Robert Hamatake

Gene Expression Profiling Indicates the Roles of Host Oxidative Stress, Apoptosis, Lipid Metabolism, and Intracellular Transport Genes in the Replication of Hepatitis C Virus

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic liver disease. The identification and characterization of key host cellular factors that play a role in the HCV replication cycle are important for the understanding of disease pathogenesis and the identification of novel antiviral therapeutic targets. Gene expression profiling of JFH-1-infected Huh7 cells by microarray analysis was performed to identify host cellular genes that are transcriptionally regulated by infection. The expression of host genes involved in cellular defense mechanisms (apoptosis, proliferation, and antioxidant responses), cellular metabolism (lipid and protein metabolism), and intracellular transport (vesicle trafficking and cytoskeleton regulation) was significantly altered by HCV infection. The gene expression patterns identified provide insight into the potential mechanisms that contribute to HCV-associated pathogenesis. These include an increase in proinflammatory and proapoptotic signaling and a decrease in the antioxidant response pathways of the infected cell. To investigate whether any of the host genes regulated by infection were required by HCV during replication, small interfering RNA (siRNA) silencing of host gene expression in HCV-infected cells was performed. Decreasing the expression of host genes involved in lipid metabolism (TXNIP and CYP1A1 genes) and intracellular transport (RAB33b and ABLIM3 genes) reduced the replication and secretion of HCV, indicating that they may be important factors for the virus replication cycle. These results show that major changes in the expression of many different genes in target cells may be crucial in determining the outcome of HCV infection.

Hepatitis C Replication Inhibitors That Target the Viral NS4B Protein

We describe the preclinical development and in vivo efficacy of a novel chemical series that inhibits hepatitis C virus replication via direct interaction with the viral nonstructural protein 4B (NS4B). Significant potency improvements were realized through isosteric modifications to our initial lead 1a. The temptation to improve antiviral activity while compromising physicochemical properties was tempered by the judicial use of ligand efficiency indices during lead optimization. In this manner, compound 1a was transformed into (+)-28a which possessed an improved antiviral profile with no increase in molecular weight and only a modest elevation in lipophilicity. Additionally, we employed a chimeric humanized mouse model of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity for NS4B can inhibit viral replication in vivo. This successful proof-of-concept study suggests that drugs targeting NS4B may represent a viable treatment option for curing HCV infection.

Preclinical Characterization of GSK2336805, a Novel Inhibitor of Hepatitis C Virus Replication That Selects for Resistance in NS5A

ABSTRACT GSK2336805 is an inhibitor of hepatitis C virus (HCV) with picomolar activity on the standard genotype 1a, 1b, and 2a subgenomic replicons and exhibits a modest serum shift. GSK2336805 was not active on 22 RNA and DNA viruses that were profiled. We have identified changes in the N-terminal region of NS5A that cause a decrease in the activity of GSK2336805. These mutations in the genotype 1b replicon showed modest shifts in compound activity (<13-fold), while mutations identified in the genotype 1a replicon had a more dramatic impact on potency. GSK2336805 retained activity on chimeric replicons containing NS5A patient sequences from genotype 1 and patient and consensus sequences for genotypes 4 and 5 and part of genotype 6. Combination and cross-resistance studies demonstrated that GSK2336805 could be used as a component of a multidrug HCV regimen either with the current standard of care or in combination with compounds with different mechanisms of action that are still progressing through clinical development.

In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides

BACKGROUND & AIMSnCurrent treatment of chronic hepatitis B virus infection (CHB) includes interferon and nucleos(t)ide analogues, which generally do not reduce HBV surface antigen (HBsAg) production, a constellation that is associated with poor prognosis of CHB. Here we evaluated the efficacy of an antisense approach using antisense oligonucleotide (ASO) technology already in clinical use for liver targeted therapy to specifically inhibit HBsAg production and viremia in a preclinical setting.nnnMETHODSnA lead ASO was identified and characterized in vitro and subsequently tested for efficacy in vivo and in vitro using HBV transgenic and hydrodynamic transfection mouse and a cell culture HBV infection model, respectively.nnnRESULTSnASO treatment decreased serum HBsAg levels ⩾2 logs in a dose and time-dependent manner; HBsAg decreased 2 logs in a week and returned to baseline 4 weeks after a single ASO injection. ASO treatment effectively reduced HBsAg in combination with entecavir, while the nucleoside analogue alone did not. ASO treatment has pan-genotypic antiviral activity in the hydrodynamic transfection system. Finally, cccDNA-driven HBV gene expression is ASO sensitive in HBV infected cells in vitro.nnnCONCLUSIONnOur results demonstrate in a preclinical setting the efficacy of an antisense approach against HBV by efficiently reducing serum HBsAg (as well as viremia) across different genotypes alone or in combination with standard nucleoside therapy. Since the applied antisense technology is already in clinical use, a lead compound can be rapidly validated in a clinical setting and thus, constitutes a novel therapeutic approach targeting chronic HBV infection.

In Vitro Characterization of GSK2485852, a Novel Hepatitis C Virus Polymerase Inhibitor

ABSTRACT GSK2485852 (referred to here as GSK5852) is a hepatitis C virus (HCV) NS5B polymerase inhibitor with 50% effective concentrations (EC50s) in the low nanomolar range in the genotype 1 and 2 subgenomic replicon system as well as the infectious HCV cell culture system. We have characterized the antiviral activity of GSK5852 using chimeric replicon systems with NS5B genes from additional genotypes as well as NS5B sequences from clinical isolates of patients infected with HCV of genotypes 1a and 1b. The inhibitory activity of GSK5852 remained unchanged in these intergenotypic and intragenotypic replicon systems. GSK5852 furthermore displays an excellent resistance profile and shows a <5-fold potency loss across the clinically important NS5B resistance mutations P495L, M423T, C316Y, and Y448H. Testing of a diverse mutant panel also revealed a lack of cross-resistance against known resistance mutations in other viral proteins. Data from both the newer 454 sequencing method and traditional population sequencing showed a pattern of mutations arising in the NS5B RNA-dependent RNA polymerase in replicon cells exposed to GSK5852. GSK5852 was more potent than HCV-796, an earlier inhibitor in this class, and showed greater reductions in HCV RNA during long-term treatment of replicons. GSK5852 is similar to HCV-796 in its activity against multiple genotypes, but its superior resistance profile suggests that it could be an attractive component of an all-oral regimen for treating HCV.

Hepatitis C genotype 1a replicon improved through introduction of fitness mutations

The use of subgenomic replicon systems has long been a valuable screening tool for the discovery of small molecule antivirals against Hepatitis C virus. While genotype 1a replicon systems have been widely used in stable systems, use in transient assays has been hampered by low signal. Here we describe the generation of a more robust genotype 1a (H77) replicon through the introduction of two fitness mutations, NS4A-K1691R and NS4B-E1726G, for use in transient transfections. While these mutations significantly improved the signal to noise ratio, leading to more robust data, they have no effect on the potency of tool compounds against various targets of HCV, thereby making this new system a powerful tool for screening of compounds against the genotype 1a replicon.

HCV Inhibition Mediated Through the Nonstructural Protein 5A (NS5A) Replication Complex

Abstract Chronic infection by hepatitis C virus (HCV) results in increased risk for cirrhosis and hepatocellular carcinoma. Current treatments of HCV infection use inhibitors of HCV NS3 protease in combination with pegylated interferon α and ribavirin resulting in approximately 70% cure rates. All oral regimens of direct acting antivirals are being developed in the clinic and have the potential for improved tolerability, wider access to patients contra indicated for interferon therapies, and higher cure rates. HCV NS5A inhibitors have recently emerged as an attractive component of an all-oral HCV therapy, though the function(s) of NS5A remains an enigma. We describe here the structures of compounds disclosed in the patent literature, trends in inhibitor design, and recent data on the clinical progression of NS5A inhibitors.

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein

ABSTRACT The hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors.

A double-blind, randomized, placebo-controlled study to assess the safety, antiviral activity and pharmacokinetics of GSK2336805 when given as monotherapy and in combination with peginterferon alfa-2a and ribavirin in hepatitis C virus genotype 1-infected treatment-naive subjects.

GSK2336805 is a HCV NS5A inhibitor for chronic hepatitis C (CHC). In a prior Phase I study, GSK2336805 was well tolerated and had an antiviral and pharmacokinetic profile suitable for once‐daily administration. This 28‐day, double‐blind, randomized, placebo‐controlled study evaluated once daily GSK2336805 60 mg alone or in combination with peginterferon alfa‐2a (180 μg per week) and ribavirin (1000–1200 mg daily) (PEG/RIBA) in treatment‐naive genotype 1 CHC subjects.

Synthesis and antiviral activity of novel HCV NS3 protease inhibitors with P4 capping groups.

We have synthesized and evaluated a series of novel HCV NS3 protease inhibitors with various P4 capping groups, which include urea, carbamate, methoxy-carboxamide, cyclic carbamate and amide, pyruvic amide, oxamate, oxalamide and cyanoguanidine. Most of these compounds are remarkably potent, exhibiting single-digit to sub-nanomolar activity in the enzyme assay and cell-based replicon assay. Selected compounds were also evaluated in the protease-inhibitor-resistant mutant transient replicon assay, and they were found to show quite different potency profiles against a panel of HCV protease-inhibitor-resistant mutants.