Towards an Understanding of Ligand Induced Functional Conformational Changes of MexB Efflux Transporter

10.26434/chemrxiv.14175383.v1 ◽

2021 ◽

Author(s):

Khondaker Miraz Rahman ◽

Shirin Jamshidi ◽

J Mark Sutton ◽

Sara Jambarsang

Keyword(s):

Conformational Changes ◽

Efflux Pump ◽

Vital Role ◽

Gram Negative Bacteria ◽

Efflux Transporter ◽

Structural Rearrangements ◽

Protonation State ◽

New Information ◽

Cytotoxic Molecules ◽

Efflux Pump Inhibitors

<p>MexB, an RND-superfamily efflux pump, plays a vital role in conferring resistance to cytotoxic molecules, including antibiotics, upon Gram-negative bacteria. Although the principal mechanistic elements of switching between the access, binding and extrusion conformers of the protomers of tripartite efflux transporters have been described previously, details surrounding the further mechanism that ends in either substrate extrusion or pump inhibition are limited to observations based on the type of ligand bound to the transporter. A central but missing link in the structure/mechanism relationship is a description of how ligand-induced conformational changes in the presence of a membrane and changing transporter protonation state lead to either substrate extrusion or inhibition of the pump. Here, we report that differences in conformational changes are governed by ligand binding to the transporter. The current study describes important new information about ligand-induced structural rearrangements and conformational changes of MexB in relation to the protonation state of critical acidic residues. We used tetracycline (TET) as a model substrate of MexB and phenylalanine-arginine beta-naphthylamide (PAβN) as a model inhibitor of MexB to study the aforementioned conformational changes. This new information will contribute to the design of new, effective and selective efflux pump inhibitors that could play key roles in reversing antimicrobial resistance.</p>

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A Tobramycin Vector Enhances Synergy and Efficacy of Efflux Pump Inhibitors against Multidrug-Resistant Gram-Negative Bacteria

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.7b00156 ◽

2017 ◽

Vol 60 (9) ◽

pp. 3913-3932 ◽

Cited By ~ 28

Author(s):

Xuan Yang ◽

Sudeep Goswami ◽

Bala Kishan Gorityala ◽

Ronald Domalaon ◽

Yinfeng Lyu ◽

...

Keyword(s):

Efflux Pump ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Efflux Pump Inhibitors

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Mode of action of the 2-phenylquinoline efflux inhibitor PQQ4R againstEscherichia coli

PeerJ ◽

10.7717/peerj.3168 ◽

2017 ◽

Vol 5 ◽

pp. e3168 ◽

Cited By ~ 14

Author(s):

Diana Machado ◽

Laura Fernandes ◽

Sofia S. Costa ◽

Rolando Cannalire ◽

Giuseppe Manfroni ◽

...

Keyword(s):

Mode Of Action ◽

Bacterial Infections ◽

Efflux Pump ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Synergistic Activity ◽

Dependent Manner ◽

Gram Negative ◽

E Coli ◽

Efflux Pump Inhibitors

Efflux pump inhibitors are of great interest since their use as adjuvants of bacterial chemotherapy can increase the intracellular concentrations of the antibiotics and assist in the battle against the rising of antibiotic-resistant bacteria. In this work, we have described the mode of action of the 2-phenylquinoline efflux inhibitor (4-(2-(piperazin-1-yl)ethoxy)-2-(4-propoxyphenyl) quinolone – PQQ4R), againstEscherichia coli,by studding its efflux inhibitory ability, its synergistic activity in combination with antibiotics, and compared its effects with the inhibitors phenyl-arginine-β-naphthylamide (PAβN) and chlorpromazine (CPZ). The results showed that PQQ4R acts synergistically, in a concentration dependent manner, with antibiotics known to be subject to efflux inE. colireducing their MIC in correlation with the inhibition of their efflux. Real-time fluorometry assays demonstrated that PQQ4R at sub-inhibitory concentrations promote the intracellular accumulation of ethidium bromide inhibiting its efflux similarly to PAβN or CPZ, well-known and described efflux pump inhibitors for Gram-negative bacteria and whose clinical usage is limited by their levels of toxicity at clinical and bacteriological effective concentrations. The time-kill studies showed that PQQ4R, at bactericidal concentrations, has a rapid antimicrobial activity associated with a fast decrease of the intracellular ATP levels. The results also indicated that the mode of action of PQQ4R involves the destabilization of theE. coliinner membrane potential and ATP production impairment, ultimately leading to efflux pump inhibition by interference with the energy required by the efflux systems. At bactericidal concentrations, membrane permeabilization increases and finally ATP is totally depleted leading to cell death. Since drug resistance mediated by the activity of efflux pumps depends largely on the proton motive force (PMF), dissipaters of PMF such as PQQ4R, can be regarded as future adjuvants of conventional therapy againstE. coliand other Gram-negative bacteria, especially their multidrug resistant forms. Their major limitation is the high toxicity for human cells at the concentrations needed to be effective against bacteria. Their future molecular optimization to improve the efflux inhibitory properties and reduce relative toxicity will optimize their potential for clinical usage against multi-drug resistant bacterial infections due to efflux.

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Proof of an Outer Membrane Target of the Efflux Inhibitor Phe-Arg-β-Naphthylamide from Random Mutagenesis

Molecules ◽

10.3390/molecules24030470 ◽

2019 ◽

Vol 24 (3) ◽

pp. 470 ◽

Cited By ~ 4

Author(s):

Sabine Schuster ◽

Jürgen Bohnert ◽

Martina Vavra ◽

John Rossen ◽

Winfried Kern

Keyword(s):

Conformational Changes ◽

Lipid A ◽

Efflux Pump ◽

Random Mutagenesis ◽

Gram Negative Bacteria ◽

Loss Of Function ◽

Efflux Pump Inhibitor ◽

Membrane Target ◽

Generation Sequencing

Phe-Arg-β-naphthylamide (PAβN) has been characterized as an efflux pump inhibitor (EPI) acting on the major multidrug resistance efflux transporters of Gram-negative bacteria, such as AcrB in Eschericha coli. In the present study, in vitro random mutagenesis was used to evolve resistance to the sensitizing activity of PAβN with the aim of elucidating its mechanism of action. A strain was obtained that was phenotypically similar to a previously reported mutant from a serial selection approach that had no efflux-associated mutations. We could confirm that acrB mutations in the new mutant were unrelated to PAβN resistance. The next-generation sequencing of the two mutants revealed loss-of-function mutations in lpxM. An engineered lpxM knockout strain showed up to 16-fold decreased PAβN activity with large lipophilic drugs, while its efflux capacity, as well as the efficacy of other EPIs, remained unchanged. LpxM is responsible for the last acylation step in lipopolysaccharide (LPS) synthesis, and lpxM deficiency has been shown to result in penta-acylated instead of hexa-acylated lipid A. Modeling the two lipid A types revealed steric conformational changes due to underacylation. The findings provide evidence of a target site of PAβN in the LPS layer, and prove membrane activity contributing to its drug-sensitizing potency.

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The Impact of Efflux Pump Inhibitors on the Activity of Selected Non-Antibiotic Medicinal Products against Gram-Negative Bacteria

Molecules ◽

10.3390/molecules22010114 ◽

2017 ◽

Vol 22 (1) ◽

pp. 114 ◽

Cited By ~ 6

Author(s):

Agnieszka Laudy ◽

Ewa Kulińska ◽

Stefan Tyski

Keyword(s):

Efflux Pump ◽

Gram Negative Bacteria ◽

Medicinal Products ◽

Gram Negative ◽

Efflux Pump Inhibitors ◽

The Impact

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Effects of Efflux Pump Inhibitors on Colistin Resistance in Multidrug-Resistant Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00248-16 ◽

2016 ◽

Vol 60 (5) ◽

pp. 3215-3218 ◽

Cited By ~ 42

Author(s):

Wentao Ni ◽

Yanjun Li ◽

Jie Guan ◽

Jin Zhao ◽

Junchang Cui ◽

...

Keyword(s):

Stenotrophomonas Maltophilia ◽

Efflux Pump ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

Resistant Strains ◽

Efflux Pump Inhibitors ◽

Time Kill

ABSTRACTWe tested the effects of various putative efflux pump inhibitors on colistin resistance in multidrug-resistant Gram-negative bacteria. Addition of 10 mg/liter cyanide 3-chlorophenylhydrazone (CCCP) to the test medium could significantly decrease the MICs of colistin-resistant strains. Time-kill assays showed CCCP could reverse colistin resistance and inhibit the regrowth of the resistant subpopulation, especially inAcinetobacter baumanniiandStenotrophomonas maltophilia. These results suggest colistin resistance in Gram-negative bacteria can be suppressed and reversed by CCCP.

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Conformational study of (Z)-5-(4-chlorobenzylidene)-2-[4-(2-hydroxyethyl)piperazin-1-yl]-3H-imidazol-4(5H)-one in different environments: insight into the structural properties of bacterial efflux pump inhibitors

Acta Crystallographica Section C Structural Chemistry ◽

10.1107/s2053229617016461 ◽

2017 ◽

Vol 73 (12) ◽

pp. 1151-1157 ◽

Cited By ~ 4

Author(s):

Ewa Żesławska ◽

Wojciech Nitek ◽

Jadwiga Handzlik

Keyword(s):

Multidrug Resistance ◽

Hydrogen Bonds ◽

Crystal Structures ◽

Efflux Pump ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Carboxylate Group ◽

Piperazine Ring ◽

Gram Negative ◽

Efflux Pump Inhibitors

The 2-amine derivatives of 5-arylidene-3H-imidazol-4(5H)-one are a new class of bacterial efflux pump inhibitors, the chemical compounds that are able to restore antibiotic efficacy against multidrug resistant bacteria. 5-Arylidene-3H-imidazol-4(5H)-ones with a piperazine ring at position 2 reverse the mechanisms of multidrug resistance (MDR) of the particularly dangerous Gram-negative bacteria E. coli by inhibition of the efflux pump AcrA/AcrB/TolC (a main multidrug resistance mechanism in Gram-negative bacteria, consisting of a membrane fusion protein, AcrA, a Resistant-Nodulation-Division protein, AcrB, and an outer membrane factor, TolC). In order to study the influence of the environment on the conformation of (Z)-5-(4-chlorobenzylidene)-2-[4-(2-hydroxyethyl)piperazin-1-yl]-3H-imidazol-4(5H)-one, (3), two different salts were prepared, namely with picolinic acid {systematic name: 4-[(Z)-4-(4-chlorobenzylidene)-5-oxo-3,4-dihydro-1H-imidazol-2-yl]-1-(2-hydroxyethyl)piperazin-1-ium pyridine-2-carboxylate, C16H20ClN4O2 +·C6H4NO2 −, (3 a )} and 4-nitrophenylacetic acid {systematic name: 4-[(Z)-4-(4-chlorobenzylidene)-5-oxo-3,4-dihydro-1H-imidazol-2-yl]-1-(2-hydroxyethyl)piperazin-1-ium 2-(4-nitrophenyl)acetate, C16H20ClN4O2 +·C8H6NO4 −, (3 b )}. The crystal structures of the new salts were determined by X-ray diffraction. In both crystal structures, the molecule of (3) is protonated at an N atom of the piperazine ring by proton transfer from the corresponding acid. The carboxylate group of picolinate engages in hydrogen bonds with three molecules of the cation of (3), whereas the carboxylate group of 4-nitrophenylacetate engages in hydrogen bonds with only two molecules of (3). As a consequence of these interactions, different orientations of the hydroxyethyl group of (3) are observed. The crystal structures are additionally stabilized by both C—H...N [in (3 a )] and C—H...O [in (3 a ) and (3 b )] intermolecular interactions. The geometry of the imidazolone fragment was compared with other crystal structures possessing this moiety. The tautomer observed in the crystal structures presented here, namely 3H-imidazol-4(5H)-one [systematic name: 1H-imidazol-5(4H)-one], is also that most frequently observed in other structures containing this heterocycle.

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