David Chi-Cheong Wan

Interaction study of two diterpenes, cryptotanshinone and dihydrotanshinone, to human acetylcholinesterase and butyrylcholinesterase by molecular docking and kinetic analysis.

Alzhemiers disease (AD) is a common form of dementia in the ageing population which is characterized by depositions of amyloids and a cholinergic neurotransmission deficit in the brain. Current therapeutic intervention for AD is primarily based on the inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level. Cryptotanshinone (CT) and dihydrotanshinone (DT) were diterpenoids extracted from Salvia miltiorrhiza Bge. having anti-cholinesterase activity. Here we characterized the inhibition property of these two diterpenoids towards human AChE and butyrylcholinesterase (BChE). Both CT and DT were found to be mixed non-competitive inhibitors for human AChE and an uncompetitive inhibitor for human BChE. The docking analyses of CT and DT into the active sites of both cholinesterases indicate that they interact with the allosteric site inside the active-site gorge mainly by hydrophobic interactions.

Enzyme kinetic and molecular docking studies for the inhibitions of miltirone on major human cytochrome P450 isozymes.

Previous studies have shown that major tanshinones isolated from Danshen (Salvia miltiorrhiza) inhibited human and rat CYP450 enzymes-mediated metabolism of model probe substrates, with potential in causing herb-drug interactions. Miltirone, another abietane type-diterpene quinone isolated from Danshen, has been reported for its anti-oxidative, anxiolytic and anti-cancer effects. The aim of this study was to study the effect of miltirone on the metabolism of model probe substrates of CYP1A2, 2C9, 2D6 and 3A4 in pooled human liver microsomes. Miltirone showed moderate inhibition on CYP1A2 (IC(50)=1.73 μM) and CYP2C9 (IC(50)=8.61 μM), and weak inhibition on CYP2D6 (IC(50)=30.20 μM) and CYP3A4 (IC(50)=33.88 μM). Enzyme kinetic studies showed that miltirone competitively inhibited CYP2C9 (K(i)=1.48 μM), and displayed mixed type inhibitions on CYP1A2, CYP2D6 and CYP3A4 with K(i) values of 3.17 μM, 24.25 μM and 35.09 μM, respectively. Molecular docking study further confirmed the ligand-binding conformations of miltirone in the active sites of these human CYP450 isoforms, and provided some information on structure-activity relationships for the CYPs inhibition by tanshinones. Taken together, CYPs inhibitions of miltirone were weaker than dihydrotanshinone, but stronger than cryptotanshinone, tanshinone I and tanshinone IIA.

Characterization of miR‐210 in 3T3‐L1 adipogenesis

Although accumulating evidences indicate that miRNA emerge as a vital player in cell growth, development, and differentiation, how they contribute to the process of adipocyte differentiation remains elusive. In the present study, we revealed that the expression level of miR‐210 was dramatically upregulated during 3T3‐L1 adipogenesis. Ectopic introduction of miR‐210 into 3T3‐L1 cells promoted terminal differentiation as well as the expression of adipogenic markers. MTT assay showed that miR‐210 significantly inhibited cell proliferation whereas the BrdU incorporation assay and flow cytometry analysis showed that miR‐210 did not impair G1/S phase transition. Further experiments demonstrated that enhanced expression of miR‐210 in 3T3‐L1 cells provoked adipocyte differentiation via activation of PI3K/Akt pathway by targeting SHIP1, a negative regulator of PI3K/Akt pathway. Moreover, blockade of endogenous miR‐210 during adipogenesis significantly repressed adipocyte differentiation. In summary, we have identified miR‐210 as an important positive regulator in adipocyte differentiation through the activation of PI3K/Akt pathway. J. Cell. Biochem. 114: 2699–2707, 2013.

Profiling of substrate specificities of 3C-like proteases from group 1, 2a, 2b, and 3 coronaviruses.

Background Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CLpro), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection. Methodology/Principal Findings Here, we profiled the substrate specificities of 3CLpro from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19×8 of variants with single substitutions at P5 to P3 positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CLpro prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1 and P2 positions. Despite 3CLpro from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CLpro prefers P4-Pro and SARS-CoV 3CLpro prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences ‘VARLQ↓SGF’ that can be cleaved efficiently by all 3CLpro with relative activity of 1.7 to 3.2, and ‘VPRLQ↓SGF’ that can be cleaved specifically by IBV 3CLpro with relative activity of 4.3. Conclusions/Significance The comprehensive substrate specificities of 3CLpro from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases.

Acetylshikonin, a Novel AChE Inhibitor, Inhibits Apoptosis via Upregulation of Heme Oxygenase-1 Expression in SH-SY5Y Cells.

Acetylcholinesterase inhibitors are prominent alternative in current clinical treatment for AD patients. Therefore, there is a continued need to search for novel AChEIs with good clinical efficacy and less side effects. By using our in-house natural product database and AutoDock Vina as a tool in docking study, we have identified twelve phytochemicals (emodin, aloe-emodin, chrysophanol, and rhein in Rhei Radix Et Rhizoma; xanthotoxin, phellopterin, alloisoimperatorin, and imperatorin in Angelicae dahuricae Radix; shikonin, acetylshikonin, isovalerylshikonin, and β,β-dimethylacrylshikonin in Arnebiae Radix) as candidates of AChEIs that were not previously reported in the literature. In addition to AChEI activity, a series of cell-based experiments were conducted for the investigation of their neuroprotective activities. We found that acetylshikonin and its derivatives prevented apoptotic cell death induced by hydrogen peroxide in human and rat neuronal SH-SY5Y and PC12 cells at 10u2009μM. We showed that acetylshikonin exhibited the most potent antiapoptosis activity through the inhibition of the generation of reactive oxygen species as well as protection of the loss of mitochondria membrane potential. Furthermore, we identified for the first time that the upregulation of heme oxygenase 1 by acetylshikonin is a key step mediating its antiapoptotic activity from oxidative stress in SH-SY5Y cells.

Design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus 3C-like proteases

n Abstractn n Coronaviral infection is associated with up to 5% of respiratory tract diseases. The 3C-like protease (3CLpro) of coronaviruses is required for proteolytic processing of polyproteins and viral replication, and is a promising target for the development of drugs against coronaviral infection. We designed and synthesized four nitrile-based peptidomimetic inhibitors with different N-terminal protective groups and different peptide length, and examined their inhibitory effect on the in-vitro enzymatic activity of 3CLpro of severe-acute-respiratory-syndrome-coronavirus. The IC50 values of the inhibitors were in the range of 4.6–49xa0μM, demonstrating that the nitrile warhead can effectively inactivate the 3CLpro autocleavage process. The best inhibitor, Cbz-AVLQ-CN with an N-terminal carbobenzyloxy group, was ∼10x more potent than the other inhibitors tested. Crystal structures of the enzyme–inhibitor complexes showed that the nitrile warhead inhibits 3CLpro by forming a covalent bond with the catalytic Cys145 residue, while the AVLQ peptide forms a number of favourable interactions with the S1–S4 substrate-binding pockets. We have further showed that the peptidomimetic inhibitor, Cbz-AVLQ-CN, has broad-spectrum inhibition against 3CLpro from human coronavirus strains 229E, NL63, OC43, HKU1, and infectious bronchitis virus, with IC50 values ranging from 1.3 to 3.7xa0μM, but no detectable inhibition against caspase-3. In summary, we have shown that the nitrile-based peptidomimetic inhibitors are effective against 3CLpro, and they inhibit 3CLpro from a broad range of coronaviruses. Our results provide further insights into the future design of drugs that could serve as a first line defence against coronaviral infection.n n

Discovery of FDA-approved drugs as inhibitors of fatty acid binding protein 4 using molecular docking screening

We first identified fluorescein, ketazolam, antrafenine, darifenacin, fosaprepitant, paliperidone, risperidone, pimozide, trovafloxacin, and levofloxacin as inhibitors of fatty acid binding protein 4 using molecular docking screening from FDA-approved drugs. Subsequently, the biochemical characterizations showed that levofloxacin directly inhibited FABP4 activity in both the in vitro ligand displacement assay and cell-based function assay. Furthermore, levofloxacin did not induce adipogenesis in adipocytes, which is the major adverse effect of FABP4 inhibitors.

Silibinin, a novel chemokine receptor type 4 antagonist, inhibits chemokine ligand 12-induced migration in breast cancer cells.

PURPOSEnC-X-C chemokine receptor type 4 (CXCR4) signaling has been demonstrated to be involved in cancer invasion and migration; therefore, CXCR4 antagonist can serve as an anti-cancer drug by preventing tumor metastasis. This study aimed to identify the CXCR4 antagonists that could reduce and/or inhibit tumor metastasis from natural products.nnnMETHODS AND RESULTSnAccording to the molecular docking screening, we reported here silibinin as a novel CXCR4 antagonist. Biochemical characterization showed that silibinin blocked chemokine ligand 12 (CXCL12)-induced CXCR4 internalization by competitive binding to CXCR4, therefore inhibiting downstream intracellular signaling. In human breast cancer cells MDA-MB-231, which expresses high levels of CXCR4, inhibition of CXCL12-induced chemomigration can be found under silibinin treatment. Overexpression of CXCL12 sensitized MDA-MB-231 cells to the inhibition of silibinin, which was abolished by CXCR4 knockdown. The inhibition of silibinin was also observed in MCF-7/CXCR4 cells rather than MCF-7 cells that express low level of CXCR4.nnnCONCLUSIONSnOur work demonstrated that silibinin is a novel CXCR4 antagonist that may have potential therapeutic use for prevention of tumor metastasis.

Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.

Discovery of a novel HIV-1 integrase inhibitor from natural compounds through structure based virtual screening and cell imaging

The interaction between HIV‐1 integrase and LEDGF/P75 has been validated as a target for anti‐HIV drug development. Based on the crystal structure of integrase in complex with LEDGF/P75, a library containing 80 thousand natural compounds was filtered with virtual screening. 11 hits were selected for cell based assays. One compound, 3‐(1,3‐benzothiazol‐2‐yl)‐8‐{[bis(2‐hydroxyethyl)amino]methyl}‐7‐hydroxy‐2H‐chromen‐2‐one (D719) inhibited integrase nuclear translocation in cell imaging. The binding mode of D719 was analyzed with molecular simulation. The anti‐HIV activity of D719 was assayed by measuring the p24 antigen production in acute infection. The structure characteristics of D719 may provide valuable information for integrase inhibitor design.