Identification of a Novel Polyamine Scaffold With Potent Efflux Pump Inhibition Activity Toward Multi-Drug Resistant Bacterial Pathogens

Abstract

We have previously reported the use of combinatorial chemistry to identify broad-spectrum antibacterial agents. Herein, we extend our analysis of this technology toward the discovery of anti-resistance molecules, focusing on efflux pump inhibitors. Using high-throughput screening against multi-drug resistant Pseudomonas aeruginosa, we identified a polyamine scaffold that demonstrated strong efflux pump inhibition without possessing antibacterial effects. We determined that these molecules were most effective with an amine functionality at R1 and benzene functionalities at R2 and R3. From a library of 188 compounds, we studied the properties of 5 lead agents in detail, observing a fivefold to eightfold decrease in the 90% effective concentration of tetracycline, chloramphenicol, and aztreonam toward P. aeruginosa isolates. Additionally, we determined that our molecules were not only active toward P. aeruginosa, but toward Acinetobacter baumannii and Staphylococcus aureus as well. The specificity of our molecules to efflux pump inhibition was confirmed using ethidium bromide accumulation assays, and in studies with strains that displayed varying abilities in their efflux potential. When assessing off target effects we observed no disruption of bacterial membrane polarity, no general toxicity toward mammalian cells, and no inhibition of calcium channel activity in human kidney cells. Finally, combination treatment with our lead agents engendered a marked increase in the bactericidal capacity of tetracycline, and significantly decreased viability within P. aeruginosa biofilms. As such, we report a unique polyamine scaffold that has strong potential for the future development of novel and broadly active efflux pump inhibitors targeting multi-drug resistant bacterial infections.

Effectiveness of Efflux Pump Inhibitors as Biofilm Disruptors and Resistance Breakers in Gram-Negative (ESKAPEE) Bacteria

“…Using HTS, Fleeman et al [67] identified five different polyamine derivatives, consisting of a core scaffold (Figure 7) with EPI activity in P. aeruginosa, A. baumannii and S. aureus. The inhibitory activity of the compounds was determined using EtBr accumulation assays in strains displaying varying level of efflux expression.…”

Niosome‐encapsulated tobramycin reduced antibiotic resistance and enhanced antibacterial activity against multidrug‐resistant clinical strains of Pseudomonas aeruginosa

“…Our results showed that niosome‐encapsulated tobramycin could reduce the EtBr MIC value by two to four times which is indicating the efflux pump inhibitory of niosome encapsulated tobramycin. Efflux pump systems in P. aeruginosa strains expel antimicrobial agents out the cells, contribute to the intrinsic resistance of bacteria 57,58 . Moreover, biofilm formation reduces the efficacy of antimicrobial agents and several studies showed the relationship between biofilm formation and antibiotic resistance 59 .…”

1-benzyl-1,4-Diazepane Reduces the Efflux of Resistance-Nodulation-Cell Division Pumps in Escherichia Coli

“…This result is encouraging because cytotoxicity is one of the major drawbacks to the clinical use of putative EPIs. For example, NMP and PAβN are toxic because of their serotonin agonist properties [41] and membrane depolarizing activity [42], respectively.…”