells and in cells infected with genotype 1a and 2a viruses. The capacity to rescue IRF-3 activation in these cells was assessed by monitoring IFN-beta promoter activity following challenge with Sendai virus. Inhibitor-induced changes in NS3 and MAVS expression were assessed in immunoblots. RESULTS Both protease inhibitors were capable of rescuing IFN-beta promoter activation but only at concentrations approximately 100-fold the antiviral 50% effective concentration (EC(50)) for genotype 1 virus. No rescue was observed with the polymerase inhibitor, even at a concentration 600-fold greater than the EC(50). IRF-3 activation did not correlate with reductions in NS3/4A levels or detection of full-length MAVS. Overexpression of the product of NS3/4A cleavage of MAVS did not result in a dominant-negative effect on signaling. CONCLUSIONS NS3/4A protease inhibitors can restore IRF-3 signaling in HCV-infected cells but only at concentrations far in excess of the antiviral EC(50).

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

ABSTRACT The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

TMC647055, a Potent Nonnucleoside Hepatitis C Virus NS5B Polymerase Inhibitor with Cross-Genotypic Coverage

ABSTRACT Hepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report the in vitro inhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase. In vitro combination studies with an HCV NS3/4A protease inhibitor demonstrated potent suppression of HCV RNA replication, confirming the potential for combination of these two classes in the treatment of chronic HCV infection. TMC647055 is a potent nonnucleoside NS5B polymerase inhibitor of HCV replication with a promising in vitro biochemical, kinetic, and virological profile that is currently undergoing clinical evaluation.

1,5-Benzodiazepine inhibitors of HCV NS5B polymerase

Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zc and (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zk as potent (replicon EC(50)=400nM and 270nM, respectively) and selective (CC(50)>20muM) inhibitors of HCV replication. These data warrant further lead-optimization efforts.

1,5-Benzodiazepines, a Novel Class of Hepatitis C Virus Polymerase Nonnucleoside Inhibitors

ABSTRACT The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as BZD receptors.

Discovery and Early Development of TMC647055, a Non-Nucleoside Inhibitor of the Hepatitis C Virus NS5B Polymerase

Structure-based macrocyclization of a 6-carboxylic acid indole chemotype has yielded potent and selective finger-loop inhibitors of the hepatitis C virus (HCV) NS5B polymerase. Lead optimization in conjunction with in vivo evaluation in rats identified several compounds showing (i) nanomolar potency in HCV replicon cells, (ii) limited toxicity and off-target activities, and (iii) encouraging preclinical pharmacokinetic profiles characterized by high liver distribution. This effort culminated in the identification of TMC647055 (10a), a nonzwitterionic 17-membered-ring macrocycle characterized by high affinity, long polymerase residence time, and broad genotypic coverage. In vitro results of the combination of 10a with the HCV protease inhibitor TMC435 (simeprevir) supported an evaluation of this combination in patients with regard to virus suppression and resistance emergence. In a phase 1b trial with HCV genotype 1-infected patients, 10a was considered to be safe and well-tolerated and demonstrated potent antiviral activity, which was further enhanced in a combination study with TMC435.

759 COMBINATION OF TMC435 WITH TWO NOVEL NS5B INHIBITORS INCREASES ANTI HCV ACTIVITY AND RESULTS IN A HIGHER GENETIC BARRIER IN VITRO

Background: MP-424 (telaprevir) is a highly selective inhibitor of the hepatitis C virus (HCV) NS3–4A protease which is currently investigated in phase 3 trial in combination with peginterferon and ribavirin. We reported the first clinical study examining telaprevir monotherapy with an extended dosing period of 24 weeks in Japan (Ozeki I, et al: EASL 2009). After completing study drug therapy, patients were treated with peginterferon alfa-2b and ribavirin. Methods: Five naive patients were treated with telaprevir monotherapy at 750mg every 8 hours for up to 24 weeks. All patients, 48 to 68 years of age, had a chronic infection with HCV genotype 1b and their baseline serum HCVRNA levels were at least 1×105 IU/ml. Patients who met the definition of viral breakthrough (>2-log increase in HCVRNA above nadir) discontinued telaprevir dosing. RNA sequence was analyzed using the clonal sequencing method. After a withdrawal of MP-424, 4 of the 5 patients were enrolled in the off-study treatment with peginterferon alfa-2b and ribavirin within 4 weeks after the last administration of study drug. Results: At the initiation of off-study treatment, major NS3 protease variants in the patients were T54A, T54S+A156T, and A156V+V158I. All patients achieved undetectable HCVRNA levels by 12 weeks regardless of selected telaprevir-resistant variants. Sustained virologic response (SVR) was achieved in 2 patients who completed the assigned treatment for 48 weeks. The other 2 patients are now receiving peginterferon alfa-2b and ribavirin beyond 48 weeks for extended treatment period to 72 weeks. HCVRNA levels in these 2 patients continue to be undetectable. Conclusions: By the off-study treatment of standard peginterferon alfa-2b and ribavirin, all patients achieved complete EVR regardless of selected drug-resistant variants. Two patients who were typically treated for 48 weeks achieved an SVR. Updated results of this study will be presented.