Understanding the mode of action of compounds that are suggested to possess efflux inhibitory properties

Grimsey, Elizabeth Mary (2021). Understanding the mode of action of compounds that are suggested to possess efflux inhibitory properties. University of Birmingham. Ph.D.

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Abstract

In Gram-negative bacteria, efflux is an important mechanism conferring multidrug resistance and the inhibition of efflux is one strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action was poorly understood. Here, these compounds potentiated the activities and increased the intracellular concentrations of AcrB substrates against S. Typhimurium and E. coli. This inhibitory activity of chlorpromazine and amitriptyline was a result of their activity as competitive inhibitors of AcrB. Both compounds bound AcrB within the distal binding pocket and clashed with the binding of the AcrB substrates ethidium bromide and norfloxacin.

Further investigation into the mechanism of chlorpromazine resistance revealed that exposure to this drug resulted in the mutation of ramR and marR. These conferred chlorpromazine resistance though the increased expression of ramA and marA by S. Typhimurium and E. coli, respectively, and a resulting increase in acrB expression and the efflux of AcrB substrates from these strains. In S. Typhimurium this was as a result of an inability of the mutant RamR protein to bind the promoter of ramA. The mutations were proposed to reflect the role of chlorpromazine as an AcrB substrate, whereby mutation of marR and ramR occured to increase the efflux of chlorpromazine. Further experiments with S. Typhimurium further supported that chlorpromazine and amitriptyline are AcrB substrates; exposure to chlorpromazine resulted in the upregulation of acrB and its regulatory genes and chlorpromazine and amitriptyline exposure resulted in the reversion of a strain of S. Typhimurium containing AcrB D408A (a a non-functional efflux pump) from the mutant acrB allele to the wild type.

This research has revealed that chlorpromazine and amitriptyline exert their efflux inhibitory activity against S. Typhmurium and E. coli as competitive substrates of AcrB.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):