Discovery of broad-spectrum bacterial polyamine detoxification inhibitors as potential antivirulence agents and antibiotic adjuvants

Abstract

The alarming rise in antimicrobial resistance reinforces an urgent need for new antimicrobial strategies. Host- or bacteria-derived chemicals, such as polyamines, present at infection sites often influence microbial virulence and antibiotic response. Polyamines are cationic small molecules overproduced by the host during infection, modulating immune responses. Polyamine detoxification by several pathogens correlates with increased virulence. We sought to uncover inhibitors of polyamine detoxification through a high-throughput whole-cell screen against the community-acquired methicillin-resistant Staphylococcus aureus (MRSA) USA300, identifying the polyamine analog OES2-0017 and the catechol derivative isoproterenol (OES1-1087), which synergized with polyamines in the low-micromolar range. Chemical genomics and enzymatic assays combined with computational studies revealed that both compounds prevented polyamine detoxification by spermine/spermidine acetyltransferase SSAT (SpeG) and another previously uncharacterized S. aureus SSAT (denoted PaiASa herein). OES2-0017 directly inhibited the enzymes, whereas OES1-1087 reduced intracellular levels of acetyl-CoA required for SSAT activity. OES2-0017 perturbed the bacterial membrane at higher concentrations, likely causing the observed growth-inhibitory effects, whereas OES1-1087 increased membrane fluidity, likely increasing susceptibility to spermine-mediated membrane perturbation. The inhibitors showed broad-spectrum activity against various Gram-positive and Gram-negative bacteria, as well as Candida albicans. OES2-0017 abolished the spermine-mediated protection of MRSA USA300 from antibiotics, including vancomycin, phenocopying the {Delta}speG mutant and suggesting its potential utility as an antibiotic adjuvant. OES2-0017 eradicated SpeG-expressing Salmonella Typhimurium inside murine macrophages, whereas OES1-1087 reduced the S. Typhimurium burden in the liver and spleen in a murine gastrointestinal infection model, demonstrating their potential as antivirulence agents. Neither inhibitor exhibited cytotoxic activity against eukaryotic cells at their respective antimicrobial ranges. Small-scale structure-activity relationship experimental and computational analyses identified OES2-0017 analogs with improved specificity for the bacterial enzyme compared to the human SAT1 and no toxicity. This study provides novel antimicrobial compounds with broad-spectrum activity and a novel mode of action for multidrug-resistant priority pathogens.