In silico identification and analysis of potential inhibitors for acid phosphatase, HppA from Helicobacter pylori.

Helicobacter pylori is the most common cause of gastric ulcers and is associated with gastric cancer. The enzyme HppA of class C nonspecific acid phosphohydrolases (NSAPs) of H. pylori plays a crucial role in the electron transport chain. Herein, we report an in silico homology model of HppA consisting of a monomeric α + ß model. A high throughput structure-based virtual screening approach yielded potential inhibitors against HppA with higher binding energies. Further analyses of molecular interaction maps and protein-ligand fingerprints, followed by molecular mechanics-generalized Born surface area (MM-GBSA) end point binding energy calculations of docked complexes, resulted in the detection of top binders/ligands. Our investigations identified potential substrate-competitive small molecule inhibitors of HppA, with admissible pharmacokinetic properties. These molecules may provide a starting point for developing novel therapeutic agents against H. pylori.

In silico analysis and characterization of potential inhibitors of MmaA3, a methoxy mycolic acid synthase from Mycobacterium tuberculosis.

The emergence of the multi-and extensively drug-resistant (MDR and XDR) strains of Mycobacterium tuberculosis (M.tb), necessitates paradigm-shifting therapeutic approaches. The impermeable waxy lipid layer, primarily composed of mycolic acids, is a key factor in conferring resistance to conventional drugs. This study introduces a novel strategy to combat drug resistance by targeting Methoxy mycolic acid synthase 3 (MmaA3), a critical enzyme in the mycolic acid biosynthesis pathway. MmaA3 is responsible for the O-methylation of hydroxymycolate precursors and emerges as a promising therapeutic target. Through homology-based modeling, we generated a three-dimensional structure of MmaA3, providing crucial insights into its structural characteristics. High throughput virtual screening was performed against the MmaA3 model, using diverse sources: knowledge-based, FDA-approved Drugbank, and Asinex-Elite libraries. Through rigorous computational analyses, including binding affinity assessments, molecular interactions analysis, and binding free energy calculations, potential inhibitors of MmaA3 have been identified. Subsequent validation studies evaluated the stability of top protein-ligand complexes, and free energy calculations using molecular dynamics simulations. The stability of complexes within the catalytic site was confirmed through RMSD and RMSF profile analyses. Furthermore, binding free energy calculations using the MM-GBSA approach revealed significant binding affinity of identified ligands for MmaA3 target protein, comparable to its substrate/cofactors. These findings underscore the potential of the proposed molecules as candidates for further experimental exploration, offering promising avenues for the development of effective inhibitors against M.tb. Overall, our research contributes to significantly advancing the formulation of progressive therapeutic strategies in combating drug-resistant tuberculosis.Communicated by Ramaswamy H. Sarma.

Molecular analysis of dUTPase of Helicobacter pylori for identification of novel inhibitors using in silico studies.

The human gastric pathogen Helicobacter pylori chronically affects the gastric mucosal layer of approximately half of world's population. The emergence of resistant strains urges the need for identification of novel and selective drug against new molecular targets. A ubiquitous enzyme, Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), is considered as first line of defense against uracil mis-incorporation into DNA, and essential for genome integrity. Lack of dUTPase triggers an elevated recombination frequency, DNA breaks and ultimately cell death. Hence, dUTPase can be considered as a promising target for development of novel lead inhibitor compounds in H. pylori treatment. Herein, we report the generation of three-dimensional model of the target protein using comparative modelling and its validation. To identify dUTPase inhibitors, a high throughput virtual screening approach utilizing Knowledge-based inhibitors and DrugBank database was implemented. Top ranked compounds were scrutinized based on investigations of the protein-ligand interaction fingerprints, molecular interaction maps and binding affinities and the drug potentiality. The best ligands were studied further for complex stability and intermolecular interaction profiling with respect to time under 100 ns classical molecular dynamic stimulation, establishing significant stability in dynamic states as observed from RMSD and RMSF parameters and interactions with the catalytic site residues. The binding free energy calculation computed using MM-GBSA method from the MD simulation trajectories demonstrated that our molecules possess strong binding affinity towards the Helicobacter pylori dUTPase protein. We conclude that our proposed molecules may be potential lead molecules for effective inhibition against the H. pylori dUTPase protein subject to experimental validation.Communicated by Ramaswamy H. Sarma.

A novel acetate-bound complex of human carbonic anhydrase II.

The enzyme human carbonic anhydrase II (hCAII) crystallized in an acetate-bound complex belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 42.3, b = 71.8, c = 74.0 A. The structure was solved by the molecular-replacement method and refined to an R value of 0.18 and an R(free) of 0.21. The acetate molecule replaced the zinc-bound water molecule in the structure, differing from previous reports regarding the site of acetate binding. This mode of binding disrupts the hydrogen-bonded solvent network required for activity of the enzyme. This mode of inhibitor binding is a novel one that has not been observed previously.

Natural variation in Brassica FT homeologs influences multiple agronomic traits including flowering time, silique shape, oil profile, stomatal morphology and plant height in B. juncea.

Natural structural variants of regulatory proteins causing quantitative phenotypic consequences have not been reported in plants. Herein, we show that 28 natural structural variants of FT homeologs, isolated from 6 species of Brassica, differ with respect to amino-acid substitutions in regions critical for interactions with FD and represent two evolutionarily distinct categories. Analysis of structural models of selected candidates from Brassica juncea (BjuFT_AAMF1) and Brassica napus (BnaFT_CCLF) predicted stronger binding between BjuFT and Arabidopsis thaliana FD. Over-expression of BjuFT and BnaFT in wild type and ft-10 mutant backgrounds of Arabidopsis validated higher potency of BjuFT in triggering floral transition. Analysis of gain-of-function and artificial miRNA mediated silenced lines of B. juncea implicated Brassica FT in multiple agronomic traits beyond flowering, consistent with a pleiotropic effect. Several dependent and independent traits such as lateral branching, silique shape, seed size, oil-profile, stomatal morphology and plant height were found altered in mutant lines. Enhanced FT levels caused early flowering, which in turn was positively correlated to a higher proportion of desirable fatty acids (PUFA). However, higher FT levels also resulted in altered silique shape and reduced seed size, suggesting trait trade-offs. Modulation of FT levels for achieving optimal balance of trait values and parsing pair-wise interactions among a reportoire of regulatory protein homeologs in polyploid genomes are indeed future areas of crop research.

Crystal structure of low-molecular-weight protein tyrosine phosphatase from Mycobacterium tuberculosis at 1.9-A resolution.

The low-molecular-weight protein tyrosine phosphatase (LMWPTPase) belongs to a distinctive class of phosphotyrosine phosphatases widely distributed among prokaryotes and eukaryotes. We report here the crystal structure of LMWPTPase of microbial origin, the first of its kind from Mycobacterium tuberculosis. The structure was determined to be two crystal forms at 1.9- and 2.5-A resolutions. These structural forms are compared with those of the LMWPTPases of eukaryotes. Though the overall structure resembles that of the eukaryotic LMWPTPases, there are significant changes around the active site and the protein tyrosine phosphatase (PTP) loop. The variable loop forming the wall of the crevice leading to the active site is conformationally unchanged from that of mammalian LMWPTPase; however, differences are observed in the residues involved, suggesting that they have a role in influencing different substrate specificities. The single amino acid substitution (Leu12Thr [underlined below]) in the consensus sequence of the PTP loop, CTGNICRS, has a major role in the stabilization of the PTP loop, unlike what occurs in mammalian LMWPTPases. A chloride ion and a glycerol molecule were modeled in the active site where the chloride ion interacts in a manner similar to that of phosphate with the main chain nitrogens of the PTP loop. This structural study, in addition to identifying specific mycobacterial features, may also form the basis for exploring the mechanism of the substrate specificities of bacterial LMWPTPases.

Molecular modeling of RecX reveals its mode of interaction with RecA.

The protein RecA is involved in homologous recombination, DNA repair and also catalyzes DNA strand exchange. RecX gene is downstream of recA and the gene product RecX is supposed to be important for RecA regulation. Recombinant RecX is purified to homogeneity, and circular dichroism (CD) and FTIR spectroscopy show the protein to exist mostly in helical conformation. The fluorescence emission maxima of the native and the denatured protein and the steady-state fluorescence quenching studies with acrylamide indicate the presence of tryptophan residues partially exposed to the bulk solvent. Denaturation studies with urea and guanidine hydrochloride by use of spectroscopic methods, fluorescence, and CD also confirm the instability of the protein and unfolding occurs following a two-state model. Mass spectrometry and gel permeation chromatography suggest the monomeric form of the protein. Molecular modeling of RecX represents the molecule as extended and helical bundle in conformity with the spectroscopic results. To understand the mechanism of RecX in the regulation of RecA the structural model of RecA-RecX has been discussed. In this proposed model, entry of RecX into hexameric RecA filament prevents binding of ssDNA and also inhibits ATPase activity.