Small Molecular Compounds that Inhibit Hepatitis C Virus Replication Through Destabilizing Heat Shock Cognate 70 Messenger RNA†

Abstract

Host heat shock cognate 70 (Hsc70) protein is packaged into hepatitis C viral (HCV) particles as a structural component of the virus in the assembly process. It helps HCV RNA release into the cytoplasm in the next infection cycle. The goal of this study is to investigate whether chemically down-regulating host Hsc70 expression could be a novel strategy to interrupt HCV replication. Compounds were screened with an Hsc70 messenger RNA (mRNA) assay. IMB-DM122 was found to be an effective and safe inhibitor for Hsc70 mRNA/protein expression in human hepatocytes. IMB-DM122 inhibited HCV replication through destabilization of Hsc70 mRNA, and the half-life of host Hsc70 mRNA was reduced by 78% after the compound treatment. The Hsc70 mRNA 3 0 untranslated region sequence is the element responsible for the effect of IMB-DM122 on Hsc70 mRNA. The compound appears to be highly efficient in inhibiting Hsc70-related HCV replication. Treatment of the HCV-infected hepatocytes with IMB-DM122 reduced the virion encapsidation of Hsc70, and therefore disrupted HCV replication and the infection cycle. IMB-DM122 showed considerable good safety in vitro as well as in vivo with no indication of harmful effect on liver and kidney functions. Conclusion: Hsc70 might be a new drug target and mechanism to inhibit HCV proliferation. (HEPATOLOGY 2010;52:845-853) H epatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flaviviridae family. 1 Current standard therapy for hepatitis C in the clinic is the combination of pegylated-interferon with ribavirin. 2,3 The regimen is effective in 40%-50% of patients infected with HCV genotype 1 and is associated with significant side effects. 3,4 Recently, telaprevir and boceprevir, two peptidomimetic inhibitors of the HCV nonstructural protein 3/4A (NS3/4A) protease, have shown great promise in clinical patients. [5][6][7][8] However, antiviral therapy targeting specific HCV proteases has caused emergence of drug-resistant mutations, 9-12 and thus, new mechanisms for anti-HCV drugs are highly desirable.Human heat shock cognate 70 (Hsc70, or heat shock protein A8) is a cytoplasm adenosine triphosphate-binding protein with 646 amino acids. 13 It is a member of the heat shock protein 70 (Hsp70) family with the gene located in chromosome 11. 14 The functions of Hsc70 protein in normal cells are complicated and remain to be clarified. 15 Virology research has shown that Hsc70 might play a role in regulating virion capsid assembly. 16 A recent study by Parent et al. demonstrated that host Hsc70 is part of the HCV particle. 17 They showed Hsc70 interaction HPD (HisPro-Asp) motif present on the E2 envelope of HCV (the J6/JFH [Japanese fulminant hepatitis] strain) and coexistence of Hsc70 with the HCV core and E2 proteins around lipid droplets in the infected cells.

N-Substituted Benzyl Matrinic Acid Derivatives Inhibit Hepatitis C Virus (HCV) Replication through Down-Regulating Host Heat-Stress Cognate 70 (Hsc70) Expression

“…It appears that 7m was effective before, at and after HCV infection, supporting its host environment-related action mode. The anti-HCV effect of compound 7m was similar to that of the positive control Intron A (interferon a-2b), consistent with our previous report [13]. Compound 7c exhibited an anti-HCV pattern close to that of 7m .…”

“…As the inhibition rate of intracellular HCV replication was basically consistent with activity in Hsc70 down-regulation [13], [16], [17], SAR analysis for the inhibiting HCV replication was conducted with 1 as the lead in the present study. We retained the butyric acid chain, and focused the SAR study on the influence of the substituents on the phenyl ring including electron-withdrawing and electron-donating, and the effect of ( S )- or ( R )-configuration of the chiral carbon at the 5-position, respectively.…”

“…All of the synthesized compounds were examined for their anti-HCV activity and cytotoxicity in Huh 7.5 cells using specific real-time RT-PCR assay, as described in our previous publication [13]. Anti-HCV activity was evaluated by measuring both EC 50 (for anti-HCV activity) and CC 50 (for cytotoxicity) values.…”

“…Using Hsc70 as a target for HCV inhibition, we found that 12- N - p -methoxybenzyl matrinic acid ( 1 , Figure 1) synthesized in our laboratory showed an anti-HCV activity [13]. It significantly down-regulates host Hsc70 expression at the post transcriptional level through destabilizing Hsc70 mRNA [13]. Its mode of action is distinctly different from that of the current anti-HCV drugs such as telaprevir and boceprevir [14], [15].…”

“…We have demonstrated that host Hsc70 was a new drug target and mechanism against HCV [12]. Using Hsc70 as a target for HCV inhibition, we found that 12- N - p -methoxybenzyl matrinic acid ( 1 , Figure 1) synthesized in our laboratory showed an anti-HCV activity [13]. It significantly down-regulates host Hsc70 expression at the post transcriptional level through destabilizing Hsc70 mRNA [13].…”