Kinetic analysis of the inhibition of the drug efflux protein AcrB using surface plasmon resonance

Abstract

Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl β-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB

“…As demonstrated previously 49 , a primary consideration before carrying out HDX-MS is to ensure close to full binding to the target protein under deuterium exchange conditions; particularly for low-affinity ligands (dissociation constants, k D , in the μM range or lower). CIP and PAβN both possess moderate affinity binding to AcrB within DDM detergent micelles (CIP with a k D of 74.1 ± 2.6 μM, as measured by fluorescence polarization 39 and PAβN with a k D of 15.72 ± 3.0 μM, as measured by surface plasmon resonance 50 ). To achieve sufficient ligand binding saturation under deuterium exchange conditions AcrB was first incubated in the presence of saturating concentrations of ligand for 30 minutes on ice, before dilution into deuterated buffer containing saturating concentrations of ligand (740 μM, ∼1000:1 ligand to AcrB ratio) for deuterium exchange experiments.…”

“…AcrB ligand binding was determined using fluorescence polarization (FP) assays as performed by Su et al 39 . An AcrB–PAβN protein complex stock was prepared; to ensure a loaded complex, AcrB and 150 μM PAβN was incubated for 2 h at 25 °C before titrating with 1.5 μM CIP ( k D of PAβN is 15.72 ± 3.0 μM, as measured by surface plasmon resonance 50 ). AcrB protein titration experiments were performed in ligand binding solution (50 mM sodium phosphate, 150 mM sodium chloride, 10% (v/v) glycerol, 1.5 μM CIP, 150 μM PAβN, 0.03% (w/v) DDM, pH 7.4).…”

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

“…As demonstrated previously 44 , a primary consideration before carrying out HDX-MS is for ligands with known dissociation constants to ensure close to full binding to the target protein under deuterium exchange conditions; particularly for low affinity ligands (dissociation constants, kD, in the μM range or lower). CIP and PAβN both possess moderate affinity binding to AcrB within DDM detergent micelles (CIP with a k D of 74.1 ± 2.6 μM, as measured by fluorescence polarization 38 and PAβN with a k D of 15.72 ± 3.0 μM, as measured by surface plasmon resonance 45 ). To achieve sufficient ligand binding saturation under deuterium exchange conditions AcrB was first incubated in the presence of saturating concentrations of ligand for 30 minutes on ice, before dilution into deuterated buffer containing saturating concentrations of ligand (740 μM, ∼1000:1 ligand to AcrB ratio) for deuterium exchange experiments.…”

“…38 . An AcrB-PAβN protein complex stock was prepared; to ensure a loaded complex, AcrB and 150 μM PAβN was incubated for 2 hours at 25 °C before titrating with 1.5 μM CIP (k D of PAβN is 15.72 ± 3.0 μM, as measured by surface plasmon resonance 45 ). AcrB protein titration experiments were performed in ligand binding solution (50 mM sodium phosphate, 150 mM sodium chloride, 10 % (v/v) glycerol, 1.5 μM CIP, 150 μM PAβN, 0.03 % (w/v) DDM, pH 7.4).…”

Reversing resistance to counter antimicrobial resistance in the World Health Organisation’s critical priority of most dangerous pathogens

“…The best studied example of an EPI against the tripartite antibiotic efflux pump of Gram-negative organisms is phenylarginyl-β-naphthylamide (PAβN), a simple naphthylamide peptide which did not progress beyond clinical trials due to toxicity [63]. Recent activity in this field by our group and others led to the design and synthesis of several compounds with increased efficacy [64][65][66][67] and low cytotoxicity [68].…”