Chemoproteomic Profiling of Signaling Metabolite Fructose-1,6-Bisphosphate Interacting Proteins in Living Cells.

Fructose-1,6-bisphosphate (FBP), a cellular endogenous sugar metabolite in the glycolytic pathway, has recently been reported to act as a signaling molecule to regulate various cellular events through the engagement of important proteins. Though tremendous progress has been made in identifying specific FBP-protein interactions, the comprehensive identification of FBP-interacting proteins and their regulatory mechanisms remains largely unexplored. Here, we describe a concise synthetic approach for the scalable preparation of a photoaffinity FBP probe that enables the quantitative chemoproteomic profiling of FBP-protein interactions based on photoaffinity labeling (PAL) directly in living cells. Using such a protocol, we captured known FBP targets including PKM2 and MDH2. Furthermore, among unknown FBP-interacting proteins, we identified a mitochondrial metabolic enzyme aldehyde dehydrogenase 2 (ALDH2), against which FBP showed inhibitory activity and resulted in cellular ROS upregulation accompanied by mitochondrial fragmentation. Our findings disclosed a new mode of glucose signaling mediating by the FBP-ALDH2-ROS axis.

Design and Synthesis of Arylamidine Derivatives as Serotonin/Norepinephrine Dual Reuptake Inhibitors.

To improve the in vivo antidepressant activity of previously reported serotonin (5-HT) and norepinephrine (NE) dual reuptake inhibitors, three series of arylamidine derivatives were designed and synthesized. The in vitro 5-HT and NE reuptake inhibitory activities of these compounds were evaluated, and compound II-5 was identified as the most potent 5-HT (IC50 = 620 nM) and NE (IC50 = 10 nM) dual reuptake inhibitor. Compound II-5 exhibited potent antidepressant activity in the rat tail suspension test and showed an acceptable safety profile in a preliminary acute toxicity test in mice. Our results show that these arylamidine derivatives exhibit potent 5-HT/NE dual reuptake inhibition and should be explored further as antidepressant drug candidates.

Bicyclol promotes toll-like 2 receptor recruiting inosine 5'-monophosphate dehydrogenase II to exert its anti-inflammatory effect.

The aim was to investigate potential targets and anti-inflammatory mechanisms of bicyclol, which has been extensively used in clinic for decades in China. Tar-Fis-Dock, virtual molecular docking system, showed that inosine 5'-monophosphate dehydrogenase II (IMPDH II) has the highest probability of binding to bicyclol. To investigate the possible role of IMPDH II in mechanisms of bicyclol, recombinant enzyme models, mice splenic lymphocytes, and human lymphocytes were used. Bicyclol (1-5 µM) significantly inhibited the proliferation of mice splenic lymphocytes stimulated by concanavalin A (conA). However, bicyclol did not show inhibitory effects on proliferation of human peripheral blood mononuclear cells (hPBMC) induced by phytohemagglutinin (PHA). IMPDH II enzyme kinetic model showed that bicyclol only had a slight regulatory effect on IMPDH II enzyme activity. These results revealed that bicyclol may be not a conventional inhibitor of IMPDH II. Further studies showed that bicyclol could promote recruitment of IMPDH II by active toll-like 2 receptor (TLR2) complex. Such effects lead to the reduction of nuclear factor κB (NF-κB) expression, increase in I-κB expression, and decrease in cytokine release, including tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). It may be a new mechanism of bicyclol for its anti-inflammatory effect.

[Design, synthesis and pharmacological investigation of isoindoline derivatives as 5-HT/NE double reuptake inhibitors].

A series of isoindoline derivatives were designed, synthesized, and evaluated for their double inhibitory activities. All of them were new compounds, and their structures were confirmed by 1H NMR and HR-MS. Preliminary in vitro pharmacological tests showed that all compounds exhibited 5-HT or NE reuptake inhibition activity. Among the tested compounds, compound I-3 exhibited potent inhibitory activity against 5-HT and NE reuptake in vitro, and exhibited potent antidepressant activity in vivo. These compounds designed can be further optimized for finding more potent 5-HT/NE dual reuptake inhibitors and antidepressant candidates as well.

Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit.

Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit.

Synthesis and inhibitory effect of piperine derivates on monoamine oxidase.

A series of piperine derivates (1-19) have been designed, synthesized and evaluated in vitro for their monoamine oxidase (MAO) A and B inhibitory activity and selectivity. It is worth noting that most of the small amine moieties substituted on the piperidine ring proved to be potent and selective inhibitors of MAO-B rather than of MAO-A. 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid n-propyl amide (3) showed the greatest MAO-B inhibitory activity (IC(50)(MAO-B)=0.045 µM) and good selectivity (IC(50)(MAO-A)=3.66 µM). The conjugated double bond and carbonyl group of piperine are proved to be an essential feature for piperine and related alkylamides to exhibit MAO-inhibitory activity. Binding mode of the titled compounds was predicted using FlexX algorithm. The design and optimization of novel small molecule monoamine oxidase inhibitors will be guided by the results of this report.

The adenosine derivative 2',3',5'-tri-O-acetyl-N6-(3-hydroxylaniline) adenosine activates AMPK and regulates lipid metabolism in vitro and in vivo.

AIMS: Our overall objective was to investigate the effect of the adenosine derivative 2',3',5'-tri-O-acetyl-N6-(3-hydroxylaniline) adenosine (WS010117) on AMP-activated protein kinase (AMPK) activation and lipid metabolism and to also assess the underlying mechanisms involved in these processes. MAIN METHODS: HepG2 cells and hamsters fed a high-fat diet were used to test the effects of WS010117 on lipid metabolism. Western blots, chemical intervention, HPLC, SAMS peptide assay, (14)C-labelled acetate and palmitate assays, molecular docking assay and siRNA targeting the AMPK γ1 subunit were used to investigate the effect of WS010117 on AMPK activation as well as the underlying mechanism involved in this activation. KEY FINDINGS: WS010117 treatment resulted in the dose-dependent activation of AMPK in HepG2 cells, increasing lipid oxidation and decreasing lipid biosynthesis. In hamsters that were fed a high-fat diet, WS010117 treatment (1.5-6 mg/kg) significantly inhibited the increase in lipid accumulation. WS010117-induced AMPK activation was essentially abolished by treatment with compound C, and the addition of WS010117 did not alter the intracellular AMP:ATP ratio. In HeLa cells endogenously lacking LKB1, WS010117-mediated AMPK activation was not impaired, even following co-treatment with STO-609, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK). The results from the molecular docking assays and experiments targeting the AMPK γ1 subunit with siRNA indicated that WS010117 may activate AMPK by binding to and regulating the γ subunit of AMPK. SIGNIFICANCE: Our data indicate that WS010117 can regulate lipid metabolism through the activation of AMPK. WS010117 may activate AMPK by binding to and regulating the AMPK γ subunit.

Synthesis and biological evaluation of novel quinazoline-derived human Pin1 inhibitors.

A series of novel 2,4-disubstituted quinazoline derivatives were prepared and their inhibitory activities on hPin1 were evaluated. Of all the synthesized compounds, eight compounds displayed inhibitory activities with IC(50) value at the level of 10(-6)mol/L. Preliminary structure-activity relationships were analyzed in details and the binding mode of the titled compounds was predicted using FlexX algorithm. The design and optimization of novel small molecule Pin1 inhibitors will be guided by the results of this report.

The synthesis of cholestane and furostan saponin analogues and the determination of sapogenin's absolute configuration at C-22.

A facile and efficient way for the synthesis of cholestane and furostan saponin analogues was established and adopted for the first time. Following this strategy, starting from diosgenin, three novel cholestane saponin analogues: (22S,25R)-3ß,22,26-trihydroxy-cholest-5-ene-16-one 22-O-[O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranoside] 11, (25R)-3ß,16ß,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranoside] 14 and (25R)-3ß,16ß,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranoside] 17, three novel furostan saponin analogues: (22S,25R)-furost-5-ene-3ß,22,26-triol 22-O-(α-D-glucopyranoside) 23, (22R,25R)-furost-5-ene-3ß,22,26-triol 22-O-(α-D-glucopyranoside) 24 and (22S,25R)-furost-5-ene-3ß,22,26-triol 22-O-[O-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranoside] 26, were synthesized ultimately. The structures of all the synthesized analogues were confirmed by spectroscopic methods. The S-chirality at C-22 of cholestane was confirmed by Mosher's method. The absolute configuration at C-22 of furostan saponin analogues was distinguished by conformational analysis combined with the NMR spectroscopy. The cytotoxicities of the synthetic analogues toward four types of tumor cells were shown also.

[Synthesis and activity of some new histone deacetylases inhibitors].

To explore novel histone deacetylase (HDAC) inhibitors with anti-tumor activity, twelve target compounds were synthesized, and their structures were confirmed by 1H NMR, MS and elemental analyses. Evaluation results in vitro showed that compound Ia exhibited potent inhibition against HDAC and is worth for further investigation. And compounds IIa, IIb, IIIa-IIIi possessed moderate HDAC inhibitory activity.

[Design of multiple targeted drugs].

Drugs designed to act on individual molecular targets usually can not combat multigenic diseases such as cancer, or diseases that affect multiple tissues or cell types such as diabetes. Increasingly, it is being recognised that a balanced modulation of several targets can provide a superior therapeutic effect and side effect profile compared to the action of a selective ligand. The multi-target drugs which impact multiple targets simultaneously are better at controlling complex disease systems and are less prone to drug resistance. Here, we compare the disadvantage of the selective ligands and the predominance of multi-targets drugs in detail and introduce the approaches of designing multiple ligands and the procedure of optimization particularly. A key challenge in the design of multiple ligands is attaining a balanced activity at each target of interest while simultaneously achieving a wider selectivity and a suitable pharmacokinetic profile. On this point, the multi-target approach represents a new challenge for medicinal chemists, pharmacologists, toxicologists, and biochemists.

[Comparative pharmacophore analysis of dual dopamine D2/5-HT(2A) receptor antagonists].

Dual dopamine D2/5-HT2A receptor antagonists have potent activity and are referred to atypical antipsychotics due to their lower propensity to elicit EPS and their moderate efficacy toward negative symptoms. However, an on-going challenge in developing atypical antipsychotics drugs is to maintain the favorable profiles and avoid of cardiovascular risk. In this paper, comparative pharmacophore analysis of dual dopamine D2/5-HT2A receptor antagonists, hERG K+ channel blockers, and alA adrenoceptor antagonists is carried out, and the results could give some insight into multi-target drug design.

[Design, synthesis and activity evaluation of novel matrix metalloproteinases inhibitors based on the structure of enzyme].

A novel inhibitor series for matrix metalloproteinases (MMPs) were designed and synthesized. Using succinate and malonate as zinc binding groups and long hydrophobic substituents to bind with S1' pockets, the compounds showed micromolar inhibition and selectivity for MMP-2 over others. And we found a better activity compound. It is a chance to find a better precursor of MMP-2 inhibitors with activity and bioavailability by further optimization of compounds.

Computational study of human phosphomannose isomerase: Insights from homology modeling and molecular dynamics simulation of enzyme bound substrate.

Phosphomannose isomerase is a zinc metalloenzyme that catalyzes the reversible isomerization of mannose-6-phosphate and fructose-6-phosphate, and the three-dimensional (3D) structure of human phosphomannose isomerase has not been reported. In order to understand the catalytic mechanism, the 3D structure of the protein is built by using homology modeling based on the known crystal structure of mannose-6-phosphate isomerase from (PDB code 1PMI). The model structure is further refined by energy minimization and molecular dynamics methods. The mannose-6-phosphate-enzyme complex is developed by molecular docking and the key residues involved in the ligand binding are determined, which will facilitate the understanding of the action mode of the ligands and guide further genetic studies. Our results suggest a hydride transfer mechanism of alpha-hydrogen between the C1 and C2 positions but do not support the cis-enediol mechanism. The detailed mechanism involves, on one side, Zn2+ mediating the movement of a proton between O1 and O2, and, on the other side, the hydrophobic environment formed in part by Tyr278 promoting transfer of a hydride ion.

Inhibitory mode of N-phenyl-4-pyrazolo[1,5-b] pyridazin-3-ylpyrimidin-2-amine series derivatives against GSK-3: molecular docking and 3D-QSAR analyses.

Glycogen synthase kinase 3 (GSK-3) inhibition is an important research topic because of its wide range of associated health implications. The interaction mode of a series of N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amine compounds with human GSK-3 has been studied using molecular docking and 3D-QSAR approaches. In the 3D-QSAR studies, the molecular alignment and conformation determination are so important that they affect the success of a model. Flexible docking (AutoDock3.0.5) was used for the determination of 'active' conformation and molecular alignment. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used to develop 3D-QSAR models of 80 N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amine compounds. The r(2) values were 0.870 and 0.861 for CoMFA and CoMSIA models, respectively. The predictive ability of these models was validated by 10 compounds of the test set. Mapping these models back to the topology of the active site of GSK-3 led to a better understanding of the vital N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amines-GSK-3 interactions. The results demonstrate that combination of ligand-based and receptor-based modeling is a powerful approach to build 3D-QSAR models. The interaction mode from this study may be helpful for the design of a novel inhibitor and guide the selection of candidate sites for further experimental studies on site-directed mutagenesis.

Synthesis and evaluation of azaindole-alpha-alkyloxyphenylpropionic acid analogues as PPARalpha/gamma agonists.

A series of azaindole-alpha-alkyloxyphenylpropionic acid analogues was synthesized and evaluated for PPAR agonist activities. Structure-activity relationship was developed for PPARalpha/gamma dual agonism. One of the synthesized compound 7a was identified as a potent, selective PPARalpha/gamma dual agonist.

Acryloylamino-salicylanilides as EGFR PTK inhibitors.

A series of acryloylamino-salicylanilides were synthesized as inhibitors of EGFR PTK. A strategy of pseudo six-membered ring formed through intramolecular hydrogen bonding in salicylanilides is employed to mimic the planar pyrimidine ring of quinazoline EGFR inhibitors. Acrylamido moiety is incorporated to target the Cys-773 of EGFR specifically. Some of the obtained compounds exhibited good activity as EGFR inhibitors.

Design, synthesis, and evaluation of 2-alkoxydihydrocinnamates as PPAR agonists.

A series of 2-alkoxydihydrocinnamates were synthesized as PPARgamma and PPARalpha dual agonists. In vitro studies in cell model showed that these compounds were efficacious. Compound 1g was found to be a potent PPARalpha/gamma dual agonist and will be further evaluated for the treatment of type II diabetes.

Exploration of a binding mode of indole amide analogues as potent histone deacetylase inhibitors and 3D-QSAR analyses.

Docking simulations and three-dimensional quantitative structure-activity relationship (3D-QSAR) analyses were conducted on a series of indole amide analogues as potent histone deacetylase inhibitors. The studies include comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Selected ligands were docked into the active site of human HDAC1. Based on the docking results, a novel binding mode of indole amide analogues in the human HDAC1 catalytic core is presented, and enzyme/inhibitor interactions are discussed. The indole amide group is located in the open pocket, and anchored to the protein through a pair of hydrogen bonds with Asp99 O-atom and amide NH group on ligand. Based on the binding mode, predictive 3D-QSAR models were established, which had conventional r2 and cross-validated coefficient values (r(cv)2) up to 0.982 and 0.601 for CoMFA and 0.954 and 0.598 for CoMSIA, respectively. A comparison of the 3D-QSAR field contributions with the structural features of the binding site showed good correlation between the two analyses. The results of 3D-QSAR and docking studies validate each other and provided insight into the structural requirements for activity of this class of molecules as HDAC inhibitors. The CoMFA and CoMSIA PLS contour maps and MOLCAD-generated active site electrostatic, lipophilicity, and hydrogen-bonding potential surface maps, as well as the docking studies, provided good insights into inhibitor-HDAC interactions at the molecular level. Based on these results, novel molecules with improved activity can be designed.

[Using distance comparison method to build pharmacophore model of epidermal growth factor receptor inhibitors].

OBJECTIVE: To build 3D-pharmacophore model of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors using distance comparisons method and design novel EGFR inhibitors. METHODS: Thirteen typical EGFR inhibitors were selected, and their biologically active conformations were obtained by using DOCK5.0 program, then 3D-pharmacophore model of EGFR inhibitors was built using distance comparisons method. RESULTS: Validation of the 3D-pharmacophore model was carried out and novel structures with potential inhibitory activity were selected by means of 3D-searching and docking method. CONCLUSION: This method can improve hit rate of lead compounds discovery and can be used to design novel EGFR inhibitors.