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2011 International Conference on Bioscience, Biochemistry and Bioinformatics IPCBEE vol.5 (2011) © (2011) IACSIT Press, Singapore Virtual Screening of Potential Drug-like Inhibitors against MexB Efflux Protein from Pseudomonas Aeruginosa Mohanalakshmi N. Aparna V. Junior Research Fellow, Dept. of Bioinformatics School of Bioengineering, SRM University Kattankulathur, Tamilnadu, India [email protected] Junior Research Fellow, Dept. of Bioinformatics School of Bioengineering, SRM University Kattankulathur, Tamilnadu, India [email protected] Waheeta Hopper Professor & Head, Dept. of Bioinformatics, School of Bioengineering, SRM University Kattankulathur, Tamilnadu, India [email protected] contributes to both intrinsic and acquired resistance in P. aeruginosa, while the MexCD-OprJ and MexEF-OprM efflux system contributes to acquired resistance. The inner membrane component MexB is responsible for the substrate specificity and uses proton electrochemical gradient to transport its substrates and virulence factors that are important for the colonization and infection of host cells [6]. A widely exploited strategy is the development of inhibitors of efflux pumps [7], which are intended for combination therapy with antibiotics. Inhibition of efflux pumps provides a better way to restore the activity of antibiotics. To date, no efflux pump inhibitors have been approved for the treatment of bacterial infections [6], hence this area affords promising role in drug development. The aim of this study was to elucidate the mode of binding of substrates to the efflux pump and also to identify the efflux inhibitory potential of compounds with structural similarity to the known inhibitor MC-207,110 using virtual screening and molecular docking approaches. Abstract—Prevalence of multidrug resistant Pseudomonas aeruginosa strains in clinical setup demands the need for developing potent drugs against efflux mechanisms. In this study, drug-like molecules with structural similarity to the known efflux inhibitor were used in order to screen for potential inhibitors. A total of 167 compounds were screened and the Glide XP shortlisted compounds were selected based on Glide score and interaction with active site residues. The virtual screening method was also applied to study the binding modes of substrates. The shortlisted compound could have a better ability to inhibit the MexB protein, hence will provide adjunctive therapy t o the antibiotics. Keywords-Pseudomonas aeruginosa; efflux pump; MC-207, 110; Glide I. virtual screening; INTRODUCTION Toxic compound efflux from cells is a general mechanism that bacteria have developed to protect themselves against the adverse environments. Efflux pumps are proteins involved in the transfer of toxic compounds from the cell which confers the resistance to drug and a wide variety of structurally unrelated compounds [1]. Pseudomonas aeruginosa is a notoriously difficult organism to control with antibiotics or disinfectants [2]. The organism’s intrinsic resistance has been attributed to the outer membrane, a barrier of limited permeability [3]. While the reduced uptake likely does limit access of antimicrobials to their targets within the cells, additional resistance mechanisms such as drug efflux, helps to reduce the threshold concentration of antibiotics within the cells [4]. The efflux pumps of P. aeruginosa belong to Resistance Nodulation Cell Division (RND) family which forms tripartite system consisting of three components: a transporter (efflux) protein in the inner membrane (MexB), a periplasmic accessory protein, (MexA) and an outer membrane protein (OMP) channel (OprM) [5]. MexABOprM is the first identified tripartite system [6], which II. METHODS A. Receptor X-ray structure The X-ray crystal structure of the inner membrane protein MexB (PDB code: 2V50) (Fig.1), in complex with non-ionic detergent n-dodecyl-D-maltoside resolved at 3.0Å was downloaded from Protein Data Bank (PDB) [8]. The asymmetric unit contained two structurally identical trimers, subunits ABC and DEF. Among the three chains of the MexB structure the B subunit was considered as the binding subunit [9]. B. Protein preparation PDB files of the protein may have various problems that need to be corrected before proceeding with Virtual Screening studies hence bond orders, ionization states, steric clashes and side chain orientations were fixed and energy minimization was performed with the force field Optimized 388 potential for liquid simulations 2005 (OPLS) using protein preparation wizard of Schrödinger suite version 9 [10]. The active site of the protein was defined and grid files were generated using receptor grid generation panel. Grid files represent physical properties of a volume of the receptor (active site) that are searched when attempting to dock a ligand. bonding mode of the inhibitors and their structurally similar compounds were compared with those of the substrates. III. C. Substrates of MexB The MexB efflux pumps of P. aeruginosa are of particular interest because of their exceptionally broad substrate specificity. The following reporter antibiotics were used as phenotypic markers of the Mex efflux pumps of interest: carbenicillin, erythromycin, levofloxacin, Fig.2 [11]. In order to compare the binding modes of the inhibitors with the substrates, docking was perfumed with the substrates like carbenicillin, erythromycin, levofloxacin using glide module of Schrodinger suite v 9. D. Ligand Database MC-207,110 (Fig.2) has been identified in Highthroughput screening of small molecule libraries as an inhibitor of the three multi-drug resistance (MDR) pumps (MexAB-OprM, MexCD-OprJ and MexEF-OprN) in clinical isolates of P. aeruginosa [12]. Similarities in structures are indicative of similarities in bioactivities, therefore, structure based searching of PubChem database (>85% similarity) was carried out. This resulted in 167 compounds. The Structure files of these compounds were obtained from PubChem Compound Database [13]. The geometries of these compounds were optimized by adding hydrogens and eliminating unwanted structures using LigPrep module and Database of chemical compounds was created using Schrodinger suite. E. Virtual Screening studies for substrates The virtual screening of substrate database with prepared protein was performed with OPLS2001 force field using virtual screening workflow (VSW) module of the Schrodinger Suite. VSW uses glide docking to rank the best compound which utilizes the scoring functions, High throughput virtual screening (HTVS), standard precision (SP) and extra precision (XP). HTVS and SP modes are used for large set of ligands and XP docking is more accurate than the above two methods. It uses the ligand poses that have a high score from SP docking. The XP GlideScore scoring function was used to order the best ranked compounds and the specific interactions like pi-cation and pi-pi stacking were analyzed using XP visualizer in Glide module. The binding mode and interactions of the substrates were ascertained to compare with the inhibitors. RESULT AND DISCUSSION Computer based strategies for structure based drug discovery presents a valuable alternative to the costly and time consuming process of random screening. In the present study, two different virtual screening approaches were used. First, MC-207,110 analogues were docked into the active site of MexB protein. For the validation of docking process, the substrates of the MexB were introduced as a control with the purpose of screening the compounds with docking score greater than the substrate. The docking of the substrates to the active site is shown in Fig.3a-c, the substrates carbenicillin, erythromycin and levofloxacin exhibited molecular docking with Glide score value of -4.271, -3.672, -3.507 and -3.025 (kcal/mol) respectively. The substrates docked near the vicinity of the amino acids Thr495, Gly461, Arg468 and Gly97 and were observed be form hydrogen bond (Table I). Out of the 167 compounds virtually screened, the shortlisted molecule [(2R)-2,5-diamino-N-[(2S)-3-(4methylphenyl)-1-(naphthalen-2-ylamino)-1-oxopropan-2yl]pentanamide (Pubchem ID: 21970537)] was obtained from the XP output based on its good interaction, forming hydrogen bonds with the active site residues Arg468, Ala37, Gln96 and Glide score of -5.344 (Fig. 4 and Table II). Hydrogen bonding measures the intermolecular interaction between the protein and ligands. The compound possessed 1.78 score in the hydrophobic map prediction, hppmap tool of Schrodinger suite (Fig. 5) proved its strong binding and interaction. The shortlisted inhibitor docked with better Glide score than the substrates. However hydrogen bonding with the active site residue Arg468 was observed dominant among both the inhibitor and substrate. It is known that MC-207,110 is a competitive efflux inhibitor which competes for the binding sites in the receptor with the substrates [14]. IV. CONCLUSION Combination therapy is a significant approach to combat drug resistance in bacteria. Efflux pump inhibitors can serve as an additional drug to alleviate the activity of the existing antimicrobials. In this study, molecular docking approach was imparted to study the binding modes and to correlate the docking score between substrates and inhibitors of efflux pump MexAB-OprM. The docking studies provide better insights onto the binding mechanism of substrate and inhibitor at the molecular level. The virtually screened and shortlisted compound could act as a potential competitive inhibitor with the substrate for MexAB-OprM efflux pump. ACKNOWLEDGMENT F. Virtual Screening studies for inhibitors The ligand based virtual screening of inhibitor compound database with prepared protein was performed with OPLS2001 force field using virtual screening workflow (VSW) module of the Schrodinger Suite. The same protocol used for the substrates was followed. The interactions and The authors are thankful to Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India for their financial assistance and SRM University for their support. 389 REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] L. E. Lawrence, and J. F. Barrett, “Efflux pumps in bacteria: overview, clinical relevance, and potential pharmaceutical target,” Exp. Opin. Invest. Drugs. vol. 7, 1998, pp. 199–217. R. E. Hancock, “Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria,” Clin. Infect. Dis. vol. 1, 1998, pp. S93–99. H. Nikaido, “Outer membrane barrier as a mechanism of antimicrobial resistance,” Antimicrob. Agents Chemother. vol. 33, 1989, pp. 1831-1836. K. Poole, K. Krebes, C. McNally and S. Neshat, “Multiple antibiotic resistance in Pseudomonas aeruginosa:evidence for involvement of an efflux operon,” J. Bacteriol. vol. 75, 1993, pp. 7363–7372. M. A. Webber, L. J. V. Piddock, “The importance of efflux pumps in bacterial antibiotic resistance,” J. Antimicrob. Chemother. vol. 51, 2003, pp. 9–11. L. J. Piddock, “Multidrug-resistance efflux pumps—not just for resistance,” Nat. Rev. Microbiol. vol. 4, 2006, pp. 629–636. J. M. Pages, L. Amaral, “Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria,” Biochim. Biophys. Acta. vol. 1794, 2009, pp.826-833. H. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, “The protein data bank,” Nucleic Acids Res. vol. 28, 2000, pp. 235–42. G. Sennhauser, M.A. Bukowska, C. Briand and M. G. Grütter, “Crystal Structure of the Multidrug Exporter MexB from Pseudomonas aeruginosa,” J. Mol. Biol. vol. 389, 2009, pp. 134–145. Schrödinger, LLC, New York, NY, 2010, (http://www.schrodinger.com/) N. Mesaros, Y. Glupczynski, L. Avrain, N. E. Caceres, P. M. Tulkens and F. Van Bambeke, “Expand+A combined phenotypic and genotypic method for the detection of Mex efflux pumps in Pseudomonas aeruginosa,” Journal of Antimicrobial Chemotherapy, vol. 59(3), 2007, pp. 378-386. M. S. Warren, J. C. Lee, A. Lee, K. Hoshino, H. Ishida, O. Lomovskaya, “An Inhibitor of the Mex Pumps from Pseudomonas aeruginosa is also a Substrate of these Pumps,” Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother, vol. 40, Sep. 2000 pp. 207. PubChem Database: http://www.ncbi.nlm.nih.gov/pccompound A. Mahamoud, J. Chevalier, S. Alibert-Franco, W. V. Kern and J. M. Pagès, “Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy,” Journal of Antimicrobial Chemotherapy, vol. 59(6), 2007, pp.1223-1229. TABLE I. D--H---A Pubchem ID: (Arg468)N--H---O (Ala37) N--H---O N--H---O (Ala37) N--H---O (Gln96) 21970537 Distance (Å) 2.883 3.128 2.891 2.733 Glide Score (Kcal/mol) -5.344 Figure 1. Structure of MexB protein from P. aeruginosa INTERACTION OF ANTIBIOTIC SUBSTRATES OF MEXABOPRM EFFLUX PUMP WITH MEXB PROTEIN D--H---A Distance (Å) Glide Score (Kcal/mol) Carbenicillin O--H---O(Thr495) (Gly461)N--H---O 2.629 2.620 -4.271 Erythromycin (Arg468)N—H--O 2.877 -3.672 Levofloxacin (Gly97) N--H---O 3.081 -3.507 Substrates TABLE II. Inhibitor Figure 2. Structure of MC-207,110, an inhibitor of MexB efflux pump and substrates INTERACTION OF XP SHORTLISTED COMPOUD (PUBCHEM ID-21970537) WITH MEXB PROTEIN 390 Figure 3. a. Interaction of MexB (B-chain) with carbenicillin (The substrate is in ball and stick model and the amino acids in lines) Figure 4. Interaction of MexB (B-chain) with XP docked inhibitor PubChem ID: 21970537 (in ball and sticks) Figure 4. b. Interaction of MexB (B-chain) with erythromycin Figure 5. Hydrophobic map of XP output for the inhibitor showing the strong hydrophobic patches (blue) Figure 3. c. Interaction of MexB (B-chain) with levofloxacin 391