Combinatorial Chemistry & High Throughput Screening - Volume 18, Issue 1, 2015

The Rho family of Ras superfamily small GTPases regulates a broad range of biological processes such as migration, differentiation, cell growth and cell survival. Therefore, the availability of small molecule modulators as tool compounds could greatly enhance research on these proteins and their biological function. To this end, we designed a biochemical, high throughput screening assay with complementary follow-up assays to identify small molecule compounds inhibiting MgcRacGAP, a Rho family GTPase activating protein involved in cytokinesis and transcriptionally upregulated in many cancers. We first performed an in-house screen of 20,480 compounds, and later we tested the assay against 342,046 compounds from the NIH Molecular Libraries Small Molecule Repository. Primary screening hit rates were about 1% with the majority of those affecting the primary readout, an enzyme-coupled GDP detection assay. After orthogonal and counter screens, we identified two hits with high selectivity towards MgcRacGAP, compared with other RhoGAPs, and potencies in the low micromolar range. The most promising hit, termed MINC1, was then examined with cell-based testing where it was observed to induce an increased rate of cytokinetic failure and multinucleation in addition to other cell division defects, suggesting that it may act as an MgcRacGAP inhibitor also in cells.

The advent of carbapenem resistance by the production of β-lectamases and mutated penicillin binding proteins (PBPs) has challenged the treatment of Enterobacteriaceae. Hence there is an urgent need to establish drugs that can fit in the pipeline by overcoming those situations. The working hypothesis of the work is based on two facts, i.e., i) design of inhibitors against mutated PBPs to which present drugs cannot bind efficiently to kill pathogen by inhibiting cell wall formation, ii) design of molecules that can bind with β-lectamases with high affinity, so that they can supplement available drugs preventing their unwanted hydrolysis. In this work, over thousands of thienamycin (first natural carbapenem) derivatives were generated and out of which non-toxic 273 molecules were used for further study. Out of which, only few followed the first hypothesis and rest obeyed the second. Ligand L5 strictly followed the first hypothesis and L1-L4 followed to a satisfactory level. Molecular dynamic simulation was performed to check post-docking stability of the pharmacophores.

Inhibition of the hepatitis C virus (HCV) non-structural protein 3 (NS3) serine protease by molecule inhibitors is an attractive strategy for the treatment of hepatitis C. We built four classification models based on a dataset of 413 HCV NS3 protease inhibitors using support vector machine method. The best performing model obtains the best prediction performance for the test set with prediction accuracy, sensitivity (SE), specificity (SP) and Matthews correlation coefficient (MCC) of 90.76%, 92.21%, 88.10% and 0.799, respectively. The number of rotatable bonds (NRotBond), charge and electronegativity related properties were found to be correlated with the bioactivity of the inhibitors. The ECFP_4 analyses of structural features were performed and it was found that the cyclopropyl with acylsulfonamide group was the unique substructure in the active inhibitors. The method with dataset split by Kohonen's self-organizing map and descriptors selected by SVMAttributeEval presented in this study can be employed in virtual screening for discovering novel inhibitors of HCV NS3 protease.

Vasoactive intestinal polypeptide (VIP) is an intrinsic 28-amino acid peptide, involved in a wide range of physiologic effects, and therefore considered as a promising drug candidate for the treatment of several diseases. But the clinical application of VIP has been limited for the easy in vivo digestion. Various researches aiming to prolong the VIP half-life, by modifying the VIP structure, have been reported. The first thing to be considered after structural modification is to know it is a VPAC agonist or antagonist. To analyze the structure-activity relationships of VIP derivatives and build classifiers to distinguish newly designed VIPs, here in this work, we collected 46 samples and two classifiers were established respectively for VPAC1 and VPAC2 receptors. The built classifiers are robust and predictive with high sensitivity, specificity and concordance for the prediction set. By analyzing the meanings of the used variables, we found that the electrostatic properties of VIP derivatives are vital in their interactions with VPAC receptors. Finally, these two classifiers were used to predict the bioactivities of novel VIPs, without experimental activities, which were suggested for experimental research groups to test their bioactivities and the possible practical applications in future.

Environmentally method for the synthesis of 1,3-thiazole has been achieved by multicomponent reactions of primary amines, isothiosyanates and alkyl bromides under solvent-free conditions using nanorod ZnO structures as catalyst. These reactions were not performed without catalyst. The catalyst showed significant reusable activity.

Some novel analogs of 4H-1,4-benzothiazines were synthesized under environmentally benign solvent free conditions by one pot oxidative cyclocondensation of substituted 2- aminobenzenthiols with compounds having active methylene group and then converted in to sulfones. These compounds were examined as antitubercular agents against Mycobacterium tuberculosis H37Rv using REMA plate method. The best results have MICs from 6.4 to 8.8 µg/mL, comparable to phenothiazines. IR, NMR and mass spectral investigations are included for structural elucidation.

A convenient green synthesis of benzopyrazines and bis-benzopyrazines has been performed by the condensation of diversely substituted diamines and dicarbonyl compounds in water using indium powder as catalyst under controlled microwave exposure. The effects of microwave power, and temperature on this reaction are investigated in detail to identify the best condition. A wide range of diversely substituted diamines and dicarbonyl compounds produce excellent yield of the corresponding benzopyrazines and bis-benzopyrazines. A comparative study has also been made between microwave-induced procedure and conventional method. The present method is atom-economical, environmentally friendly, and affords synthetically useful and challenging products very rapidly.

The pharmacophore modeling and 3D-QSAR studies were performed on a series of amino alkyl rupestonates (Rupestonic Acid) derivatives reported for H1N1, H3N2 and Influenza B virus, NA inhibition. In order to improve the efficacy of amino alkyl rupestonates derivatives, a four point pharmacophore model with one acceptor and three hydrophobic regions was developed. Furthermore, the 3D-QSAR model was generated based on the pharmacophore hypothesis (AHHH) for each subtype. The hypothesis was more significant with R2=0.9204, Q2=0.917 for H1N1, R2=0.8911, Q2=0.8905 for H3N2 and R2=0.8385, Q2=0.7043 for Influenza B virus. The 3D-QSAR results provided an invaluable insight into structure activity correlation and it was shown that the hydrophobic regions were crucial for inhibitory activity. CoMFA and COMSIA validation had been done by leave one out and no validation methods.

The activation of T cell immune responses, which relies on peptide antigens transported by TAP and bound to major histocompatibility complex (MHC) molecules, is recognized by T cell receptors (TCR). The quantitative prediction of MHC-epitope binding affinity can facilitate epitope screening and reduce cost and experimental efforts greatly. In this study, a comprehensive quantitative prediction method of binding affinity was established using quantitative structureactivity relationship (QSAR) modeling derived from amino acid physicochemical information. Firstly, the epitope was characterized by a set of amino acid physicochemical parameters. Secondly, the structural variables were optimized by the stepwise regression (STR). Finally, the robust quantitative models with were built by multiple linear regressions (MLR) for 31 MHC Class I subtypes. The normalized regression coefficients (NRCs) of QSAR model could demonstrate the mechanism of interaction of MHC, epitope, and TCR very well. The contribution of amino acid at each position of epitope, which was calculated by NRC, could determine which one was favorable for binding affinity or not. Therefore, the quantitative models established by STR-MLR could be used to guide virtual combinational design and high throughout screening of CTL epitope. Besides, they have many advantages, such as definite physiochemical indication, easier calculation and explanation, and good performances.

In the present work, we report characterization studies of antigen(Ag)-antibody (Ab) interaction based on the multi-walled carbon nanotube array-modified gold electrode for label-free electrochemical immunosensor. -COOH functionalization of MWCNT was suggested by FTIR spectroscopy. Images from atomic force microscopy (AFM) and RAMAN spectroscopy confirmed the interaction of Ab on the gold interface based on the MWCNT array. A cyclic voltammetric study exhibited a linear response in phosphate buffered saline (PBS) solution including 1mM K3Fe (CN) 6 at pH 7.4 with PSA concentrations over a range of 2 to 40 ngmL-1, and a calculated detection limit of 0.56 ngmL-1.