Evaluation of toxicity and efflux inhibitory activity of candidate drugs for repurposing against Neisseria gonorrhoeae

Background: Neisseria gonorrhoeae (NG) is the etiological agent of gonorrhea, a common sexually transmitted infection, with 82.4 million estimated cases in 2020. NG isolates resistant to the available therapeutic options have emerged, highlighting the threat of untreatable gonorrhea. Thus, it is crucial to develop new therapeutic alternatives. Efflux inhibitors potentially increase the intracellular concentration of currently used antimicrobials, thus restoring their activity in resistant strains. In this work, we studied the activity of repurposed candidate drugs, predicted to target membrane transporters or energy metabolism, in NG.
Methods: Minimum inhibitory concentrations (MICs) of acetazolamide, amlodipine, atovaquone, chlorpromazine, clomipramine, dequalinium, thioridazine and verapamil were determined by microdilution against NG ATCC 49226. Each drug was tested (at ¼ their MIC) for the ability to reduce the MICs of antimicrobials such as azithromycin, ciprofloxacin, gentamicin, kanamycin, and tetracycline. Efflux inhibitory activity was evaluated for each candidate drug by ethidium bromide (EtBr) accumulation fluorometric assays. The effect of each drug on the membrane potential of NG was assessed by fluorometry and fluorescence microscopy using DiOC2(3). Drug cytotoxicity of drugs at 1×, 0.1×, 0.01× and 0.001× of their MIC was evaluated against Vero and HepG2 cell lines.
Results: Dequalinium showed the highest antimicrobial activity. Most drugs caused a slight reduction of the MIC of the tested antimicrobials. Amlodipine and verapamil promoted MIC reduction and accumulation of EtBr, suggesting an efflux inhibitory activity. Atovaquone, chlorpromazine, thioridazine and verapamil decreased the membrane potential of NG. Cell viability above 90% was observed for most drugs up to 0.1× and 0.01× of their MICs for HepG2 and Vero cell lines, respectively. In particular, verapamil did not show cytotoxicity at the concentration corresponding to its MIC in the Vero cell line.
Conclusions: This study identified promising candidate drugs that could be further explored for the development of new therapeutic approaches for NG infections, in particular efflux inhibitors that may be used to combat antimicrobial resistance in NG.