Combinatorial Chemistry & High Throughput Screening - Volume 16, Issue 9, 2013

RNA interference (RNAi) has opened promising avenues to better understand gene function. Though many RNAi screens report on the identification of genes, very few, if any, have been further studied and validated. Data discrepancy is emerging as one of RNAi main pitfalls. We reasoned that a systematic analysis of lethality-based screens, since they score for cell death, would examine the extent of hit discordance at inter-screen level. To this end, we developed a methodology for literature mining and overlap analysis of several screens using both siRNA and shRNA flavors, and obtained 64 gene lists censoring an initial list of 7,430 nominated genes. We further performed a comparative analysis first at a global level followed by hit re-assessment under much more stringent conditions. To our surprise, none of the hits overlapped across the board even for PLK1, which emerged as a strong candidate in siRNA screens; but only marginally in the shRNA ones. Furthermore, EIF5B emerges as the most common hit only in the shRNA screens. A highly unusual and unprecedented result was the observation that 5,269 out of 6,664 nominated genes (∼80%) in the shRNA screens were exclusive to the pooled format, raising concerns as to the merits of pooled screens which qualify hits based on relative depletions, possibly due to multiple integrations per cell, data deconvolution or inaccuracies in intracellular processing causing off-target effects. Without golden standards in place, we would encourage the community to pay more attention to RNAi screening data analysis practices, bearing in mind that it is combinatorial in nature and one active siRNA duplex or shRNA hairpin per gene does not suffice credible hit nomination. Finally, we also would like to caution interpretation of pooled shRNA screening outcomes.

Molecular imprinting is an interesting technique for preparation of molecular recognition materials with discriminating similar molecules from complex systems. In particular, imprinting more than one molecule has immense application in remediation of industrial waste. Major difficulty in molecular imprinting is the selection of suitable polymer precursors. In this article, authors have proposed a new computational approach for combinatorial screening of polymer precursor library to select appropriate polymer precursors to prepare imprinted polymer capable of selectively binding carcinogenic polycyclic aromatic hydrocarbons (PAHs). Molecular Dynamics (MD) and Quantum Mechanics (QM) models were used to compute interaction energy scores between polymer precursors and PAHs in a simulated solvent box. A self-designed virtual library of functional monomers has been prepared, and then used for MD simulations to screen the best functional monomers. Initially, molecules used in the study were geometrically optimized and then interaction energies were computed using density functional theory (DFT) in Becke 3-Parameter Exchange Correlation Function (B3LYP) level with 6-31G*basis set on Gaussian 4.1 Ver. software. Complimentary to theoretical predictions, selected polymers were prepared in laboratory and compared theoretically computed binding score with the binding capacity of the polymer on spectrofluorimetry. The computer simulations used in this research paper are rapid and reliable for the combinatorial screening of polymer precursors in experimental-free way to design of multi-template imprinted polymers.

Glutathione (GSH) is a major endogenous antioxidant highly active in human tissues and plays a key role in controlling cellular thiol redox system, maintaining the immune and detoxification system. The determination of GSH levels in tissue is important to estimate endogenous defenses against oxidative stress. In our study, the multi-walled carbon nanotube modified screen-printed electrodes (MWCNT-SPEs) were used to determine the levels of GSH in trichloroacetic acid (TCA)-treated or untreated samples of rat plasma. It was found that the deproteinization of samples with TCA improved the electrochemical detection of GSH particularly in plasma. The oxidation of GSH was measured by using differential pulse voltammetry (DPV) method in combination with MWCNT-SPE (n=3), and the detection limit of GSH was found to be 0.47 μM (S/N=3). The GSH levels in plasma samples were also measured spectrophotometrically in order to compare the effectiveness of electrochemical method and we obtained a high correlation between the two methods (R2=0.976).

Thrombin plays a key role in the regulation of hemostasis and thrombosis. Inhibition of thrombin is therefore an effective therapeutic target to prevent the formation of blood clots and related thromboembolism disorders. Hence, we have developed chemical feature based pharmacophore models of thrombin inhibitors. The best hypothesis, Hypo1, is characterized with two hydrogen bond acceptors (A), one hydrophobic (H) and one ring aromatic (R) feature. Hypo1 was cross validated using several techniques to prove its validity and statistical significance. The well validated model Hypo1 was used as a 3D query to perform virtual screening. The scores obtained from virtual screening were sorted by applying drug-like filters and molecular docking studies. Finally, 4 compounds were obtained as drug-like leads based on scoring functions, binding modes and molecular interactions at the active site. These 4 molecules were further optimized by adding different substitutions in their side chains. When compared to the original database hits, optimized molecules showed high scoring function, good binding modes and molecular interactions. Hence, we suggest that, upon optimization, these four database hits can act as potential virtual leads to design novel thrombin inhibitors. Also, our model could be useful to retrieve the structurally diverse compounds from various databases.

Mycobacterium tuberculosis (Mtb), due to its unusual organization crosses different immune barriers and causes tuberculosis. The advent of multidrug resistance tuberculosis (MDR-TB) has attained alarming situation. Hence, computational drug design has been performed in this work to find potent molecules for this purpose. Isoniazid is a widely used frontline drug against tuberculosis. But reports justified the inactivity of isoniazid on acetylation by Arylamine N-acetyltransferase (NAT). 35 countries were highlighted to have isoniazid resistance from survey in 1998. Hence, Mtb NAT has been selected as the target in the present case and hundred compounds were screened in order to find potent NAT inhibitor to raise the efficacy of isoniazid. Molecular docking with Biosolveit LeadIT and Autodock 4.2 simulation was performed. The result showed 7- methylpicene-1, 2-diol to have -26.77 and -8.26 kcal/mol score in LeadIT and Autodock 4.2. The work validated 7- methylpicene-1, 2-diol to be a potent NAT inhibitor to supplement isoniazid.

4-Benzothiazol-2-ylamino-4-oxo-2-butenoic acid, prepared from reaction of 2-aminobenzothiazole and maleic anhydride, are used as an acid component in Passerini three-component reaction, under solvent-free media, to produce unsaturated α-benzothiazole acyloxyamides in good yields.

In the present study, receptor induced 3D-QSAR model was developed for a set of 46 thieno[2,3-b]pyridine-5- carbonitrile PKC- inhibitors, to explore the structural requirements of the molecules necessary for PKC- inhibition. Since the chemical nature of the studied molecules was different from the crystal ligand of the selected protein, induced fit docking (IFD) protocol was employed to induce the conformational changes in the active site of the selected protein. Thereafter, all molecules were docked into the newly generated active site environment of the selected protein using glide docking program, and the 3D-QSAR analysis was performed in PHASE program utilizing the docking based alignment of the molecules. The best 3D-QSAR model was selected on the basis of the highest value of Q2test (0.600), and the selected model also showed high values of R2train, 0.915, Pearson-r, 0.801 and low value of SD, 0.241. The contour maps corresponding to the selected 3D-QSAR model, in combination with docking analysis, helped to explore the essential amino acid residues involved in binding, and structural requirements of the ligand molecules necessary for complementary fit with the active site of the protein. Therefore, the information revealed from the generated model can further be explored as a novel tool for the designing of new congener molecules that can serve as potential therapeutics for the treatment of various disease conditions associated with abnormal PKC- signalling.

Multi-drug resistance to the available antimalarial drugs is a major threat for malaria treatment. Due to the recent characterization of human and parasite genome sequences, both ligand and target based drug design strategies may be helpful for the design of potential antimalarial compounds with reduced degree of resistance. The present work deals with quantitative structure-activity relationship (QSAR) modeling, pharmacophore mapping and docking studies of a series of 95 nucleoside analogs as inhibitors of Plasmodium falciparum deoxyuridine-5'-triphosphate nucleotidohydrolase (PfdUTPase), an enzyme involved in nucleotide metabolism that acts as a first line of defence against uracil incorporation into DNA. The QSAR and pharmacophore models were validated both internally and externally showing good statistical results. The docking study was performed and validated using three different software tools namely Discovery Studio 2.1 (Accelrys), Maestro 9.3 (Schrodinger) and MOE (Chemical Computing Group). The QSAR studies revealed that compounds containing substituted aromatic carbons (aasC fragment) and those bearing hydroxyl groups without an noxolane ring exert potent PfdUTPase inhibitory activity. The best pharmacophore hypothesis (hypothesis 1) possessed four features: (i) one hydrogen bond donor (HBD), (ii) one hydrogen bond acceptor (HBA), (iii) one hydrophobic (HYD) and (iv) one ring aromatic (RA). The docking studies revealed that the PfdUTPase inhibitors interact with a pocket containing Phe46, Lys48, Leu88, Asn103, Gly106, Leu107, Ile108, Tyr112, Ile116, Ile117, Ala118 and Ala119 amino acid residues. The interaction pattern of all the PfdUTPase inhibitors was almost same in case of docking using Discovery Studio 2.1, Maestro 9.3 and MOE software. This work thus presents the first QSAR report for nucleoside analogs which may serve as an efficient tool to address the increasing threat of malaria in the developing countries.

An efficient synthesis of thiazoles is described via an one-pot reaction between acid chlorides, ammonium thiocyanate tetramethylguanidine and α-bromocarbonyl compounds under solvent-free conditions without using any catalyst. The method offers several advantages including high yields of products and an easy work-up procedure at room temperature in excellent yield.