Li-Ping Shi

Anti-hepatitis B virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro.

Chlorogenic acid and its related compounds are abundant plant polyphenols that have a diverse antiviral activity. In this study, HepG2.2.15 cells and duck hepatitis B virus infection model were used as in vitro and in vivo models to evaluate their anti-HBV activity. In the cell model, all the three compounds inhibited HBV-DNA replication as well as HBsAg production. Chlorogenic acid and caffeic acid also reduced serum DHBV level in DHBV-infected duckling model. Moreover, the anti-HBV activity of crude extracts of coffee beans, which have a high content of chlorogenic acid, was studied. Both the extracts of regular coffee and that of decaffeinated coffee showed inhibitory effect on HBV replication.

Investigation of the immunosuppressive activity of artemether on T-cell activation and proliferation

Artemisinin and its derivatives exhibit potent immunosuppressive activity. The purpose of the current study was to examine the immunosuppressive activity of artemether directly on T lymphocytes and to explore its potential mode of action.

The new water-soluble artemisinin derivative SM905 ameliorates collagen-induced arthritis by suppression of inflammatory and Th17 responses

Our previous study showed that SM905, a novel artemisinin derivative, exhibited potent immunosuppressive activity. In this study, we evaluate preventive and therapeutic effect of SM905 on collagen‐induced arthritis (CIA) in DBA/1 mice, and investigate its mechanisms both in inflammatory and autoimmune aspects of the disease.

A reversible S-adenosyl-L-homocysteine hydrolase inhibitor ameliorates experimental autoimmune encephalomyelitis by inhibiting T cell activation

The reversible S-adenosyl-l-homocysteine hydrolase inhibitor DZ2002 [methyl 4-(adenin-9-yl)-2-hydroxybutanoate] suppresses antigen-induced-specific immune responses, particularly type 1 helper T cell (Th1)-type responses. Experimental autoimmune encephalomyelitis (EAE) is thought to be a Th1 cell-mediated inflammatory demyelinating autoimmune disease model of human multiple sclerosis (MS). In this study, we examined the effects of DZ2002 on active EAE induced by myelin oligodendrocyte glycoprotein (MOG) 35-55 in female C57BL/6 mice. Administration of DZ2002 (50 mg/kg/day i.p.) significantly reduced the incidence and severity of EAE, which was associated with the inhibition of MOG35-55-specific T cell proliferation and Th1-type cytokine production. In vitro studies also demonstrated that DZ2002 inhibited anti-CD3/28-induced naive T cell activation concomitant with the down-regulation of cyclin-dependent kinase (CDK) 4, CDK6, cyclin D3, and the up-regulation or protection of the CDK inhibitor p27. These findings highlight the fact that DZ2002 likely prevents EAE by suppressing T cell activation and suggest its utility in the treatment of MS and other Th1-mediated inflammatory diseases.

WSS45, a sulfated α- D -glucan, strongly interferes with Dengue 2 virus infection in vitro

AbstractAim:To investigate the mode of action of WSS45, one sulfated derivative of an α-D-glucan from the Gastrodia elata Bl, on the multiplication cycle of dengue virus serotype 2 (DV2), including initial infection and intracellular replication.Methods:Virus multiplication in BHK cells were monitored by qRT-PCR, plaque reduction assay, and flow cytometry. Initial virus infection was dissected into adsorption and penetration steps by converting temperature and treating by acid glycine. Surface bound virions were detected by immunofluorescence staining for Evelope protein.Results:WSS45 effectively inhibited DV2 infection in BHK cells with an EC50 value of 0.68±0.17 μg/mL , mainly interfered with virus adsorption, in a very early stage of the virus cycle. However, WSS45 showed no viricidal effect. Moreover, WSS45 could increase the detaching of virus from cell surface in BHK cell line.Conclusion:WSS45 exerted potent inhibitory effect on DV2 through interfering with the interaction between viruses and targeted cells. This activity was related to its molecular size.

Isothiafludine, a novel non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation.

Aim:To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo.Methods:HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg−1·d−1) for 15 d.Results:NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 μmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 μmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, 20 μmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks.Conclusion:NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.

(5R)-5-Hydroxytriptolide Attenuated Collagen-Induced Arthritis in DBA/1 Mice via Suppressing Interferon-gamma Production and Its Related Signaling

(5R)-5-Hydroxytriptolide (LLDT-8) displays strong immunosuppressive activities both in vitro and in vivo in our previous studies. This study aims to investigate whether LLDT-8 has antiarthritic potential in a murine model of type II bovine collagen (CII)-induced arthritis (CIA) and to show the mechanism(s) of LLDT-8 action. DBA/1 mice were immunized with CII to induce arthritis and administered with LLDT-8. The severity of arthritis was evaluated according to the clinical score and joint damage. The effects of LLDT-8 on immune responses were determined by measurement of serum antibody levels, lymphocyte proliferation assay, cytokine assay, nitric oxide (NO) production, arginase activity assays, fluorescence-activated cell sorting analysis of splenic Mac-1+ cells, as well as polymerase chain reaction analysis for interferon-γ (IFN-γ)-related gene expression. We showed that LLDT-8 treatment significantly reduced the incidence and severity of CIA. The preventive and therapeutic effects of LLDT-8 are associated with 1) reduction of serum anti-CII immunoglobulin (Ig) G, IgG2a, and IgG1 levels; 2) inhibition of CII-specific lymphocyte proliferation, IFN-γ and interleukin-2 production; 3) blockade of gene expressions in IFN-γ signaling, including IFN-γ production pathways [signal transducer and activator of transcription (STAT) 1, T-box transcription factor, interleukin 12Rβ2, and STAT4] and IFN-γ-induced chemokine transcription [macrophage inflammatory protein (Mip)-1α, Mip-1β, regulated on activation normally T cell expressed and secreted, and inducible protein 10]; and 4) retardation of the abnormal increase of NO via IFN-γ/STAT1/interferon regulatory factor 1/inducible nitric-oxide synthase pathway and arginase activity. Moreover, the mRNA transcription of chemokine receptors was also suppressed [including C-C chemokine receptor (CCR) 1, CCR5, and C-X-C chemokine receptor 3]. In conclusion, our data suggest that the antiarthritic effect of LLDT-8 is closely related to the blockade of IFN-γ signaling. LLDT-8 may have a therapeutic value in the treatment of rheumatoid arthritis.

Rational Design and Synthesis of 2,2-Bisheterocycle Tandem Derivatives as Non-Nucleoside Hepatitis B Virus Inhibitors

Despite the existence of an effective vaccine against the hepatitis B virus (HBV), chronic infection still poses a huge health burden on the global community. Current clinical therapies for the treatment of chronic hepatitis B include interferon a, and the nucleoside-derived, viral polymerase inhibitors lamivudine (3TC), adefovir, entecavir, and telbivudine. Unfortunately, resistance of the virus to nucleoside-derived inhibitors, and the adverse effects of interferon a, limit the clinical application of these drugs. Therefore, the development of potent non-nucleoside anti-HBV agents is urgently required. Natural products have been a rich source of medicines; their structural diversity has served as a source of inspiration in the search for pharmacologically active molecules. Our strategy focuses on the discovery of therapeutic agents inspired by small-molecule natural products with unique structural motifs, such as heterocyclic tandem subunits. Heterocyclic tandem pairs have been found in a few natural products, and play a pivotal role in their bioactivity through specific interactions with DNA or other targets. Leucamide A is a cyclic heptapeptide containing a mixed 4,2-bisheterocycle tandem pair isolated from the Australian marine sponge Leucetta microraphis, and is moderately cytotoxic in several tumor cell lines. The mixed 4,2-bisheterocycle tandem pair represents a novel scaffold worthy of further investigation. Following our first total synthesis of leucamide A, we synthesized a library of 4,2-bisheterocycle tandem derivatives consisting of a methyloxazole and a thiazole subunit (Figure 1). These compounds were screened in several in vitro assays to determine their antiviral activities against influenza virus, herpes simplex virus (HSV), and HBV. Several compounds showed moderate activity against influenza A virus, HSV-2, and HBV, whereas leucamide A itself showed no antiviral activity. Of these derivatives, compound 1, which has an IC50 value of 76.4 mm against HBV DNA replication, represents a novel chemical scaffold unlike any known non-nucleoside inhibitors. Though compound 1 showed relatively weak inhibitory activity against HBV DNA replication, it was considered to be a good lead for further optimization. To diversify the bisheterocyclic scaffold, several series of compounds were designed and synthesized, including 2,4 and 2,2 tandem pairs in which the latter proved more potent (Figure 1). Figure 1. Discovery of the anti-HBV lead compound (1) from the natural product Leucamide A and the design of a focused library of tandem substituted 2,2’-bisheterocycles.

Effect of a hepatitis B virus inhibitor, NZ-4, on capsid formation.

During the hepatitis B virus (HBV) life cycle, nucleocapsid assembly is essential for HBV replication. Both RNA reverse transcription and DNA replication occur within the HBV nucleocapsid. HBV nucleocapsid is consisted of core protein (HBcAg), whose carboxy-terminal domain (CTD) contains an Arg-rich domain (ARD). The ARD of HBcAg does contribute to the encapsidation of pregenomic RNA (pgRNA). Previously, we reported a small-molecule, NZ-4, which dramatically reduced the HBV DNA level in an in vitro cell setting. Here, we explore the possible mechanisms by which NZ-4 inhibits HBV function. As an HBV inhibitor, NZ-4 leads to the formation of genome-free capsids, including a new population of capsid that runs faster on agarose gels. NZ-4s activity was dependent on the presence of the ARD I, containing at least one positively charged amino acid. NZ-4 might provide a new option for further development of HBV therapeutics for the treatment of chronic hepatitis B.

Anti-Hepatitis B Virus Drugs in Clinical and Preclinical Development

Up to date, there are two types of drugs approved to treat hepatitis B: interferons and nucleos (t) ide analogues. However, the therapies are limited in the clinical context because of the negative side effects of interferon-α and the development of substantial viral resistance to nucleos (t) idic inhibitors. Therefore, new drugs with novel structures and mechanisms are needed. In this article, the drugs approved by FDA or the European Commission for treating chronic hepatitis B virus infection, as well as those under clinical trials, and several compounds in preclinical studies are reviewed. Additionally, some potential targets and strategies to combat chronic hepatitis B virus infection are discussed.