Speaker
Mr
Branko J. Drakulić
(Department of Chemistry-IChTM, University of Belgrade, Njegoševa 12, Belgrade, Serbia)
Description
Cysteine proteases involved in degradation of proteins, widespread in plants, parasites and vertebrates, are an important medicinal chemistry target implicated in the diseases ranging from immunological processes to cancer. Due to conserved cysteine residue in their active site, unsaturated ketones and their analogs represent one of the major chemotypes used for inhibitors design [1]. In this communication the dynamics of the papain-like cysteine protease isolated from the fruit, uninhibited and inhibited with covalent inhibitor E-64 (Scheme 1), on non-physiological pH, were reported merging results and experiences from our biochemical and medicinal chemistry laboratories.The aim of our study is to explain some experimental findings. Proteins are modeled using similar ones with the known 3D structure, taken from Protein Data Bank [2]. After sequence alignment residues that differentiate templates from the experimental proteins were manually changed. Afterward the eventual existence of close contacts, bumps or similar was carefully checked. The protonation states of the aminoacid residues and the inhibitor ionizable groups were adjusted to pH 1.5, using empirical function [3]. Systems were neutralized with explicit counterions, than embed in explicit water, obtaining the sphere having ~ 100 Å radius. Systems under simulations were minimized during 30000 steps, than heated to 300 K during 10000 steps. After equilibration, the 5 ns unconstrained and unbiased molecular dynamics simulation, on 300 ± 10 K, were performed on the each system. CHARMm22 force field and Geisteiger charges were used. Electrostatics was treated by Particle Mesh Ewald method. The periodic boundary conditions were applied, and 12 Å cut-off (8 Å switching), with pair list distances set to 13.5 Å. Each simulation was performed in duplicate, using different random seeds and giving comparable results. The root-mean-square deviation of the backbone atoms and the energy profiles of the systems under the study proved stable, converged simulation. The movement of the loops and the (flexible) inhibitor, as well as radius of gyration of the selected amino-acid side chains was analyzed and conclusion derived on the influence of the covalently bound inhibitors on the dynamics of the enzyme on pH 1.5. All calculations were performed by NAMD 2.8 [4] on the multimode Linux cluster. For the preparation of the systems, and analysis of the results VegaZZ 2.4.0 were used [5].
Acknowledgement: The work is supported by the European Commission under EU FP7 project HP-SEE, http://www.hp-see.eu/. The Ministry of Education and Science of Serbia support this work. Grants 172035 and 172049.
References: [1] Mini-Rev. Med. Chem. 7 (2007) 1040; [2] J. Mol. Biol. 112 (1977) 535; [3] Proteins 73 (2008) 765; [4] J. Compt. Chem. 26 (2005) 1781; [5] J. Comp. Aided Mol. Des. 18 (2004) 167
Primary author
Mr
Branko J. Drakulić
(Department of Chemistry-IChTM, University of Belgrade, Njegoševa 12, Belgrade, Serbia)
Co-author
Dr
Marija Gavrović-Jankulović
(Faculty of Chemistry, University of Belgrade, Studentski Trg 12-16, Belgrade, Serbia)
