Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump

Abstract

Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors. IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.

New Multidrug Efflux Inhibitors for Gram-Negative Bacteria

“…This suggests that AcrB activity is particularly important for the intrinsic resistance to these compounds. Compound RB16, which caused most potentiation of antibiotic activity, had an MIC of 128 μM for the wild-type strain and an MIC of 32 μM for the mutant lacking AcrB activity, suggesting that this compound may be a “preferential substrate” over the tested antibiotics, similarly to the mode of action proposed for chlorpromazine ( 55 ).…”

“…As other efflux inhibitors, including chlorpromazine and PAβN, are substrates of AcrB ( 26 , 55 ), we sought to determine if the efflux inhibitors identified by this study are also substrates of the efflux pumps. If a compound is a substrate only of AcrB, then loss of AcrB function via a mutation conferring D408A causes hypersusceptibility to the compound.…”

“…Furthermore, E. coli cells lacking a functional AcrB (due to a D408A substitution inhibiting the proton relay of AcrB) were more susceptible to some of the efflux inhibitors than the wild-type cells, suggesting that they are substrates of AcrB. A recent molecular dynamics study revealed that amitriptyline and chlorpromazine are also efflux substrates that appear to inhibit efflux of other compounds ( 55 ). The molecular dynamics study also indicated that amitriptyline and chlorpromazine have different efflux-inhibitory effects in combination with ethidium bromide or norfloxacin due to the differences in binding locations within AcrB of the inhibitors and substrates.…”

Determination of Drug Efflux Pump Efficiency in Drug-Resistant Bacteria Using MALDI-TOF MS

“…In addition, Soto et al [ 31 ] indicated that no modulation activity of PAβN was observed for norfloxacin in Salmonella Enteritidis isolates expressing AcrB-like efflux transporter. Previous docking studies indicated that norfloxacin could bind to the CH3 site of AcrB [ 32 ], and PAβN could bind to the distal binding pocket (DBP) of AcrB [ 33 ]. Nakashima et al [ 34 ] indicated that erythromycin/rifampicin bound to the proximal binding pocket (PBP) of AcrB and concluded that a glycine-rich swinging loop located between the proximal binding pocket and distal binding pocket acts as a swinging valve during the drug’s translocation by peristaltic motion [ 34 ], which might suggest that competitive inhibitor PAβN could possess a better inhibitory effect on the efflux of PBP-binding substrates than the efflux of CH3-binding substrates.…”

Antibiotic Resistant, Virulence-associated Genes, Biofilm and Efflux Pump Gene Expression and Molecular Typing of Klebsiella Pneumoniae Strains Recovered from Clinical Samples

“…One of the important mechanisms for creating the MDR is e ux pump systems and bio lm formation capacity [7]. E ux pumps are protein based structure which capable extrude the different toxic substances out of cells and the AcrAB-TolC e ux pump system which belongs to the Resistance Nodulation Division (RND) e ux pump which is an important cause of MDR of K. pneumoniae strains occurrence [8][9][10][11]. The AcrAB-TolC e ux pump is composed a periplasmic component (AcrA), a transporter located in the inner membrane (AcrB) and an outer membrane compartment (TolC).…”