Combinatorial Chemistry & High Throughput Screening - Volume 19, Issue 4, 2016

The addition of palmitoyl moieties to proteins regulates their membrane targeting, subcellular localization, and stability. Dysregulation of the enzymes which catalyzed the palmitoyl addition and/or the substrates of these enzymes have been linked to cancer, cardiovascular, and neurological disorders, implying these enzymes and substrates are valid targets for pharmaceutical intervention. However, current chemical modulators of zDHHC PAT enzymes lack specificity and affinity, underscoring the need for screening campaigns to identify new specific, high affinity modulators. This report describes a mixture based screening approach to identify inhibitors of Erf2 activity. Erf2 is the Saccharomyces cerevisiae PAT responsible for catalyzing the palmitoylation of Ras2, an ortholog of the human Ras oncogene proteins. A chemical library developed by the Torrey Pines Institute for Molecular Studies consists of more than 30 million compounds designed around 68 molecular scaffolds that are systematically arranged into positional scanning and scaffold ranking formats. We have used this approach to identify and characterize several scaffold backbones and R-groups that reduce or eliminate the activity of Erf2 in vitro. Here, we present the analysis of one of the scaffold backbones, bis-cyclic piperazine. We identified compounds that inhibited Erf2 auto-palmitoylation activity using a fluorescence-based, coupled assay in a high throughput screening (HTS) format and validated the hits utilizing an orthogonal gel-based assay. Finally, we examined the effects of the compounds on cell growth in a yeast cell-based assay. Based on our results, we have identified specific, high affinity palmitoyl transferase inhibitors that will serve as a foundation for future compound design.

The N,N-dimethylbenzylamine (DMBA), as an efficient and commercially available organocatalyst was employed for the one-pot synthesis of dihydropyrano[3,2-c]chromene derivatives in ethanol medium. In this work, the one-pot Knoevenagel-Michael-Thorpe-Ziegler type cascade heterocyclization has been developed for the synthesis of dihydropyrano[3,2-c]chromene-containing heterocycles from the one-pot multicomponent condensation reaction of 4-hydroxycoumarin, malononitrile/ethyl cyanoacetate, and various aldehydes at 60°C. The salient features of this basecatalyzed reaction are mild reaction conditions, reusability of the reaction medium, shorter reaction times, and easy separation of the reaction mixture.

A random 12-peptide library was screened against Erysipelothrix rhusiopthiae and porcine circovirus 2 recombinant Cap protein and the selected peptides were used for detecting the corresponding pathogens quickly and effectively. To our surprise, seven peptides, P1 (WHWNAP WWNGVY), P2 (FHWTWQFPYTST), P3 (GAMHLPWHMGTL), P4 (HWNIWWQHHPSP), P5 (HFFKWHTRTNDQ), P6 (HFFRWHPSAHLG) and P7 (HFAYWWNGVRGP) with the characteristics of polystyrene plate (PS) binding target-unrelated peptides (TUPs), were selected from the library. It has been found that P2 and P4 shared common motif of plastic binding peptide, moreover, P2, P3, P5 and P7 have been isolated repeatedly in other research groups using different targets. Then, the seven peptide phage clones were identified as the PS binding TUP phages by phage-ELISA and elution titration, particularly, P1 and P2 showed strong PS binding affinity which can not be inhibited by usual blocking buffers. In addition, all of the phages were not propagation-related TUP, but P3 showed the similar propagation rate with M13KE (vector phage). We also found that the seven PS-TUPs are rich in W, H, F, P and G, particularly, both W and H are contained in all PS-TUPs. It deduced that they may play a potential role in peptide binding to plastic. Although it is difficult to eliminate the TUP phages in phage display completely, these PS-TUPs can be used to exclude the false positive peptides rapidly and effectively and help us to obtain truly interesting peptides more accurately.

Michael addition of indole derivatives with various substituted nitrostyrenes to yield β- indolylnitroalkanes is accomplished effectively under solvent free conditions using TiO2-SO42- as efficient catalyst at 60 º C. All the synthesized compounds were screened for their antibacterial activity through in silico and in vitro methods. The molecular docking studies against FabH enzyme, a potential drug target of bacterial fatty acid biosynthetic pathway indicated the scope of developing them a new class of antimicrobial agents. Among the title compounds, 5h exhibited the highest dock score and the highest antibacterial activity when compared with other compounds and the standard drug Ampicillin. In addition, the compounds 5d, 5e, 5g, 5h, 5i, 5j and 5l showed significant inhibitory activity at different dose concentrations under in vitro conditions against the specified bacterial strains thus qualifying for further clinical evaluation so that they can be used as effective anti-bacterial agents.

Glycogen synthase kinase-3β (GSK-3β) is a promising target for therapeutic invasion of Alzheimer’s disease (AD). The kinase enzyme plays major role in pathological process for the formation of β-amyloid plaques and neurofibrillary tangles in AD. In the present study, structure-based pharmacophore and ligand-based 3D QSAR, HQSAR and pharmacophore mapping studies have been emphasized to explore the possible structural requirement of this potential kinase inhibitors using a structurally diverse set of compounds. The developed models were validated with the interaction study at the catalytic cleft. The 3D QSAR studies yield robust models of CoMFA R2 = 0.965, se = 0.212, Q2 = 0.525, R2pred = 0.709, r2m = 0.579 and CoMSIA: R2 = 0.935, se = 0.289, Q2 = 0.581, R2pred = 0.723, r2m = 0.935, that explain the importance of steric, electrostatic, hydrogen bond (HB) acceptor of the molecule for inhibition of GSK-3β. The HQSAR study (R2 = 0.871, se = 0.400, Q2 = 0.639, R2pred = 0.721, r2m = 0.664) indicated the fragments of the molecular fingerprints that might be important for inhibition. Both structure- and ligand-based pharmacophore mapping proposed that acceptor and donor features of the molecule are essential for receptor–ligand interactions. Molecular diversity provides an opportunity on wide range of applicability for the GSK-3β inhibitors, and depicts information on the structural and properties requirement for effective binding at the active site selectivity that minimize the side effects with therapeutic benefits.

The Human Pregnane X Receptor (hPXR) is a regulator of drug metabolising enzymes (DME) and efflux transporters (ET). The prediction of hPXR activators and non-activators has pharmaceutical importance to predict the multiple drug resistance (MDR) and drug-drug interactions (DDI). In this study, we developed and validated the computational prediction models to classify hPXR activators and non-activators. We employed four machine learning methods support vector machine (SVM), k-nearest neighbour (k-NN), random forest (RF) and naïve bayesian (NB). These methods were used to develop molecular and fingerprint based descriptors for the prediction of hPXR activators and non-activators. Total 529 molecules consitsting of 317 activators and 212 non-activators were used for model development. The overall prediction accuracy of models was 69% to 99% to classify hPXR activators and nonactivators using RDkit descriptors. In case of 5 and 10-fold cross validation the prediction accuracy for training set is 74% to 82% and 79% to 83% for hPXR activators respectively and 50% to 62% and 49% to 65% non-activators, respectively. The external test prediction is between 59% to 73% for hPXR activators and 55% to 68% for hPXR non-activators. In addition, consensus models were developed in which the best model shows overall 75% to 83% accuracy for fingerprint and RDkit descriptors, respectively. The best developed model will be utilized for the prediction of hPXR activators and non-activators.

Camptothecin (CPT) and its analogs as inhibitors of topoisomerase I are anticancer compounds. Their antitumor potency is seriously limited due to hydrolysis of lactone form of camptothecins in solutions at pH>5.5, which leads to the formation of inactive carboxylate form with open lactone ring. Furthermore, the clinical application of CPT is also restricted by strong affinity of its carboxylate form to human serum albumin which destabilizes the active lactone form. By UV irradiation of the CPT carboxylate authors of this paper received camptothecin compound which has biophysical properties similar to the lactone form. The specific objective of the project is to determine the properties using the methods of absorption and steady-state fluorescence spectra analysis, fluorescence lifetime measurements as well as steady-state fluorescence anisotropy. The results suggest that the UV exposed camptothecin carboxylate changes the chemical structure. The high-throughput assays based on the steady state fluorescence anisotropy measurements proved that the form obtained as a result of UV irradiation of CPT carboxylate exhibits weaker affinity to albumin than CPT carboxylate however stronger than CPT lactone. This property is very desirable from the point of view of clinical applications.

Global demand for bioethanol is increasing tremendously as it could help to replace the conventional fossil fuel and at the same time supporting the bioremediation of huge volume of cellulosic wastes generated from different sources. Ideal genetic engineering approaches are essential to improve the efficacy of the bioethanol production processes for real time applications. A locally isolated fungal strain Aspergillus fumigatus NITDGPKA3 was used in our laboratory for the hydrolysis of lignocellulose with good cellulolytic activity when compared with other contemporary fungal strains. An attempt is made to sequence the cellobiohydrolases (CBHs) of A. fumigatus NITDGPKA3, model its structure to predict its catalytic activity towards improving the protein by genetic engineering approaches. Herein, the structure of the sequenced Cellobiohydrolases (CBHs) of A. fumigatus NITDGPKA3, modelled by homology modelling and its validation is reported. Further the catalytic activity of the modelled CBH enzyme was assessed by molecular docking analysis. Phylogenetic analysis showed that CBH from A. fumigatus NITDGPKA3 belongs to the Glycohydro 6 (Cel6A) super family. Molecular modeling and molecular dynamics simulation suggest the structural and functional mechanism of the enzyme. The structures of both the cellulose binding (CBD) and catalytic domain (CD) have been compared with most widely studied CBH of Trichoderma reesei. The molecular docking with cellulose suggests that Gln 248, Pro 287, Val236, Asn284, and Ala288 are the main amino acids involved in the hydrolysis of the β, 1-4, glycosidic bonds of cellulose.

During World War II, organophosphorus compounds with neurotoxic action were developed and used as the basis for the development of structures currently used as pesticides in the agricultural industry. Among the nerve agents, Tabun, Sarin, Soman and VX are the most important. The factor responsible for the high toxicity of organophosphorus (OP) is the acetylcholinesterase inhibition. However, one of the characterized enzymes capable of degrading OP is Phosphotriesterase (PTE). This enzyme has generated considerable interest for applications of rapid and complete detoxification. Due to the importance of bioremediation methods for the poisoning caused by OP, this work aims to study the interaction mode between the PTE enzyme and organophosphorus compounds, in this case, Sarin, Soman, Tabun and VX have been used, which are potent acetylcholinesterase inhibitors, taking into account the enantiomers "Rp" and " Sp" of each compound, with the Sp-enantiomers presenting the higher toxicity. With that, we were able to demonstrate the existence of the stereochemical preference by PTE in these compounds. With the purpose of increasing the speed of the hydrolysis mechanism, we have proposed a modification in the enzyme active site structure, where Zn2+ ions were substituted by Al3+ ions. To analyze the stability of Al3+ ions in the wild-type PTE active site, MD simulations were also performed. This mutation brought relevant results; in this case, there was a reduction of the reaction energy barrier for all the compounds, mainly for VX in which the reaction presented lower activation energy values, and consequently, a faster hydrolysis process.