Protein Ligand Binding Site Of A Flexible Development And Application System Analysis And Flexible Molecular Docking Method

Receptor-based virtual screening utilizes molecular docking method to place small molecular compounds into the receptor binding site, and rank the compounds according to their scores computed in force field-based or empirical-based scoring functions. Despite the successful applications of molecular docking, there still exists big challenges. Neglecting protein flexibility is known to cause errors in structure-based drug design. Treating binding-site flexibility in molecular docking is critical in certain cases, but not yet realized in rigorous fashion. Since protein-ligand binding will cause multiple levels of conformational change, computationally assessing ligand binding-site flexibility and reducing the sampling space are critical issues in structure-based drug design. Fortunately, the advance of the structural biology provides an opportunity for better understanding molecular recognition and improving the performance of docking programs. Here, we assembled a comprehensive, yet specialized relationship database, Flex-Site DB (free available at http://flexsite. huanglab. org. cn/flexsite), containing5,525unique ligand binding-site conformational ensembles derived from a total of30,313protein crystal structures. Flex-Site DB provides the community a rich resource for systematically investigating protein binding-site flexibility and protein-ligand interaction, including but not limited to binding-site conformational ensembles, the structural properties of binding-site residues and variety of descriptors of protein-ligand interaction. We mainly investigated the magnitude of protein conformational change at residue level via structure statistical analysis and Monte Carlo sampling method. Interestingly, many ligand binding pockets do not embody significant conformational rearrangement, especially among holo structures bound with structurally similar ligands. Remarkably, for the same residue type comparison, the binding-site residues forming hydrogen bond interaction with ligand can confer structural rigidity and low mutation propensity. It is likely that the specific protein-ligand interaction intends to be pre-organized to minimize the entropic loss upon ligand binding. Our analysis suggests that sampling a few predetermined binding-site residues may be adequate in majority of lead discovery and lead optimization applications. Nevertheless, the generated protein binding-site conformational ensembles and identified flexible residues in present study can directly facilitate our development of flexible-receptor docking algorithm.Besides exploring the residue flexibility information in Flex-Site DB to identify flexible residue in binding-site, we used the multiple "time step" Monte Carlo algorithm to sampling residue side chain conformational change without ligand and then used modified version of DOCK3.5.54for flexible molecular docking, based on "conformational selection" model. We benchmarked this method against10different proteins,20docking test cases.11out of20perform well in flexible docking while only6out of20perform well in rigid docking. We applied this method to screen the LOPAC database against PIM1(proviral integration site in Moloney) kinase, a drug target related with leukemia and prostate cancer. Interestingly, among the top ranked docking poses, a dopamine D1/D2receptor antagonist, Thioridazine, was identified to form favorable interactions with binding site residues. Subsequently, we experimentally validated Thioridazine as low micromolar inhibitor of PIM1kinase. One drug can simultaneously interact with G-protein coupled receptor and protein kinase, expanding our knowledge on drug off-target effect. In addition, we further improved our flexible docking protocol by sampling and minimizating the side chains along with the docked ligand based on "induced fit" theory. Encouragingly, after further side chain sampling and minimization, both docking poses and docking enrichment were improved. Therefore, we screened our in-house database containing200,000compounds against PIM1kinase using our improved flexible docking strategy. Finally, we selected70diverse compounds with good docking poses and will test their bioactivity.