Purification and characterization of arginine deiminase from Pseudomonas putida: Structural insights of the differential affinities of l-arginine analogues.

Arginine deiminase (ADI) from Pseudomonas putida was purified using ammonium sulphate precipitation, anion exchange and hydrophobic interaction chromatography. Influence of various chemical compounds (metal ions, reducing agents, sulphydryl agents, and surfactants) on the catalytic activity of ADI was determined was evaluated on the purified ADI. The enzyme displayed high sensitivity towards thiol binding metal ions, chemicals acting on sulfhydryl group, and most of the surfactants. Substrate specificity studies exhibited that among the eight substrate analogues tested, canavanine had the highest affinity for ADI, followed by d-arginine and guanidine. Canavanine decreased the ADI activity up to 50% at its lowest concentration tested (10 mM), while d-arginine decreased the ADI activity up to ∼4% at its highest tested concentration (200 mM). Differential affinities of the structural analogues of arginine towards ADI were further studied by molecular modeling methods, which included homology modeling, molecular docking and molecular dynamic simulations. The molecular docking studies revealed the critical importance of residues Arg 243, Asp 166, Asp 280, Gly 299 and His 278. RMSDs for protein-ligand complexes were within a range of 1-3 Å, suggesting that the complexes were stable throughout the molecular dynamic simulation. The formation of strong hydrogen bonds by residues Asn 160, Asp166, Arg 185, Arg243, Asp280 and Gly 399 in l-arginine were preserved in the case of d-arginine and canavanine and was responsible for higher affinity towards ADI. Calculations of the substrate binding energies revealed that binding energies ΔGbind and ΔGvdw play a critical role for the differential affinities of various substrate analogues towards P. putida ADI.

Identification of leads for antiproliferative activity on MDA-MB-435 human breast cancer cells through pharmacophore and CYP1A1-mediated metabolism.

CYP1A1 is a potential target for anticancer drug development due to its overexpression in certain cancer cells and role in cancer progression. To identify new leads for CYP1A1 mediated anticancer action, we attempted ligand based pharmacophore mapping, virtual screening of databases, molecular docking, MetaSite based filtering, and molecular dynamics simulations. Initial computational and in vitro screening identified 11 compounds from which we identified two lead compounds, ZINC33468944 and ZINC32101539, showed potential antitumor activity on MDA-MB-435 cell lines (GI50 < 0.1 µM) and CYP1A1 inhibition of 0.13 and 0.3 µM, respectively. Furthermore, the lead compounds were evaluated for CYP1A1 mediated metabolism, showing N-hydroxylated metabolites, which have potential of DNA adduct formation and cause cancerous cell death. Analysis of molecular dynamics simulations provided important guidelines for the further modification of the lead compounds. Hence, we claim the lead molecules for further development in anticancer drug discovery.