Docking studies of AcrB efflux pump inhibitors on Escherichia coli .pdf

ĨJ it MINISTRY OF EDUCATION AND TRAINING NGUYEN TAT THANH UNIVERSITY 0O0 - DISSERTATION FINAL REPORT SCIENTIFIC RESEARCH PROJECT OF STUDENT IN 2020 NAME OF DISSERTATION: DOCKING STUDIES OF ACRB EFFLUX PUMP INHIBITORS ON ESCHERICHIA COLI Code of dissertation: Supervisor of dissertation: DAO NHUT LINH Scientific instructor: M.S.Pharm PHAN THIEN VY, NGUYEN THI THU HIEN Faculty: Faculty of Pharmacy Student’s name: DAO NHUT LINH Student ID: 151140717 Class: 15DDS6B Ho Chi Minh City- 2020 ÌI TABLE OF CONTENTS LIST OF ACRONYMS LIST OF FIGURE LIST OF TABLE CHAPTER LITERATURE REVIEW 1.1 Mechanisms of antibiotic resistance 1.2 Overview of efflux pump families 1.3 Overview of AcrB efflux pump 1.3.1 Structure 1.3.2 Extrusion mechanism of AcrB 13 1.4 Efflux pump inhibitors (EPIs) 15 1.5 Docking method .17 1.5.1 Docking method overview 17 1.5.2 Previous docking studies on E.coli 20 CHAPTER RESEARCH METHOD 23 2.1 Research subject 23 2.1.1 Protein 23 2.1.2 Database 23 2.2 Docking process 24 2.2.1 Preparing Protein 24 2.2.2 Determining binding site by LeadIT 2.0.2 25 2.2.3 Redocking with Doxorubicin 25 2.2.4 Preparing ligand 25 2.2.5 Docking ligand into binding site by Lead IT 2.0.2 26 2.2.6 Analyze and evaluate the result 27 2.2.7 Screening compounds of Drugbank,Traditional Chinese Medicine (TCM), natrual compounds database 28 CHAPTER RESULTS AND DISCUSSION 29 3.1 Determining binding sites 29 3.2 Redocking results 30 3.3 Docking results into Site 30 3.3.1 Docking results of Dataset 30 3.3.2 Docking results of Dataset2 34 3.3.3 Docking results of Dataset3 40 3.4 Docking results into Site II 42 3.4.1 Docking results of Dataset 42 3.4.2 Docking results of Dataset2 44 3.4.3 Docking results of Dataset 50 3.5 Virtual screening result of Drugbank, Traditional Chinese Medicine Compounds database and Natural compounds 52 CHAPTER CONCLUSION AND SUGGESTION 54 4.1 Conclusion 54 4.2 Suggestion 54 REFERENCES APPENDIX AP-1 LIST OF ACRONYMS Abbreviation Explanation ABC AcrB Ala Arg Asp DBP EPI Gin Gly HAE-RND HME-RND He Leu Lys MATE MDR Met MFS PACE PBP Phe Pro RND Ser SMR TCM Vai WHO ATP binding cassette Acriflavine resistance protein B Alanine Arginine Aspartic acid Distal binding pocket Efflux pump inhibitor Glutamine Glycine Hydrophobic and amphiphilic efflux RND Heavy metal efflux RND (HME-RND) Isoleucine Leucine Lysine The multidrug and toxic compound extrusion Multidrug resistant Methionine The major facilitator Proteobacterial antimicrobial compound efflux Proximal binding pocket Phenylalanine Proline The resistance-nodulationcell division Serine The small multidrug resistance Traditional Chinese Medicine Valine World Health Organization LIST OF FIGURE Figure 1.1 Efflux pumps family Functional diversity among efflux proteins (Del­ mar et al., 2014) Figure 1.2 Structure of AcrB (Murakami et al., 2002) Figure 1.3 The structure of a single protomer (Anes et al., 2015) 12 Figure 1.4 A cutaway stereo view displaying the solvent-accessible surface of AcrB (Anes et al., 2015) 12 Figure 1.5 A model representation of AcrAB-TolC efflux pump (Anes et al., 2015) 15 Figure 2.1 Preparing protein process 25 Figure 2.2 Illustrating the types of interaction 27 Figure 3.1 Location of two binding site 29 Figure 3.2 Images of binding pocket and some residues 30 Figure 3.3 Residues in Site I interact with compounds of Dataset 31 Figure 3.4 The interactions between BBA_2018_1860_878_Hoechst33342, BBA_ 2018_1860_878_PApN and surrounding residues in Site I 32 Figure 3.5 Interactions of BBA_2020_1864_6_58997260 compound and the super­ imposing image of six (3 - aminocyclobutyl) pyrimidin - - amine compounds in Site 34 Figure 3.6 Residues in Site I interact with compounds of Dataset 35 Figure 3.7 The interaction and superimposing image of Piperazine Arylideneimidazolones derivatives compounds and Site 36 Figure 3.8 The interaction of PJ_2017_5_PQQ4R, IV_2014_28_1071_rac3i, EJMC_2018_143_699_6b, EJMC_2018_143_699_12i compounds and Site 38 Figure 3.9 Interactions and the superimposing image of Nitrothiophene carboxami­ de compounds and Site 39 Figure 3.10 Residues in Site I interact with compounds of Dataset 40 Figure 3.11 Residues in site II interact with compounds in Dataset 42 Figure 3.12 Interactions between lowest docking score compounds of Dataset and residues in Site II 44 Figure 3.13 Residues in site II interact with compounds in Dataset 45 Figure 3.14 Interactions between Nitrothiophene carboxamides compound and re­ sidues in Site II 46 Figure 3.15 Interactions between - subtituted - naphthamide derivatives and residues in Site II 47 Figure 3.16 Interactions between MO_2014_3_6_885_Mangiferin, IV_2014_28_ 1071_rac3i, PR_2018_l_4 compounds and residues in Site II 49 Figure 3.17 Interaction and the superimposing image of Piperazine Arylideneimidazolones and Site II 49 Figure 3.18 Residues in Site II interact with compounds of Dataset 51 LIST OF TABLE Table 1.1 Table identifying the residues (Vargiu et al., 2014) 14 Table 1.2 X-ray diffraction structures of AcrB 19 Table 2.1 Database 23 Table 3.1 The top lowest docking score compounds of Dataset into Site 30 Table 3.2 Structure of substituted (3-aminocyclobutyl) pyrimidin - - amines compounds and docking score in both sites 33 Table 3.3 Top 10 lowest docking score compounds in Dataset into Site 34 Table 3.4 Top lowest docking score compounds in Dataset into Site 40 Table 3.5 Top lowest docking score compounds of Dataset into Site II 42 Table 3.6 Top 10 lowest docking score compounds of Dataset into Site II 45 Table 3.7 Top lowest docking score compounds of Dataset into Site II 50 Table 3.8 The lowest docking score compounds from each database into Site I and Site II 53 CHAPTER LITERATURE REVIEW Mechanisms of antibiotic resistance 1.1 Gram-negative bacteria, like E coli, have several mechanisms of resistance when it comes to surviving the selective pressure exerted by antimicrobial agents Some mechanisms can be definitive whereas others may reverse when the selective pressure is released or completely removed (Anes et al., 2015) Resistance can occur due to (Anes et al., 2015): Accumulation of mutations involved in specific antimicrobial targets (e.g., - mutations in quinolone resistance-determining regions (QRDRs) in gyrA, gyrB, parE, andparC genes); Antimicrobial inactivation/modification (e.g., production of p-lactamase enzy­ - mes); Acquisition of mobile genetic elements such as plasmids, transposons, or - integrons acquired by HGT; - Alteration in the cell wall composition (e.g., lipopolysaccharide modification); - Reduced expression of cell wall porins, resulting in decreased influx of antimicrobials); Over-expression of efflux pumps - The most important mechanism among the above mechanisms is the over­ expression of efflux pump 1.2 Overview of efflux pump families Based on sequence similarity, transport function, substrate specificity, and energy coupling, efflux pumps can be classified into six superfamilies (Figure 1.1) (Delmar et al., 2014) - The Adenosine Triphosphate (ATP)-Binding Cassette (ABC) superfamily, - The Major Facilitator Superfamily (MFS), - The Small Multidrug Resistance (SMR) superfamily, - The Resistance-Nodulationcell Division (RND) superfamily - The Multidrug And Toxic Compound Extrusion (MATE) superfamily - Proteobacterial Antimicrobial Compound Efflux (PACE) ABC transporters use ATP as an energy source to drive toxins from the cell, whereas the other four superfamilies rely on an electrochemical gradient (Figure 1.1) Specifically, MFS, RND, and SMR proteins employ the proton-motive force, and the MATE superfamily is characterized by either Na+- or H+-substrate antiport Gram-negative bacteria have been found to contain members of all five superfamilies, and this diversity contributes to their intrinsic resistance to diverse antimicrobials RND transporters are capable of forming powerful, cooperative, multiprotein structures that bridge both the inner and outer membranes They are essential to the multidrug resistance observed in many pathogens (Delmar et al., 2014) Figure 1.1 Efflux pumps family Functional diversity among efflux proteins (Delmar et al., 2014) RND transporters operate as part of a tripartite system composed of the RND pump located in the inner membrane, a periplasmic adaptor protein from the MFP family and an OMP belonging to the outer membrane factor (OMF) family located in the outer membrane The OMP TolC, for example, works in combination with other RND, ABC, and MFS efflux pump The absence of any of these components renders the entire complex non-functional Nonetheless, the efflux systems show a cooperative interaction between them and can act sequentially when one fails RND transporters can be classified into two different subfamilies according to their substrates, the hydrophobic and amphiphilic efflux RND (HAE-RND) family and the heavy metal efflux RND (HME-RND) family In E coli there are five efflux transporters that belong to the HAE-RND subfamily, AcrAB, AcrAD, AcrEF, MdtAB, and MdtEF In contrast, there is only one efflux transporter that belongs to the HME-RND, the CusCFBA (Anes et al., 2015) The most important tripartite system belonging to E.coli RND superfamily is AcrAB-TolC efflux system This system is known to be responsible for the extrusion of a broad range of compounds such as lipophilic antimicrobial drugs, i.e., penicillin G, cloxacillin, nafcillin, macrolides, novobiocin, linezolid, and fusidic acid; antibiotics (such as fluoroquinolones, cephalosporins, tetracyclines); various dyes (i.e., crystal violet, acridine, acriflavine, ethidium, 6-rhodamine 6G); detergents [sodium dodecyl sulfate (SDS) and Triton X-100]; organic solvents (hexane, cyclohexane); steroid hormones (bile acids, estradiol, and progesterone); essential oils; and others AcrB resides in the inner membrane and is the energy transducing and substrate specificity determinant of the entire three-component pump assembly AcrA is the adapter component that associates the inner membrane pump with the TolC outer membrane channel Importantly, all three components are necessary to obtain the multidrug resistance phenotype (Eicher et al., 2012) AcrB also efflux pump on Enterobacter aerogenes and Klebsiella pneumoniae 1.3 Overview of AcrB efflux pump 1.3.1 Structure The RND protein AcrB is composed of 1,049 amino acids and is distributed throughout the transmembrane domain and the large periplasmic domain (Figure 1.2 A) The first symmetrical crystal structure for AcrB protein was resolved by Murakami et al (2002) at a 3.5 Ả resolution in which three AcrB protomers were organized as a homotrimer Co-crystallization of AcrB with several ligands (including 6-rhodamine 6G, ethidium, dequalinium and ciprofloxacin) showed that these ligands bind near the transmembrane domain and in various positions within the binding pocket The binding is established primarily through hydrophobic, aromatic stacking, and van der Waals interactions Asymmetric crystal structures of AcrB were later resolved using minocycline, doxorubicin, ethidium, dequalinium, and designed ankyrin repeat proteins (DARPins), the latter being an inhibitor designed specifically for AcrB, as a substrate (Anes et al., 2015) The symmetric structure at 3.5 Ả resolution is used to describe the structure of AcrB in this dessetation It is divided into domains: transmembrane domain, pore domain and TolC docking domain (Figure 1.2) (Murakami et al., 2002) Figure 1.2 structure of AcrB (Murakami et al., 2002) (A) Side view of a ribbon representation Three protomers are individually coloured (blue, green and red) The N-terminal and C-terminal halves of the protomers are depicted as dark and pale colours, respectively The extra-membrane (periplasmic) headpiece (TolC docking domains and pore domains) is at the top, and the transmembrane region is at the bottom (B) Top view of a ribbon representation The protomers are individually coloured as in A c, Structure within a slab (~23 Â) of the transmembrane domain parallel to the membrane plane near the periplasmic surface The protomers are individually coloured as in a and b Three-fold and pseudo-two-fold rotation axes are indicated The label numbers indicate the transmembrane helix numbers a) Transmembrane domain structure The transmembrane domain of each protomer contains twelve transmembrane ahelices (Figure 1.2A,C) The number of the transmembrane segments is consistent with the hydropathy plot-based prediction and topology studies by site-directed chemical modification of cysteine mutants A pseudo-two-fold symmetry axis exists in each transmembrane domain, that is, the six N-terminal helices are symmetrically arranged with the six C-terminal helices The membrane domain contains an additional extra-membrane a-helix (la) located between TM6 and TM7 attached to the cytoplasmic membrane surface (Figure 1.3A,B) The inter-protomer interaction in the transmembrane region is restricted to the surface buried between TM1 and TM8 (Figure 1.2C) TM4 and TM10 form the centre of the transmembrane helix bundle (Figure 1.2C) These helices are long and protrude beyond the cytoplasmic surface of the membrane (Figure 1.3B) TM2 is also a long helix that protrudes upwards across the boundary of the membrane (Figure 1.3B) TM8 corresponds to TM2 in the C-terminal half Except for the asymmetry of the top of TM2 and TM8, pseudo- two-fold symmetry of the N- and C-terminal six helix bundles can be observed in Figure 1.2C According to the results of site-directed mutagenesis studies on MexB and AcrB, the transmembrane region contains three functionally essential charged residues: Asp407, Asp480 and Lys940 (Figure 1.3C) When these residues are replaced with any other aminoacid residues, the resulting mutants completely lose the drug resistance These residues are located in the middle of TM4 and TM10, and form ion pairs (Figure 1.3C) These residues are possible candidates for the proton-translocating pathway (Murakami et al., 2002) b) Pore domain structure The pore domain is composed of four subdomains: PN1, PN2, PCI and PC2 (Figure 1.3A, B) PN1 and PN2 comprise the polypeptide segment between TM1 and TM2, and PCI and PC2 comprise the segment between TM7 and TM8 All of these subdomains contain a characteristic structural motif That is, two p-strand-a- helix-p-strand motifs are directly repeated and sandwiched with each other This motif forms a structure in which two a- helices are located on a four-stranded antiparallel P-sheet As found by a database search additional antiparallel p-strand from the other half of the protomer (Figure 1.3A) Thus, the antiparallel p-sheets in 10 ... 1.2 Overview of efflux pump families 1.3 Overview of AcrB efflux pump 1.3.1 Structure 1.3.2 Extrusion mechanism of AcrB 13 1.4 Efflux pump inhibitors (EPIs)... only occurs when additional substrate has bound to the adjacent monomer The functional rotation mechanism suggests interdependence of the monomers, which means that inactivation of only one of. .. (Eicher et al., 2012) AcrB also efflux pump on Enterobacter aerogenes and Klebsiella pneumoniae 1.3 Overview of AcrB efflux pump 1.3.1 Structure The RND protein AcrB is composed of 1,049 amino acids