Jian-Rui Li

Small molecular compounds that inhibit hepatitis C virus replication through destabilizing heat shock cognate 70 messenger RNA

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′ 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;)

Effects of simvastatin within two weeks on anti-inflammatory cytokine interleukin 10 in patients with unstable angina

Evidence has shown that the inflammatory process is involved in all stages of atherosclerosis in various clinical settings and that an imbalance between anti-inflammatory mechanisms and proinflammatory factors, in favour of the proinflammatory factors, results in rupture of atherosclerotic plaque.1–3 Previous data also showed that proinflammatory cytokines are important in acute coronary events and that decreased plasma concentrations of anti-inflammatory cytokine are associated with acute coronary syndromes.1 In addition to their cholesterol lowering activity, statins have been shown to have pleiotropic effects, including anti-inflammatory effects.2,3 Recent studies have shown that the anti-inflammatory cytokine interleukin 10 has a protective role in both atherosclerotic formation and stability.4–6 However, the potential effects of statins on anti-inflammatory cytokines in patients with acute coronary disease has not been investigated. In the present study, we investigated whether a statin would affect interleukin 10 concentration within two weeks in patients with unstable angina (UA). Forty two patients with typical UA were enrolled and randomly assigned to either standard treatment plus 20 mg/day or 80 mg/day of simvastatin immediately after admission. The standard treatment comprised aspirin, β blocker, heparin or low molecular weight heparin, angiotensin converting enzyme inhibitors, and oral nitrates. Patients with evidence of myocardial infarction consisting of ST elevation, formation of Q waves, and increased entry concentration of troponin T or I, congestive heart failure, poorly controlled hypertension, statin treatment before hospitalisation, valvar heart disease, a history of dysphagia, swallowing and intestinal motility disorders, and untreated thyroid disease were excluded from the study. Selective coronary angiography was performed with the standard Judkins technique. The severity of coronary stenosis was evaluated with the incremental score method as in …

Host apolipoprotein b messenger RNA‐editing enzyme catalytic polypeptide‐like 3G is an innate defensive factor and drug target against hepatitis C virus

Host cellular factor apolipoprotein B messenger RNA (mRNA)‐editing enzyme catalytic polypeptide‐like 3G (hA3G) is a cytidine deaminase that inhibits a group of viruses including human immunodeficiency virus‐1 (HIV‐1). In the continuation of our research on hA3G, we found that hA3G stabilizing compounds significantly inhibited hepatitis C virus (HCV) replication. Therefore, this study investigated the role of hA3G in HCV replication. Introduction of external hA3G into HCV‐infected Huh7.5 human hepatocytes inhibited HCV replication; knockdown of endogenous hA3G enhanced HCV replication. Exogenous HIV‐1 virion infectivity factor (Vif) decreased intracellular hA3G and therefore enhanced HCV proliferation, suggesting that the presence of Vif might be an explanation for the HIV‐1/HCV coinfection often observed in HIV‐1(+) individuals. Treatment of the HCV‐infected Huh7.5 cells with RN‐5 or IMB‐26, two known hA3G stabilizing compounds, increased intracellular hA3G and accordingly inhibited HCV replication. The compounds inhibit HCV through increasing the level of hA3G incorporated into HCV particles, but not through inhibiting HCV enzymes. However, G/A hypermutation in the HCV genome were not detected, suggesting a new antiviral mechanism of hA3G in HCV, different from that in HIV‐1. Stabilization of hA3G by RN‐5 was safe in vivo. Conclusion: hA3G appears to be a cellular restrict factor against HCV and could be a potential target for drug discovery. (HEPATOLOGY 2011;)

CD36 is a co-receptor for hepatitis C virus E1 protein attachment.

The cluster of differentiation 36 (CD36) is a membrane protein related to lipid metabolism. We show that HCV infection in vitro increased CD36 expression in either surface or soluble form. HCV attachment was facilitated through a direct interaction between CD36 and HCV E1 protein, causing enhanced entry and replication. The HCV co-receptor effect of CD36 was independent of that of SR-BI. CD36 monoclonal antibodies neutralized the effect of CD36 and reduced HCV replication. CD36 inhibitor sulfo-N-succinimidyl oleate (SSO), which directly bound CD36 but not SR-BI, significantly interrupted HCV entry, and therefore inhibited HCV replication. SSO’s antiviral effect was seen only in HCV but not in other viruses. SSO in combination with known anti-HCV drugs showed additional inhibition against HCV. SSO was considerably safe in mice. Conclusively, CD36 interacts with HCV E1 and might be a co-receptor specific for HCV entry; thus, CD36 could be a potential drug target against HCV.

N-substituted benzyl matrinic acid derivatives inhibit hepatitis C virus (HCV) replication through down-regulating host heat-stress cognate 70 (Hsc70) expression.

Heat-stress cognate 70 (Hsc70) is a host factor that helps hepatitis C virus (HCV) to complete its life cycle in infected hepatocytes. Using Hsc70 as a target for HCV inhibition, a series of novel N-substituted benzyl matrinic/sophoridinic acid derivatives was synthesized and evaluated for their anti-HCV activity in vitro. Among these analogues, compound 7c possessing N-p-methylbenzyl afforded an appealing ability to inhibit HCV replication with SI value over 53. Furthermore, it showed a good oral pharmacokinetic profile with area-under-curve (AUC) of 13.4 µM·h, and a considerably good safety in oral administration in mice (LD50>1000 mg/kg). As 7c suppresses HCV replication via an action mode distinctly different from that of the marketed anti-HCV drugs, it has been selected as a new mechanism anti-HCV candidate for further investigation, with an advantage of no or decreased chance to induce drug-resistant mutations.

Synthesis and biological evaluation of N-substituted sophocarpinic acid derivatives as coxsackievirus B3 inhibitors.

A series of novel N‐substituted sophocarpinic acid derivatives was designed, synthesized, and evaluated for their anti‐enteroviral activities against coxsackievirus type B3 (CVB3) and coxsackievirus type B6 (CVB6) in Vero cells. Structure–activity relationship analysis revealed that the introduction of a benzenesulfonyl moiety on the 12‐nitrogen atom in (E)‐β,γ‐sophocarpinic acid might significantly enhance anti‐CVB3 activity. Among the derivatives, (E)‐12‐N‐(m‐cyanobenzenesulfonyl)‐β,γ‐sophocarpinic acid (11 m), possessing a meta‐cyanobenzenesulfonyl group, exhibited potent activity against CVB3 with a selectivity index (SI) of 107. Furthermore, compound 11 m also showed a good oral pharmacokinetic profile, with an AUC value of 7.29 μM h−1 in rats, and good safety through the oral route in mice, with an LD50 value of >1000 mg kg−1; these values suggest a druggable characteristic. Therefore, compound 11 m was selected for further investigation as a promising CVB3 inhibitor. We consider (E)‐β,γ‐N‐(benzenesulfonyl)sophocarpinic acids to be a novel class of anti‐CVB3 agents.

Host APOBEC3G Protein Inhibits HCV Replication through Direct Binding at NS3

Human APOBEC3G (hA3G) is a cytidine deaminase that restricts replication of certain viruses. We have previously reported that hA3G was a host restriction factor against hepatitis C virus (HCV) replication, and hA3G stabilizers showed a significant inhibitory activity against HCV. However, the molecular mechanism of hA3G against HCV remains unknown. We show in this study that hA3G’s C-terminal directly binds HCV non-structural protein NS3 at its C-terminus, which is responsible for NS3’s helicase and NTPase activity. Binding of hA3G to the C-terminus of NS3 reduced helicase activity, and therefore inhibited HCV replication. The anti-HCV mechanism of hA3G appeared to be independent of its deamination activity. Although early stage HCV infection resulted in an increase in host hA3G as an intracellular response against HCV replication, hA3G was gradually diminished after a long-term incubation, suggesting an unknown mechanism(s) that protects HCV NS3 from inactivation by hA3G. The process represents, at least partially, a cellular defensive mechanism against HCV and the action is mediated through a direct interaction between host hA3G and HCV NS3. We believe that understanding of the antiviral mechanism of hA3G against HCV might open an interesting avenue to explore hA3G stabilizers as a new class of anti-HCV agents.

Dual Roles of Two Isoforms of Autophagy-related Gene ATG10 in HCV-Subgenomic replicon Mediated Autophagy Flux and Innate Immunity

Autophagy and immune response are two defense systems that human-body uses against viral infection. Previous studies documented that some viral mechanisms circumvented host immunity mechanisms and hijacked autophagy for its replication and survival. Here, we focus on interactions between autophagy mechanism and innate-immune-response in HCV-subgenomic replicon cells to find a mechanism linking the two pathways. We report distinct effects of two autophagy-related protein ATG10s on HCV-subgenomic replication. ATG10, a canonical long isoform in autophagy process, can facilitate HCV-subgenomic replicon amplification by promoting autophagosome formation and by combining with and detaining autophagosomes in cellular periphery, causing impaired autophagy flux. ATG10S, a non-canonical short isoform of ATG10 proteins, can activate expression of IL28A/B and immunity genes related to viral ds-RNA including ddx-58, tlr-3, tlr-7, irf-3 and irf-7, and promote autophagolysosome formation by directly combining and driving autophagosomes to perinuclear region where lysosomes gather, leading to lysosomal degradation of HCV-subgenomic replicon in HepG2 cells. ATG10S also can suppress infectious HCV virion replication in Huh7.5 cells. Another finding is that IL28A protein directly conjugates ATG10S and helps autophagosome docking to lysosomes. ATG10S might be a new host factor against HCV replication, and as a target for screening chemicals with new anti-virus mechanisms.

Differential Effects of Autophagy-Related 10 Protein on HCV Replication and Autophagy Flux Are Mediated by Its Cysteine44 and Cysteine135

Autophagy-related 10 (ATG10) is essential for autophagy since it promotes ATG5-ATG12 complex formation. Our previous study found that there are two isoforms of the ATG10 protein, ATG10 (a longer one) and ATG10S, which have identical sequences except an absence of a 36-amino acid fragment (peptide B) in ATG10S, yet exhibit distinct effects on HCV genome replication. Here, we report the existence of two amino acids, cysteine at residue 44 and 135 (Cys44 and Cys135, respectively), in ATG10 being related to differential effects of ATG10 on HCV replication and autophagy flux. Through a series of ATG10 mutation experiments and protein modeling prediction, we found that Cys44 was involved in the dual role of the two isoforms of ATG10 protein on HCV replication and autophagy flux, and that Cys135 plays similar roles as Cys44, but the disulfide bond of Cys44-Cys135 was not verified in the ATG10 protein. Further analyses by full HCV virion infection confirmed the roles of -SH of Cys44 and Cys135 on HCV replication. ATG10 with deleted or mutated Cys44 and/or Cys135 could activate expression of innate immunity-related genes, including il28a, irf-3, irf-7, and promote complete autophagy by driving autophagosomes to interact with lysosomes via IL28A-mediation. Subcellular localization assay and chromatin immunoprecipitation assay showed that ATG10 with the sulfydryl deletion or substitution of Cys44 and Cys135 could translocate into the nucleus and bind to promoter of IL28A gene; the results indicated that ATG10 with Cys44 and/or Cys135 absence might act as transcriptional factors to trigger the expression of anti-HCV immunological genes, too. In conclusion, our findings provide important information for understanding the differential roles on HCV replication and autophagy flux between ATG10 and ATG10S, and how the structure-function relationship of ATG10 transformed by a single -SH group loss on Cys44 and Cys135 in ATG10 protein, which may be a new target against HCV replication.

Internal driving factors leading to extrahepatic manifestation of the hepatitis C virus infection

The hepatitis C virus (HCV) infection is associated with various extrahepatic manifestations, which are correlated with poor outcomes, and thus increase the morbidity and mortality of chronic hepatitis C (CHC). Therefore, understanding the internal linkages between systemic manifestations and HCV infection is helpful for treatment of CHC. Yet, the mechanism by which the virus evokes the systemic diseases remains to be elucidated. In the present study, using gene set enrichment analysis (GSEA) and signaling pathway impact analysis (SPIA), a comprehensive analysis of micro-array data of mRNAs was conducted in HCV-infected and -uninfected Huh7.5 cells, and signaling pathways (which are significantly activated or inhibited) and certain molecules (which are commonly important in those signaling pathways) were selected. Forty signaling pathways were selected using GSEA, and eight signaling pathways were selected with SPIA. These pathways are associated with cancer, metabolism, environmental information processing and organismal systems, which provide important information for further clarifying the intrinsic associations between syndromes of HCV infection, of which seven pathways were not previously reported, including basal transcription factors, pathogenic Escherichia coli infection, shigellosis, gastric acid secretion, dorso-ventral axis formation, amoebiasis and cholinergic synapse. Ten genes, SOS1, RAF1, IFNA2, IFNG, MTHFR, IGF1, CALM3, UBE2B, TP53 and BMP7 whose expression may be the key internal driving molecules, were selected using the online tool Anni 2.1. Furthermore, the present study demonstrated the internal linkages between systemic manifestations and HCV infection, and presented the potential molecules that are key to those linkages.