Docking studies on MexB efflux pump inhibitors of Pseudomonas aeruginosa.pdf

MINISTRY OF EDUCATION AND TRAINING NGUYEN TAT THANH UNIVERSITY DISSERTATION FINAL REPORT SCIENTIFIC RESEARCH PROJECT OF STUDENT IN 2020 NAME OF DISSERTATION: DOCKING STUDIES ON MEXB EFFLUX PUMP INHIBITORS OF PSEUDOMONAS AERUGINOSA Code of dissertation: Author of dissertation: LE THI KHANH VY Scientific instructor: M.S Pharm PHAN THIEN VY Faculty: Faculty of Pharmacy Student’s name: LE THI KHANH VY Student’s ID: 1511541065 Class: 15DDS7B Ho Chi Minh City, October 2020 TABLE OF CONTENTS LIST OF ACRONYMS iii LIST OF FIGURE iv LIST OF TABLE vii CHAPTER LITERATURE REVIEW 1.1 Antibiotic resistance and multidrug efflux systems 1.1.1 Introduction 1.1.2 Antibiotic resistance 1.1.3 Multidrug efflux systems 1.2 Pseudomonas aeruginosa .4 1.2.1 Structure of MexAB - OprM 1.2.2 Functional rotation mechanism MexB 1.2.3 Drug efflux mechanism 10 1.3 Inhibition of MexB efflux pump 12 1.3.1 Synthetic compounds 12 1.3.2 Natural compounds 14 1.3.3 The methods determined efflux pumps inhibitors activity 15 1.4 Virtual screening 16 1.4.1 Structural - based virtual screening 16 1.4.2 FlexX software 17 CHAPTER SUBJECTS - RESEARCH METHOD 19 2.1 Subjects 19 2.1.1 Data sets .19 2.1.2 MexB pump 23 2.2 Docking study process 23 2.3 Docking molecular 24 2.3.1 Preparation of the protein 24 2.3.2 Preparation of the ligands 24 2.3.3 Binding site determination 25 2.3.4 Docking 26 2.3.5 Analyzing the docking results 26 2.4 Virtual screening 27 CHAPTER RESULT AND DISCUSSION .28 3.1 The result of preparing protein 28 3.2 The binding site result 28 3.3 Redock .32 3.4 The docking results 33 3.4.1 The first binding site (the distal pocket) 33 3.4.2 The second binding site 38 3.5 The virtual screening results 42 3.5.1 The virtual screening results of the first binding site (the distal pocket) 43 3.5.2 The virtual screening results of the second binding site 45 CHAPTER CONCLUSION AND SUGGESTION 47 4.1 Conclusion 47 4.2 Suggestion 48 REFERENCES APPENDIX Appendix-1 11 LIST OF ACRONYMS Abbreviation Explanation 2D Dimensions 3D Dimensions ABC The ATP binding cassette ABI-pp Inhibitors Pryridopyrimidine derivative EPIs Efflux pump inhibitors IMP Inner membrane protein LMNG Lauryl Maltose Neopentyl Glycol MATE The multidrug and toxic compound extrusion family MDR Multi-drug resistance MESs Multi-drug efflux systems MFS The major facilitator superfamily MIC The minimum inhibitory concentration MPC8 Concentration that decreases the MIC by 8-fold PACE The proteobacterial antimicrobial compound efflux family RND Resistance - Nodulation - Division SMR The small multidrug resistance family TM Transmembrane TMHs Transmembrane helices 111 LIST OF FIGURE Figure 1.1 Six major families of efflux pump transporters (Chapman et al., 2019)4 Figure 1.2 The overall structure of MexAB - OprM (Ding et al., 2014) Figure 1.3 MexB efflux pump structure (Sennhauser et al., 2009) Figure 1.4 Proximal pocket (red) and distal pocket (blue) in the porter domain (Ramaswamy et al., 2018) Figure 1.5 (A) Functional rotation mechanism and (B) Substrate transport pathway from the AP to the Exit Gate going through the DP in RND transporters (Ramaswamy et al., 2018) Figure 1.6 Drug access and pathway in each step of the functional rotation mechanism 10 Figure 1.7 Complex formation and drug efflux by MexAB-OprM (Tsutsumi et al., 2019) 11 Figure 1.8 2Dstructure of some synthetics EPIs 14 Figure 1.9 FlexX software 17 Figure 1.10 Incremental Construction Algorithm (Moitessier et al., 2008) 18 Figure 2.1 3D structure of 6IIA in MOE 2008.10 23 Figure 2.2 Docking study process 23 Figure 2.3 The virtual screening process 27 Figure 3.1 Chain B of the 6IIA protein in MOE 2008.10 28 Figure 3.2 Amino acids are determined at binding cavity by docking function in MOE 2008.10 28 Figure 3.3 Two binding sites determined by docking results in MOE 2008.10 29 Figure 3.4 Amino acids are determined at the first binding site 30 Figure 3.5 Three important amino acids at the first binding site 30 Figure 3.6 Amino acids are determined at the second binding site 31 Figure 3.7 Three important amino acids at the second binding site 32 Figure 3.8 Lauryl Maltose Neopentyl Glycol Molecular weight: 1,005.19 33 Figure 3.9 Amino acids are determined at the distal pocket in the docking result 34 Figure 3.10 3D & 2D poseview images of BMRJ 201616 PA4MBN (-42.49 KJ/mol) at the distal pocket 34 IV Figure 3.11 3D & 2D poseview images of BMCL_2006_14_8506_4C (-42.04 KJ/mol) at the distal pocket 35 Figure 3.12 The BMCL 2006 14 8506 BMCL_2006_14_8506_4B derivated compounds: BMCL_2006_14_8506_4C (pink); (blue); BMCL_2006_14_8506_4P (red); BMCL_2006_14_8506_4W (yellow) 35 Figure 3.13 2D poseview images of BMCL_2006_14_l 993-2004-37 and BMCL_2006_14_1993_2004_20 at the distal pocket 36 Figure 3.14 2D poseview of MCR_2017_26_414_430_PRENAG20 at the distal pocket 36 Figure 3.15 2D poseview images of BMCL-2004-14-2493-2497-32 (tetrazole substituents) and BMCL_2004_14_2493_2497_26 (carboxylic acid substituents) at the distal pocket 37 Figure 3.16 3D & 2D poseview images of BMCL_2007_15_7087_D139001 at the distal pocket 37 Figure 3.17 Acid mins are determined at the second binding site in the docking result 39 Figure 3.18 3D & 2D poseview images of BMCL-2003-13-2755-2758-9 (-29.732 KJ/mol) at the second binding site 39 Figure 3.19 3D & 2D poseview images of PLOS_2014_9_LANATOSIDE (7.72 KJ/mol) at the second binding site 39 Figure 3.20 2D poseview images of BMRJ_2016_16_PA4MBN and JMC-l999-42-4928-4931-PaPN at the second binding site 40 Figure 3.21 3D & 2D poseview images of BMCL_2007_15_7087_D 139001 at the second binding site 42 Figure 3.22 The virtual screening process .42 Figure 3.23 3D & 2D viewpose images of DB01145 (-34.32 KJ/mol) at the distal pocket 43 Figure 3.24 3D & 2D viewpose images of TCM4613 (-27.92 KJ/mol) at the distal 44 pocket Figure 3.25 3D & 2D viewpose images of TCM18451 (-31,87 KJ/mol) at the distal pocket 44 V Figure 3.26 3D & 2D viewpose images of DB07325 (-21.75 KJ/mol) at the second 45 binding site Figure 3.27 3D & 2D viewpose images of TCM4613 (-19.63 KJ/mol) at the second binding site 46 Figure 3.28.2D & 3D viewpose images of TCM18471 (-21.03 KJ/mol) at the second binding site 46 VI LIST OF TABLE Table 2.1 Potential compounds, which were tested by in vitro biological assay (MIC, MPC8) 19 Table 2.2 Set A, created from the agent have to inhibit the transport of an efflux pump substrate 20 Table 3.1 Interaction of Set A at the first pocket 30 Table 3.2 Interaction of Set A at the second pocket 32 Table 3.3 10 compounds have good docking score at the distal pocket 33 Table 3.4 10 compounds have good docking score at the second binding site 38 Table 3.5 Comparing docking score at two binding of BMCL 2006 14 8506 derivatives 41 vii CHAPTER LITERATURE REVIEW 1.1 Antibiotic resistance and multidrug efflux systems 1.1.1 In traduction Antibiotics have been used extensively during several decades and we are now facing the emergence of multidrug resistant strains Furthermore, multidrug - resistant (MDR) in Gram - negative pathogens, poses a significant threat to our ability to effectively treat infections caused by organisms The inability of existing therapies to treat multidrugresistant pathogens has been recognized as an important challenge of the 21st century A major component in the development of the MDR phenotype in Gram-negative bacteria is overexpression of Resistance - Nodulation - Division (RND) - type efflux pumps, which actively pump antibacterial agents and biocides from the periplasm to the outside of the cell Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of multidrug resistance bacteria (Reza et al., 2019) A particularly problematic pathogen in the clinical setting is Pseudomonas aeruginosa (P aeruginosa), an opportunistic Gram - negative pathogen characterized by intrinsic resistance to a wide variety of antimicrobial agents At present, MexAB - OprM play a very important role in extruding antibiotics from the cell in p aeruginosa It is widely overexpressed in clinical isolates and contributes to the resistance of wild - type strains to antibiotic drugs MexAB - OprM exhibits the broadest substrate specificity of all known multidrug efflux system of p aeruginosa The list of substrates includes various antimicrobial agents (Chloramphenicol, Macrolides, B-lactams, Trimethoprim, older and newer Fluoroquinolones), dyes (Acriflavine, Acridine orange, Crystal violet, and Ethidium bromide), detergents, tea tree oil components (a-terpineol), organic solvents, and triclosan (Lamut et al., 2019) In addition, elimination of RND pumps in p aeruginosa by inhibition with a potent efflux pump inhibitor decreases the frequency of resistance to Levofloxacin (Griffith et al., 2001) With the development of science and technology, computational therapeutics is a dynamic and rapid growing in the global drug research and development because these method can save a lot of time and money So that, the direction of research is - Building database of MexB efflux pump inhibitors (EPIs) in p aeruginosa from scientific research articles; - Building a molecular docking based on the X - ray crystal structure of MexB from p aeruginosa; - Analyzing and giving the connection between structure and binding potentiation; - Screening and finding out potential EPIs from Drugbank, ZINC, Traditional Chinese Medicine, Natural plant 1.1.2 Antibiotic resistance Antibiotic resistance has become a serious threat to human health (Organization, 2017) According to an exhaustive review commissioned by the UK government, globally, approximately 700,000 deaths can be attributed to antibiotic resistance each year Furthermore, based on increasing incidence of drug resistance among bacterial infections, this toll is expected to exceed 10 million by 2050, at a cumulative global cost of 100 trillion US dollars Additionally, global surveillance data from the WHO reported a widespread occurrence of antibiotic resistance among 500,000 people with suspected bacterial infections across 22 countries Moreover, resistance to penicillin, P-lactam antibiotic widely used for decades to treat a range of different bacterial infections, was reported amongst 51% of the surveyed countries (Reza et al., 2019) p aeruginosa is an opportunistic pathogen that is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals Eradication of p aeruginosa has become increasingly difficult due to its remarkable capacity to resist antibiotics Strains of p aeruginosa are known to utilize their high levels of intrinsic and acquired resistance mechanisms to counter most antibiotics (Pang et al., 2019) Antibiotic resistance p aeruginosa accounts for 13-19% of healthcare-associated infections each year in the US The increasing level of resistance in antibiotic resistance p aeruginosa is often attributed to patient-to-patient transmission of resistant strains as well as newly acquired resistance owing to previous antibiotic ... from more than one family and / or more than one type of efflux pump belonging to the same family Furthermore, efflux pumps can consist of either single components, namely Nor A of s aureus, or... membrane On top of that, a synergy between a low-level expression of the porins and an increase of the efflux pump? ??s activity leads to a very efficient expulsion of the antibiotics outside of the... a functional rotation mechanism When the concentration of drugs in this tunnel becomes higher than their concentration outside the cell, they diffuse out of the cell via the concentration gradient