Structure Analysis of Indoles as Inhibitors of the NorA in Staphylococcus Aureus

The human pathogen Staphylococcus aureus has become increasingly resistant to antibiotics. One mechanism of resistance is through the overexpression of efflux pump proteins that actively transport antibiotic out of the bacterial cell. In the case of S. aureus, the NorA efflux pump is known to be responsible for the active efflux of the antibiotic ciprofloxacin and other compounds. Research has been dedicated to the discovery of Efflux Pump inhibitors (EPIs), which could block the effects of the transporter proteins and restore susceptibility of the bacteria towards the antibiotic. Indoles have shown great potential as EPI’s. The objective of this study is to discover indole compounds that can inhibit the NorA in S. aureus using structure-activity relationships as a model of EPI identification. An In silico study of the NorA was used to extend the model. To determine the modifications that increase the inhibitory effect of indoles, nine different indoles were analyzed: (1) modification only on the aromatic ring: F, C, N; (2) modification only on the 5-membered heterocyclic ring: CA, O, I, and (3) modification on both rings: 1CO, 2CO and 1NO. The S. aureus strains used were 1199B (NorA overexpressed) and K1758 (norA knock-out). A checkerboard assay was used with each compound to test for synergy with ciprofloxacin. An Ethidium Bromide (EtBr) efflux assay was used to determine the retention of EtBr in the cell after 30 minutes of incubation. Chlorpromazine was used as a control for both experiments. F, O and CA had no effect in the EtBr efflux assay at concentrations ≤100 mg/l. However, the compounds N, 1NO, 1CO, 2CO and I presented ≥80% EtBr retention at concentrations ≥50 mg/l. In the checkerboard assay, 1NO, 2CO, and C were able to lower the minimum inhibitory concentration (MIC) of ciprofloxacin from 8 to 2 mg/l, while N and 1CO were able to lower the MIC of ciprofloxacin to susceptible levels (≤1 mg/l). A modification to the 5^th position of the aromatic ring was able to increase the inhibitory effect of indole compounds towards the NorA efflux pump in S. aureus 1199B. Modifications to the positions of the heterocyclic ring increased the inhibitory effect of the compound if done in combination with modification to the 5^th position. Our results showed that two indoles, N and 1CO, have a great promise as EPI’s. This study is the first reporting of these compounds as EPI’s.