Drug-repurposing approaches to target bacterial respiratory complexes

Antimicrobial resistance is a global health problem that resulted in an estimated 1.2 million deaths in 2019. It is predicted that antimicrobial resistance will result in an estimated 10 million deaths annually by 2050 if urgent action is not taken (Pulingam et al. 2022). This has fuelled various research aimed at discovering new antimicrobials to combat this issue. Within the past few decades, a myriad of drug targets has surfaced such as cytochrome bd. Cytochrome bd is an oxidoreductase that is found only in prokaryotes and archaea and is a promising drug candidate for numerous reasons. Cytochrome bd has been proven to increase virulence in some of the most pathogenic strains of bacteria such as Mycobacterium species, as deletion of the enzyme causes severe attenuation. The oxidase is also highly expressed and induced by the innate immune response during infection. High resolution structures of cytochrome bd from various organisms have been published which means that the oxidase can be studied computationally. These characteristics make cytochrome bd an attractive drug candidate and therefore forms the basis of this study.

Lambda-red mutagenesis was previously employed by the host lab to create E. coli mutant strains that express only a single respiratory oxidase (i.e., cytochrome bd-I or bo′) and numerous attempts were made to engineer an E. coli 'cytochrome bd-II only' strain but these were unsuccessful. The current work aimed to characterise E. coli WT and respiratory mutant strains and develop an assay to measure oxygen consumption activity in membranes of these strains. Plasmid-based approaches were used to express E. coli cytochrome bd-I and cytochrome bd-II in an E. coli 'EcoM4' strain that lacked all respiratory oxidases. The strains were successfully engineered with a CO difference spectral analysis showing peaks at 640 nm. Further genetic work introduced amino acid mutations to the quinol site of E. coli cytochrome bd-I to perform future studies on antibiotic resistance. Of five amino acids that were mutated, only strains harbouring mutations in residues F269A and L253A were able to grow. Interestingly, spectral analysis revealed a distinctive peak at 640 nm for strain harbouring F269A mutation which is representative of the assembly of cytochrome bd-I. However, strain harbouring L253A mutant did not have any peaks that suggested the assembly of cytochrome bd-I.

In this project both in silico and in vitro microbiological approaches were used to search for novel inhibitors of cytochrome bd. Initially, reductive approaches were undertaken to test the natural compounds madecassic acid 1 and 2-hydroxy-1,4-naphthoquinone for inhibition of E. coli cytochrome bd-I and bo′. Both compounds were inhibitory to E. coli cytochrome bd-I and bo′ but madecassic acid 1 exhibited significantly lower IC50 of 23.62 ± 7.9 µg/mL (46.80 ± 15.7 µM) and 1019 ± 260 µg/mL (6443 ± 1644 µM), respectively. Three derivatives of madecassic acid 1 were further tested to assess whether derivatisation could improve binding of the compound to cytochrome bd-I. The addition of an acetoxy group to madecassic acid 2 and 3 have shown to improve binding to cytochrome bd-I with significantly lower IC50 of 8.8 ± 3.5 µM (5.6 ± 2.2 µg/mL) and 10.2 ± 1.1 µM (8 ± 0.9 µg/mL), respectively.

A drug repurposing pipeline was set-up to screen a library of FDA-approved drugs for their ability to bind to E. coli cytochrome bd-I. Steroid compounds (ethinylestradiol, quinestrol and mestranol) were identified within the top hits, and these steroid compounds were docked to the quinol site of S. aureus CydA subunit of cytochrome bd. Preliminary oxygen consumption experiments with isolated E. coli membranes identified ethinylestradiol and quinestrol as inhibitors of E. coli cytochrome bd-I. Quinestrol was the more potent compound causing inhibition of 'bd-I only' membranes with an IC50 of 0.2 ± 0.04 µg/mL (0.5 ± 0.1 µM), so this drug was selected for further analyses. Similar oxygen consumption experiments also confirmed quinestrol as an inhibitor of MRSA USA300 cytochrome bd. Growth assays showed that quinestrol completely abolished growth of MRSA USA300 WT and single oxidase mutant strains, while the growth of E. coli strains was inhibited with low IC50 values, but complete growth inhibition could not be achieved at higher concentrations of quinestrol. Survival assays demonstrated that quinestrol was lethal to MRSA bd-only cells with a median lethal concentration (LC50) of 5.6 ± 0.3 µg/mL (13.7 ± 0.7 µM). However, viability assays showed that E. coli was completely resistant to the quinestrol. This study identified novel inhibitors of cytochrome bd which will pave the way for future studies on steroid drugs as antimicrobials.