A conserved polar residue plays a critical role in mismatch detection in A-family DNA polymerases.

In A-family DNA polymerases (dPols), a functional 3'-5' exonuclease activity is known to proofread newly synthesized DNA. The identification of a mismatch in substrate DNA leads to transfer of the primer strand from the polymerase active site to the exonuclease active site. To shed more light regarding the mechanism responsible for the detection of mismatches, we have utilized DNA polymerase 1 from Aquifex pyrophilus (ApPol1). The enzyme synthesized DNA with high fidelity and exhibited maximal exonuclease activity with DNA substrates bearing mismatches at the -2 and - 3 positions. The crystal structure of apo-ApPol1 was utilized to generate a computational model of the functional ternary complex of this enzyme. The analysis of the model showed that N332 forms interactions with minor groove atoms of the base pairs at the -2 and - 3 positions. The majority of known A-family dPols show the presence of Asn at a position equivalent to N332. The N332L mutation led to a decrease in the exonuclease activity for representative purine-pyrimidine, and pyrimidine-pyrimidine mismatches at -2 and - 3 positions, respectively. Overall, our findings suggest that conserved polar residues located towards the minor groove may facilitate the detection of position-specific mismatches to enhance the fidelity of DNA synthesis.

In silico Binding Profile Analysis and In vitro Investigation on Chitin Synthase Substrate and Inhibitors from Maize Stem Borer, Chilo partellus.

INTRODUCTION: Insect growth and metamorphosis are strictly dependent on the structural changes that occur in chitin containing tissues and organs. Chitin synthase catalyzes chitin polymerization by ß-(1, 4) glycosidic linkage of N-acetyl-D-glucosamine (GlcNAc) monomers; the major component of insect cuticles. Targeting this enzyme could be a promising strategy to control insect pests while avoiding adverse effects on coexisting populations. Nikkomycin Z and polyoxins are commercially available fungal inhibitors known to bind to the nucleotide-binding sites of insects and fungal chitin synthase. But the binding mode of chitin synthase has not been explored to date as its structure is not available yet. METHODS: To understand the structural features of the Chilo partellus chitin synthase enzyme (CpCHS), the three-dimensional (3D) structure of the CpCHS catalytic domain was modeled using ROBETTA webserver. The obtained model was used to investigate the binding mode of its substrate, uridine diphosphate-N-acetyl-D-glucosamine (UDP-GlcNAc), and inhibitors (nikkomycin Z and polyoxins) by molecular docking approach using Schrödinger Suite-Maestro v9.2. The docked complexes were further investigated for their interaction stability by performing molecular dynamics (MD) simulations using GROMACS v5.1.2. RESULTS: Our study highlighted the significance of various interactions made by CHS residues present in the Walker-B loop and donor-binding motifs with the substrate (UDP-GlcNAc), and GEDR motif with an acceptor (GlcNAc). Also, the interactions of the QRRRW motif while forming chitin polymer were explored. We observed that the inhibitors exhibited good binding affinity with these motifs, indicated by their docking and binding affinity scores. CONCLUSION: In vitro analysis suggested that nikkomycin Z showed higher inhibition of chitin synthase activity at a concentration of 2.5 µg.L-1. Our study provided insights into the crucial interactions of chitin synthase while designing inhibitors against insect pests.

Molecular cloning, gene expression analysis, and in silico characterization of UDP-N-acetylglucosamine pyrophosphorylase from Bombyx mori.

The present study was aimed to explore the molecular and structural features of UDP-N-acetylglucosamine pyrophosphorylase of Bombyx mori (BmUAP), an essential enzyme for chitin synthesis in insects. The BmUAP cDNA sequence was cloned and expression profiles were monitored during the molting and feeding stages of silkworm larvae. The effect of 20-hydroxyecdysone (20E) on BmUAP expression, and on silkworm molting was studied, which revealed that 20E regulates its expression. Multiple sequence alignment of various pyrophosphorylases revealed that the residues N223, G290, N327, and K407 of human UAP (PDB ID: 1JV1) were found to be highly conserved in BmUAP and all other eukaryotic UAPs considered for the study. Phylogenetic analysis inferred that the UAPs possess discrete variations in primary structure among different insect Orders while sharing good identity between species of the Order. The structure of BmUAP was predicted and its interactions with uridine triphosphate, N-acetylglucosamine-1-phosphate, and UDP-N-acetylglucosamine were analyzed. Virtual screening with a library of natural compounds resulted in five potential hits with good binding affinities. On further analysis, these five hits were found to be mimicking substrate and product, in inducing conformational changes in the active site. This work provides crucial information on molecular interactions and structural dynamics of insect UAPs.

Screening and analysis of acetyl-cholinesterase (AChE) inhibitors in the context of Alzheimer's disease.

Acetyl-cholinesterase enzyme (AChE) is a known target for identifying potential inhibitors against Alzheimer diseases (AD). Therefore, it is of interest to screen AChE with the CNS-BBB database. An AChE enzyme is a member of hydrolase family is activated by acetylcholine (ACh), so, targeting the AChE enzyme with the potential inhibitor may block the binding of the ACh. In this study we carried out virtual screening of drug-like molecules from Chemical Diversity Database particularly CNS-BBB compounds, to identify potential inhibitors using Glide docking program. Top ranking ten compounds, which have lower Glide Score when compared to known drugs (Tacrine and Galantamine) for AChE. For top three molecules MD simulation was carried out and calculated binding free energy. We report the best binding compounds with AChE compared to known drugs (Taine and Galantamine) for AD. We further document the salient features of their molecular interaction with the known target. Three molecules (1-benzyl-3-(2- hydroxyethyl)-N-[2-(3-pyridyl)ethyl]-3-pyrrolidinecarboxamide, N-{3[benzyl(methyl)amino]propyl}-1,5-dimethyl-4-oxo-4,5-dihydro- 1H-pyrrolo[3,2-c]quinoline-2-carboxamide, and 6-chloro-N-[2-(diethylamino)-2-phenylethyl]-4-oxo-4H-chromene-2-carboxamide) have -196.36, -204.27, -214.40 kJ/mol, binding free energy values respectively which are much lower than values calculated for the reference ligands Tacrine and Galantamine having -119.65 and -142.18 kJ/mol respectively. Thus these molecules can be very novel potential inhibitors against AChE involved in Alzheimer's disease.

Identifying novel small molecule antagonists for mLST8 protein using computational approaches.

Mammalian lethal with SEC13 protein 8 (mLST8), is an indispensable protein subunit of mammalian target of rapamycin (mTOR) signaling pathway that interacts with the kinase domain of mTOR protein, thereby stabilizing its active site. Experimental studies reported the over expression of mLST8 in human colon and prostate cancers by activation of both mTORC1/2 complexes and subsequent downstream substrates leading to tumor progression. Considering its role, targeting mLST8 protein would be a therapeutic approach against tumor progression in colon and prostate cancers. Hence, using in silico structure based drug design approach, the comparative binding patterns of 1,1'-binapthyl-2,2'diol (BINOL), 1-(2-carboxynaphth-1yl)-2-naphthoic acid (SCF-12) and their analogs in the cavity of mLST8 were explored. ADME and binding energy calculations led to the identification of five compounds with favorable Glide (G) scores and implicated the importance of Asn132 and Gln225 as key binding residues. Molecular dynamics (MD) simulations and free energy landscape (FEL) approaches helped in elucidating the binding mechanism and suggested the possibility of ligands 1-3 namely, ZINC01765622, ZINC62723702 and ZINC02576980 to be promising antagonists for mLST8. Thus, this study substantiates the prospect of targeting mLST8 protein using potent hits which could hinder tumor progression in colon and prostate cancers.