Wen-Long Wang

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.

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.

Synthesis and biological evaluation of novel bis-aromatic amides as novel PTP1B inhibitors.

A series of bis-aromatic amides was designed, synthesized, and evaluated as a new class of inhibitors with IC50 values in the micromolar range against protein tyrosine phosphatase 1B (PTP1B). Among them, compound 15 displayed an IC50 value of 2.34±0.08 μM with 5-fold preference over TCPTP. More importantly, the treatment of CHO/HIR cells with compound 15 resulted in increased phosphorylation of insulin receptor (IR), which suggested extensive cellular activity of compound 15. These results provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

Synthesis and SAR Studies of Praziquantel Derivatives with Activity against Schistosoma japonicum

The synthesis and structure-activity relationship (SAR) studies of praziquantel derivatives with activity against adult Schistosoma japonicum are described. Several of them showed better worm killing activity than praziquantel and could serve as leads for further optimization.

1H-2,3-Dihydroperimidine Derivatives: A New Class of Potent Protein Tyrosine Phosphatase 1B Inhibitors

A series of 1H-2,3-dihydroperimidine derivatives was designed, synthesized, and evaluated as a new class of inhibitors of protein tyrosine phosphatase 1B (PTP1B) with IC50 values in the micromolar range. Compounds 46 and 49 showed submicromolar inhibitory activity against PTP1B, and good selectivity (3.48-fold and 2.10-fold respectively) over T-cell protein tyrosine phosphatases (TCPTP). These results have provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

Benzo[c][1,2,5]thiadiazole derivatives: A new class of potent Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) inhibitors

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) is an oncogenic phosphatase linked to various kinds of cancers. Consequently, SHP2 has emerged as a promising target for novel anti-cancer agents. Using scaffold-hopping strategy, a series of benzo[c][1,2,5]thiadiazole derivatives was designed from PTP1B inhibitors with 1H-2,3-Dihydroperimidine motif, synthesized and evaluated their biological activities against PTP1B and SHP2. Among them, the representative compound 11g displayed SHP2 inhibitory activity with IC50 of 2.11 ± 0.99 μM, exhibited 2.02-fold and 25-fold selectivity for SHP2 over SHP1 and PTP1B respectively and had no visible activity against TCPTP. These preliminary results could provide a possible opportunity for the development of novel SHP2 inhibitors with optimal potency and improved pharmacological properties.

Synthesis of novel 3-aryl-1-oxa-2,8-diazaspiro[4.5]dec-2-ene derivatives and their biological evaluation against protein tyrosine phosphatase 1B.

A series of novel 3‐aryl‐1‐oxa‐2,8‐diazaspiro[4.5]dec‐2‐ene derivatives were designed, synthesized, and evaluated as a new class of inhibitors against protein tyrosine phosphatase 1B. Among them, compound 6f displayed moderate inhibitory activity with IC50 of 2.87 ± 0.24 μm and can be used as a novel lead compound for the design of inhibitors of protein tyrosine phosphatase 1B.

Synthesis and Antiviral Activity of Conformational Analogues of Leucamide A

In order to study the effect of heterocyclic core conformational state of leucamide A on its anti-influenza virus A activity, five conformational analogues were prepared by replacing the Pro-Leu dipeptide in the molecule with various amino acids. The amino acids used were of 2 to 6 carbons. The results showed that these replacements not only changed the conformational relationship between the 4,2-bisheterocycle tandem pair and the third heterocycle, but also had dramatic effect on its activity against influenza virus A.