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Chikungunya virus nsP2 replication protein is a cysteine protease, which cleaves the nonstructural nsP1234 polyprotein into functional replication components. The cleavage and processing of nsP1234 by nsP2 protease is essential for the replication and proliferation of the virus. Thus, ChikV nsP2 protease is a promising target for antiviral drug discovery. In this study, the crystal structure of the C-terminal domain of ChikV nsP2 protease (PDB ID: 4ZTB) was used for structure based identification and rational designing of peptidomimetic inhibitors against nsP2 protease. The interactions of the junction residues of nsP3/4 polyprotein in the active site of nsP2 protease have been mimicked to identify and design potential inhibitory molecules. Molecular docking of the nsP3/4 junction peptide in the active site of ChikV nsP2 protease provided the structural insight of the probable binding mode of nsP3/4 peptide and pigeonholed the molecular interactions critical for the substrate binding. Further, the shape and pharmacophoric properties of the viral nsP3/4 substrate peptide were taken into consideration and the mimetic molecules were identified and designed. The designed mimetic compounds were then analyzed by docking and their binding affinity was assessed by molecular dynamics simulations.

The human protozoan pathogens possess the essential metalloenzyme arginase (Arg) which catalyses the catabolism of l-arginine to l-ornithine and urea. This being the first committed step in polyamine biosynthesis is a potential drug target for protozoan diseases. In pathogenic organisms, arginase plays a crucial role in depleting host l-arginine, a substrate for nitric oxide synthase (NOS) that participates in protective immunity, thereby evading host immune response. In this study, the metal binding spectrum of EhArg has been determined. This study focuses on the biochemical and biophysical characterization of arginase from Entamoeba histolytica (EhArg), majorly characterizing the bivalent metal selectivity and metal binding kinetics of purified EhArg using Surface Plasmon Resonance and inductively coupled plasma mass spectroscopy. Investigation of the active site chemistry and total metal content using molecular docking and ICP-MS unraveled the fact that two Mn2+ ions are required for the enzyme to be fully functional. However, chelating loosely bound Mn2+ and replacing it with a variety of bivalent metal ions including Mg2+, Zn2+, Ni2+, Hg2+, Cu2+, Co2+, Ca2+ and Cd2+ retains its enzymatic activity. Further, the role of nine bivalent ions in the activation of EhArg was studied thermodynamically and biochemically. Phylogenetic and sequence analysis and oligomerization studies of EhArg show that unlike other eukaryotic arginases, EhArg exists in monomeric and dimeric form in solution and shows the highest similarity with bacterial arginase. This study unveiled interesting facts about EhArg that the enzyme has evolved to utilize available metal ion cofactors and survive the inhospitable environment within the host.