3D pharmacophore model studies on ACRB efflux pump inhibitors of Escherichia coli.pdf

MINISTRY OF EDUCATION AND TRAINING NGUYEN TAT THANH UNIVERSITY -0O0 DISSERTATION FINAL REPORT SCIENTIFIC RESEARCH PROJECT OF STUDENT IN 2020 NAME OF DISSERTATION: 3D- PHARMACOPHORE MODELS STUDIES ON ACRB EFFLUX PUMP INHIBITORS OF ESCHERICHIA COLI Code of dissertation: Supervisor of dissertation: TRAN THUY VY Scientific instructor: M.S.Pharm PHAN THIEN VY Faculty: Faculty of Pharmacy Student’s name: TRAN THUY VY Class: 15DDS6B Student ID: 1511541131 Ho Chi Minh City- 2020 TABLE OF CONTENTS LIST OF ACRONYMS LIST OF FIGURE LIST OF TABLE CHAPTER LITERATURE REVIEW 1.1 Antibiotic resistance .7 1.1.1 Antibiotic resistance crisis I.1.2 Multi- drug efflux systems .7 1.2 E.Coli AcrAB-Toic efflux pump 10 1.2.1 Structure of E coll AcrAB-TolC efflux pump 10 1.2.2 Substrates and mechanism of efflux pump 10 1.3 Inhibition of AcrAB-TolC efflux pump 11 1.3.1 Synthetic compounds 11 1.3.2 Natural compounds 12 1.4 Methods of the efflux pump inhibitors assay 14 1.4.1 Efflux pump inhibitors ability assay 14 1.4.2 Binding affinity assay 15 1.5 Virtual screening 16 1.5.1 3D-Pharmacophore definition 16 1.5.2 Building 3D-Pharmacophore model by MOE 2008.10 17 1.6 Pharmacophore studies on E coli inhibitors 20 CHAPTER SUBJECTS - RESEARCH METHOD 21 2.1 Data sets .21 2.1.1 Training set 21 2.1.2 Testing set 22 2.2 3D-Pharmacophore method .23 2.2.1 Preparation of the data 23 2.2.2 Energy minimization 23 2.2.3 Conformation Import 25 2.2.4 Building 3D-pharmacophore model 25 2.2.5 Applying queries on testing set 26 2.2.6 Validation pharmacophore query 26 2.3 Virtual screening 28 CHAPTER RESULT AND DISCUSSION 30 3.1 Building 3D- Pharmacophore Models 30 3.2 Validation models 31 3.3 Discussion 36 3.3.1 Doroxubicin 36 3.3.2 Lanatoside c .38 3.3.3 MBX2319 .39 3.3.4 CCCP and PA/3N 40 3.4 Screening on TCM database 41 3.5 Screening on Drugbank 44 3.6 Screening on Chromolaena odorata Asteraceae, Solarium torvuni Solanaceae, Vernonia amygdalina Asteraceae and Glinus oppossitiflius Molluginaceae 47 CHAPTER CONCLUSION AND SUGGESTION 52 4.1 Conclusion 52 4.2 Suggestion 52 REFERENCE APPENDIX AP-1 LIST OF ACRONYMS Abbreviation Explanation 3D Dimension Acc2 H-bond acceptor projection Aro Aromatic center EPIs Efflux pump inhibitors FP Fluorescence polarization Hyd Hydrophobic centroid ITC Isothermal titration calorimetry MATE The multidrug and toxic compound extrusion MDR Multi-drug resistant MES Multi-drug efflux systems MFS The major facilitator MIC Minimum inhibitory concentration MOE Molecular Operating Environment MPC4 The value of minimal concentration of an EPIs required to decrease the MIC of an antibiotic by 4-fold PACE Proteobacterial Antimicrobial Compound Efflux PH4 Pharmacophore PiR Pi ring center RMSD Root mean square deviation RND The resistance-nodulation-division family SMR The small multidrug resistance SPR Surface plasmon resonance TCM Traditional Chinese Medical database WHO World Health Organization LIST OF FIGURE Figure 1.1 Six superfamilies of efflux pumps found in bacteria Figure 1.2 Proposed model of the AcrA-AcrB-TolC complex 11 Figure 1.3 Structure of some synthetic efflux pump inhibitors 12 Figure 1.4 The methodology flow of the Pharmacophore Elucidation application 19 Figure 2.1 The pharmacophore process .24 Figure 2.2 The virtual screening process .28 Figure 3.1 Pharmacophore models 33 Figure 3.2 Pharmacophore models PH4_4, PH4_5, PH4_8, PH4_11 with their distances 34 Figure 3.3 Top three pharmacophore models of inhibitors of AcrB with BMC-2015_23_92024_22k 36 Figure 3.4 Ligand interaction of 4DX7 (AcrB in complex with doxorubicin) 37 Figure 3.5 Pharmacophore model with the best Lanatoside c conformations (having the lowest RMSD) (A)Lanatoside c structure (B) HHHa model with Lanatoside c (C) HHaa model with Lanatoside c 39 Figure 3.6 Pharmacophore model with the best MBX2319 conformation (having the lowest RMSD) (A) MBX2319 structure, (B) PH4-11 with MBX2319 40 Figure 3.7 Virtual screening result of TCM 42 Figure 3.8 Pharmacophore models with the best Nagarine conformations 44 Figure 3.9 Virtual screening result of Drugbank 45 Figure 3.10 Pharmacophore models with the best Dexamethasone isonicotinate conformations 47 Figure 3.11 Pharmacophore models with the best S-2 conformations 51 LIST OF TABLE Table 2.1 Table 2.2 Table 3.1 Table 3.2 Table 3.3 Table 3.4 Table 3.5 Training set 22 Testing set 23 Pharmacophore model with descriptions 30 11 best pharmacophore modelsof AcrB inhibitors with descriptions 32 Virtual screening on TCM 43 Virtual screening on Drugbank 46 Virtual screening on extracted compounds 48 CHAPTER 1.LITERATURE REVIEW 1.1 Antibiotic resistance 1.1.1 Antibiotic resistance crisis An antibiotic is a type of antimicrobial substance active against bacteria With the discovery of penicillin and streptomycin in the early 20th century, we entered the antibiotic era when about half of the antibiotics in use today were discovered However, overuse and overprescribing of antibiotics accelerated the evolution of bacteria, resulting in selection of antibiotic-resistant bacteria (Sharma et al., 2019) Antibiotic resistance is the ability of microorganisms, such as bacteria, viruses, fungi or parasites to grow in the presence of a drug that would normally kill them or limit their growth As a result, normal treatments become ineffective Infections therefore become more serious, leading to longer illness, higher treatment cost and greater risk of death Because of antibiotic resistance, a growing number of common infections, such as pneumonia, urinary tract infections, tuberculosis and food-borne illness are becoming harder and sometimes impossible to treat The World Health Organization (2014) has classified antibiotic resistance as a widespread "serious threat is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country" 1.1.2 Multi- drug efflux systems Bacteria develop resistance to antibiotics through mechanisms: reducing the permeability of the outer membrane to avoid the antibiotic entry into the bacterial cell, modifying the molecular targets of the antibiotics so that they can no longer act on them, enzymatic modification of antibiotics to inactivate them, bacterial plasmids spreading antibiotic resistance genes and expression of efflux pumps to pump out antibiotics from the cellular environment (Blair et al., 2015) Efflux pump is one of the major mechanisms allowing the microorganisms to regulate their internal environment by removing toxic substances and antimicrobial agents There are six major families of efflux transporters: the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, the resistance-nodulationdivision (RND) family, the small multidrug resistance (SMR) family, the major facilitator superfamily (MFS), and the multidrug and toxic compound extrusion (MATE) family In addition to these carriers, another efflux protein has been described in Acinetobacter baumannii (A baumannii) called Proteobacterial Antimicrobial Compound Efflux (PACE) superfamily PACE is structurally close to the SMR family (Seukep et al., 2019) Efflux pumps ATB I _ External membrane OMP ToicZotxM MATE SMR PACE MFS ABC RND Cyloptavn Figure 1.1 Six superfamilies of efflux pumps found in bacteria: MATE, SMR, MFS, ABC, RND and PACE (Seukep et al., 2019) The MFS is the largest superfamily that facilitates transport of diverse molecules like sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria and eukaryotes (Pao et al., 1998) The MFS proteins consist of a sequence of 400-600 amino acids in their primary structure arranged in 12 or 14 membrane-spanning a-helical Widely found in the two groups of bacteria, they are mainly involved in the uniport, the antiport and the symport of several substances They can adopt a tripartite pump structure in Gram negative bacteria due to the presence of the outer membrane (Seukep et al., 2019) However, this family is the main vector of Gram-positive bacteria NorA, QacA, and QacB from s aureus and LmrP from Lactococcus lactis are the well- characterized MFS pumps in Gram-positive bacteria involved in MDR Moreover, some other examples including CraA in A baumannii, MdfA in E coll, KpnGH in K pneumoniae mediated resistance to several antibiotics comprising chloramphenicol, norfloxacin, tetracycline, ceftazidime, cefepime, and streptomycin (Seukep et al., 2019) Compared to other carriers, SMR transport proteins are the smallest (100-120 amino acids and transmembrane helices) This family has three subclasses: small multidrug pumps, groEL mutation proteins suppressor, and paired SMR proteins The first, small multidrug pumps, has been identified as having the ability to confer MDR in both groups of bacteria from the expression of a single gene The EmrE protein from E coli is the best characterized SMR model, consists of 110 amino acid residues, and usually extrudes acriflavine and quaternary ammonium compounds Other well-known models of the SMR efflux transporters include SepA and QacC from s aureus and EbrAB from Bacillus subtilis (Seukep et al., 2019) Active multidrug efflux processes, usually involving secondary transporters belonging to the major facilitator superfamily, small multidrug resistance family, and RND superfamily, are now known to be important, especially in the baseline or intrinsic resistance of many bacteria to antimicrobial agents More recently, a new family, the multidrug and toxic compound extrusion MATE family, has been discovered , but its contribution to drug resistance has been known only for a few isolated cases (Fernandez et al., 2012) Although efflux pumps are present in both Gram positive and Gram-negative, the resistance in Gram-negative is more complicated because the cell of former group are surrounded by an extra membrane layer, the outer membrane which acted as a general barrier for the influx of agents Efflux pumps of the RND superfamily, such as AcrB of Escherichia coli (E.coli) and MexB of Pseudomonas aeruginosa (P aeruginosa) play an important role in producing multidrug resistance (both intrinsic and elevated) in Gram-negative bacteria This is because these pumps become associated with two other classes of proteins, the outer membrane channel such as TolC of and OprM of p aeruginosa, belonging to the OMF (outer membrane factor) family of proteins, and the periplasmic “adaptor” protein such as AcrA of E coli and MexA of p aeruginosa Importantly, each of these three component proteins is essential for drug efflux, and the absence of even one component makes the entire complex totally nonfunctional The construction of this tripartite complex suggested that the drugs are here exported directly into the external medium, rather than into the periplasm This is a huge advantage for bacterial cells, because once exported into the external space, drug molecules must traverse the outer membrane barrier to reenter the cells Thus these pumps work synergistically with the outer membrane barrier Wild-type strains of most Gram-negative bacteria are resistant to most lipophilic antibiotics (for E coli they include penicillin G, oxacillin, cioxacillin, nafcillin, macrolides, novobiocin, linezolid, and fusidic acid), and this “intrinsic resistance” was often thought to be caused by the exclusion of drugs by the outer membrane barrier Indeed breaching the outer membrane barrier does sensitize E coli cells to the drugs However, the inactivation of the major and constitutively expressed RND pump AcrB makes the bacteria almost completely susceptible to these agents (the minimal inhibitory concentration [MIC] of a lipophilic penicillin, cloxacillin, going down from 512 pg/ml in the wild type to only pg/ml even in the presence of the intact outer membrane barrier Thus the characteristic intrinsic resistance of gram­ negative bacteria owes as much to the RND pumps as to the outer membrane barrier (Blanco Torres, 2019) 1.2 E.Coli AcrAB-TolC efflux pump 1.2.1 Structure of E coli AcrAB-TolC efflux pump The RND superfamily is specific to Gram-negative microorganisms which the major clinically relevant efflux systems belong to AcrAB-TolC of E.coli It comprises a tripartite structure consisting of the integral membrane transporter AcrB; the outer membrane channel TolC; and the periplasmic protein adaptor AcrA, which stabilizes the interaction between AcrB and TolC (Zgurskaya et al., 2015) The proposed model of the AcrA-AcrB-TolC complex is shown in Figure 1.2 1.2.2 Substrates and mechanism of efflux pump The AcrB is the active part of the AcrAB-TolC drug export complex, pumping out tetracycline, chloramphenicol, p-lactams, novobiocin, fusidic acid, nalidixic acid, and fluoroquinolones among antibiotics and chemotherapeutic agents, and bile salts among detergents, various cationic dyes and disinfectants, and even solvents (Edward et al., 2003) 10 ... effect of a potential efflux inhibitor on a bacterial strain expressing efflux activity In particular, there are two kinds of studying the activity of efflux pumps inhibitors: efflux pump inhibitors. .. by the efflux pump, at the different concentrations of the tested compound The result of assay is either the minimum inhibitory concentration (MIC) or the value of minimal concentration of an... 23 Pharmacophore model with descriptions 30 11 best pharmacophore modelsof AcrB inhibitors with descriptions 32 Virtual screening on TCM 43 Virtual screening on Drugbank