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.
Highlights
The continued increase of antimicrobial resistant bacterial infections is an ongoing public health crisis in the United States, with mortality rates increasing yearly (Boucher et al, 2009)
The 81 Torrey Pines scaffold samples were screened for their ability to decrease the 90% effective concentration (EC90) of the known efflux pump substrate, tetracycline, toward a tetracycline resistant strain of P. aeruginosa
The capacity for potentiation of all scaffolds was determined by mathematical modeling to identify those molecules that possessed antibacterial activity themselves
Summary
The continued increase of antimicrobial resistant bacterial infections is an ongoing public health crisis in the United States, with mortality rates increasing yearly (Boucher et al, 2009). There is a clear need to identify new efflux pump inhibitors with enhanced properties, and limited toxicity This is true for Gram negative species, such as Pseudomonas aeruginosa, which have impermeable outer membranes and commonly overexpress efflux systems (Palmer and Whiteley, 2015; Tommasi et al, 2015). Using high throughput combinatorial scaffold library screening against multi-drug resistant P. aeruginosa isolates we identified a polyamine scaffold derived from a reduced acyl peptide that demonstrated strong efflux pump inhibition and limited cytotoxicity toward eukaryotic cells. We suggest that these molecules possess excellent potential for future development as anti-resistance agents targeting bacterial efflux pumps
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