Computational investigation of Locked Nucleic Acid (LNA) nucleotides in the active sites of DNA polymerases by molecular docking simulations

Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site

Mapping transmembrane residues of proteinase activated recpetor 2 (PAR2) that influence ligand-modulated calcium signaling

Proteinase-activated receptor 2 (PAR(2)) is a G protein -coupled receptor involved in metabolism, inflammation, and cancers. It is activated by proteolysis, which exposes a nascent N -terminal sequence that becomes a tethered agonist. Short synthetic peptides corresponding to this sequence also activate PAR(2), while small organic molecules show promising PAR(2) antagonism. Developing PAR(2) ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR(2). Guided by PAR(2) homology modeling and ligand docking based on bovine rhodopsin, followed by cross-checking with newer PAR(2) models based on ORL-1 and PART, site-directed mutagenesis of PAR(2) was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin-exposed tethered ligand) and one antagonist for modulation of PAR(2) signaling. Effects of 28 PAR2 mutations were examined for PAR(2)-mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty-eight PAR(2) mutations reduced the potency of at least one ligand by>10-fold. Key residues mapped predominantly to a cluster in the transmembrane (TM) domains of PAR(2), differentially influence intracellular Ca2+ induced by synthetic agonists versus a native agonist, and highlight subtly different TM residues involved in receptor activation. This is the first evidence highlighting the importance of the PAR(2) TM regions for receptor activation by synthetic PAR(2) agonists and antagonists. The trypsin-cleaved N-terminus that activates PAR(2) was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR(2) physiology. (C) 2017 Elsevier Ltd. All rights reserved