Docking study of molecular mechanism behind the quercetin inhibition of Mycobacterium tuberculosis urease

Abstract

Introduction. Mycobacterium tuberculosis urease (MTU), being an important factor of the bacterium viability and virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of urease activity. However, known urease inhibitors (phosphorodiamidates, hydroxamic acid derivatives and imidazoles) are toxic and/or unstable, that prevent their clinical use. Therefore, the development of novel efficient and safe MTU inhibitors is necessary. To achieve this goal, we have chosen flavonoid quercetin as a scaffold to develop new MTU inhibitors. Methods.Homology modeling. The target amino acid sequence of M. tuberculosis H37Rv urease was taken from GenBank at NCBI. Homology model of M. tuberculosis urease was built as described earlier by using molecular modeling program YASARA Structure. Amongst the top-scoring templates, five high-resolution X-ray structures were selected For each template five stochastic alignments were created and for each alignment a three-dimensional model was built. Each model was energy minimized with explicit water molecules using Yasara2 force field, and the models were ranked by quality Z-score. From these 25 three-dimensional models obtained, there was selected a model based on the template X-ray high-resolution structure for S. pasteurii urease (5G4H) which contained the flap in open state and had the highest quality score amongst the nonamer structures (i.e. (αβγ)₃ macromolecular ensembles). Search of inhibitor binding sites on the surface of MTU. The search of inhibitor binding sites on the surface of MTU was carried out by two steps. At first step, we used computational solvent mapping method FTSite to identify a ligand binding sites on MTU surface. At second step, docking of quercetin on MTU surface by AutoDock Vina implemented in YASARA Structure was carried out within the ligand binding sites revealed by FTSite. Mapping of protein surface by FTSite method.Computational solvent mapping method FTSite was used through the online server. FTSite server outputs the protein residues delineating the first three binding sites. Molecular docking by AutoDock VINA. Docking of quercetin on the surface of M. tuberculosis urease by AutoDock VINA was carried within the binding sites previously revealed by FTSite server. Docking was performead by using default parameters within a cubic 30Å × 30Å × 30 Å simulation cell centered on S atom of Cys 532 residue. The M. tuberculosis urease structure was kept rigid while the ligand structure was flexible. The best hit of 25 runs having the lowest binding energy was chosen as a final binding pose. An analysis of molecular interactions and a representation of the results by molecular graphics were done by YASARA Structure, LigPlot+ and PyMol. Results and discussion. In the best binding pose, quercetin molecule is situated deep in the MTU cavity which leads to the active site channel and near the active site flap. The circle B of quercetin is directed to the active center, while the circle A is directed towards the exit from the cavity. Binding energy and dissociation constant of quercetin complex with urease is 8.7 Kcal/mol and 0.4 µМ, correspondingly. Ligand efficiency equals 0.4. The binding of quercetin is provided by tight van der Waals contacts with eleven residues two of which (Cys 532 andHis 533) belong to the active site flap modulating transit of substrate and products of catalysis through the active site channel. The binding of quercetin is additionally stabilized by six hydrogen bonds with residues Glu 376, Lys 379, Thr 380, Gly 490 and Ala 576. These intermolecular interactions (through the tight contact with flap residues Cys 532 and, especiallyHis 533) cause steric hindrance for the flap transition from open to closed conformation thus fixing it in the open state that blocks catalysis. Our model of quercetin binding to MTU corresponds to the results of Xiao Z.-P. et al. which showed by enzyme kinetics and molecular docking that quercetin is a noncompetitive inhibitor of Helicobacter pylori urease and it is positioned near the active site flap as well blocking it in the open conformation. As well, our model of quercetin binding to urease corresponds to the results of MacomberL. et al. which showed by docking that quercetin binds to the flap region of Klebsiella aerogenes urease. However, our model disagrees with the proposed general mechanism of urease inhibition by aromatic poly-hydroxylated inhibitors through the covalent binding with Cys residue of the flap covering the active site. It may be a consequence of the limitation of molecular docking methods used in our study that can explore only non-bound interactions. Conclusions. Because of the absence of experimental structure of M. tuberculosis urease its homology model was built and used in further studies of ligand-urease interactions. It was revealed that quercetin molecule is situated in the MTU cavity leading to the active site channel, near the active site flap. The binding of quercetin is provided by van der Waals contacts with eleven residues and by six hydrogen bonds with urease residues. Based on the analysis of peculiarities of quercetin binding with MTU, molecular mechanism of MTU inhibition by quercetin was proposed. The model of quercetin binding with MTU corresponds well to the results of docking studies on quercetin binding to Helicobacterpylori and Klebsiella aerogenes ureases. The results obtained expand the knowledge on the molecular mechanisms of urease inhibition and contribute to the development of new anti-tuberculosis immunomodulators.