Yangming Zhang

Combinatorial augmentation of voltage-gated KCNQ potassium channels by chemical openers

Noninactivating potassium current formed by KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) subunits resembles neuronal M-currents which are activated by voltage and play a critical role in controlling membrane excitability. Activation of voltage-gated potassium channels by a chemical opener is uncommon. Therefore, the mechanisms of action are worthy further investigation. Retigabine and zinc pyrithione are two activators for KCNQ channels but their molecular interactions with KCNQ channel remain largely elusive. Here we report that retigabine and zinc pyrithione recognize two different sites of KCNQ2 channels. Their agonistic actions are noncompetitive and allow for simultaneous binding of two different activators on the same channel complex, hence giving rise to combinatorial potentiation with characteristic properties of both openers. Examining their effects on mutant channels, we showed zinc pyrithione is capable of opening nonconductive channels and coapplication of zinc pyrithione and retigabine could restore a disease mutant channel similar to wild type. Our results indicate two independent activator binding sites present in KCNQ channels. The resultant combinatorial potentiation by multiple synthetic chemical openers indicates that KCNQ channels are accessible to various types and combinations of pharmacological regulation.

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.

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 immunosuppressive agent DZ2002 and its stereoisomers

DZ2002 and its related stereoisomers were efficiently synthesized. The optical data of (R)- and (S)-DZ2002 were disclosed here for the first time. Their inhibitory potency was evaluated on SAHase and MLR assay in the mean time. In accordance with respective inhibitory potency of SAHase, the immunosuppressive potency order was demonstrated as (S)-DZ2002>(Rac)-DZ2002>(R)-DZ2002>(Keto)-DZ2002. These results indicate (S)-configuration of 2-chiral center in DZ2002 is important for binding with SAHase.

Synthesis and revision of stereochemistry of rubescensin S

An effective two step transformation of oridonin to 15,16-seco-ent-kaurane skeleton is reported. We also achieved the conversion of one intermediate to natural product rubescensin S and revised its structure as a 13S configuration although 13R is reported in the literature.

Targeting the γ-/β-secretase interaction reduces β-amyloid generation and ameliorates Alzheimer's disease-related pathogenesis.

Despite decades of intense global effort, no disease-modifying drugs for Alzheimer’s disease have emerged. Molecules targeting catalytic activities of γ-secretase or β-site APP-cleaving enzyme 1 (BACE1) have been beset by undesired side effects. We hypothesized that blocking the interaction between BACE1 and γ-secretase subunit presenilin-1 (PS1) might offer an alternative strategy to selectively suppress Aβ generation. Through high-throughput screening, we discovered that 3-α-akebonoic acid (3AA) interferes with PS1/BACE1 interaction and reduces Aβ production. Structural analogs of 3AA were systematically synthesized and the functional analog XYT472B was identified. Photo-activated crosslinking and biochemical competition assays showed that 3AA and XYT472B bind to PS1, interfere with PS1/BACE1 interaction, and reduce Aβ production, whereas sparing secretase activities. Furthermore, treatment of APP/PS1 mice with XYT472B alleviated cognitive dysfunction and Aβ-related pathology. Together, our results indicate that chemical interference of PS1/BACE1 interaction is a promising strategy for Alzheimer’s disease therapeutics.

Potent and orally efficacious bisthiazole-based histone deacetylase inhibitors.

Inspired by the thiazole-thiazoline cap group in natural product largazole, a series of structurally simplified bisthiazole-based histone deacetylase inhibitors were prepared and evaluated. Compound 8f was evaluated in vivo in an experimental autoimmune encephalomyelitis (EAE) model and found to be orally efficacious in ameliorating clinical symptoms of EAE mice.

Design, synthesis and biological evaluation of bisthiazole-based trifluoromethyl ketone derivatives as potent HDAC inhibitors with improved cellular efficacy.

Histone deacetylases (HDACs) are a class of epigenetic modulators with complex functions in histone post-translational modifications and are well known targets for antineoplastic drugs. We have previously developed a series of bisthiazole-based hydroxamic acids as novel potent HDAC inhibitors. In the present work, a new series of bisthiazole-based compounds with different zinc binding groups (ZBGs) have been designed and synthesized. Among them is compound 7, containing a trifluoromethyl ketone as the ZBG, which displays potent inhibitory activity towards human HDACs and improved antiproliferative activity in several cancer cell lines.

P-Retigabine: An N-Propargyled Retigabine with Improved Brain Distribution and Enhanced Antiepileptic Activity

Retigabine (RTG, [ethyl N-[2-amino-4-[(4-fluorophenyl)methyl]amino] phenyl] carbamate]) is a first-in-class antiepileptic drug that acts by potentiating neuronal KCNQ potassium channels; however, it has less than optimal brain distribution. In this study, we report that P-RTG (ethyl N-[2-amino-4-((4-fluorobenzyl)(prop-2-ynyl)amino)phenyl]carbamate), an RTG derivative that incorporates a propargyl group at the N position of the RTG linker, exhibits an inverted brain distribution compared with RTG. The brain-to-plasma concentration ratio of P-RTG increased to 2.30 compared with 0.16 for RTG. However, the structural modification did not change the drug’s potentiation potency, subtype selectivity, or RTG molecular determinants on KCNQ channels. In addition, in cultured hippocampal neurons, P-RTG exhibited a similar capability as RTG for suppressing both induced and spontaneous action potential firing. Notably, P-RTG antiepileptic activity in the maximal electroshock (MES)-induced mouse seizure model was significantly enhanced to a value 2.5 times greater than that of RTG. Additionally, the neurotoxicity of P-RTG in the rotarod test was comparable with that of RTG. Collectively, our results indicate that the incorporation of a propargyl group significantly improves the RTG brain distribution, supporting P-RTG as a promising antiepileptic drug candidate. The strategy for improving brain-to-plasma distribution of RTG might be applicable for the drug development of other central nervous system diseases.

Novel substituted pyrazolone derivatives as AMP-activated protein kinase activators to inhibit lipid synthesis and reduce lipid accumulation in ob/ob mice

Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome characterized by hepatic steatosis. NAFLD is closely linked to obesity, insulin resistance and dyslipidemia. AMP-activated protein kinase (AMPK) functions as an energy sensor and plays a central role in regulating lipid metabolism. In this study, we identified a series of novel pyrazolone AMPK activators using a homogeneous time-resolved fluorescence assay (HTRF) based on the AMPKα2β1γ1 complex. Compound 29 (C29) is a candidate compound that directly activated the kinase domain of AMPK with an EC50 value of 2.1–0.2 μmol/L and acted as a non-selective activator of AMPK complexes. Treatment of HepG2 cells with C29 (20, 40 μmol/L) dose-dependently inhibited triglyceride accumulation. Chronic administration of C29 (10, 30 mg/kg every day, po, for 5 weeks) significantly improved lipid metabolism in both the liver and the plasma of ob/ob mice. These results demonstrate that the AMPK activators could be part of a novel treatment approach for NAFLD and associated metabolic disorders.