(533) Small molecule interference of the Pseudomonas aeruginosa glyoxylate pathway

Background: Pseudomonas aeruginosa has been shown to utilize the glyoxylate pathway during chronic cystic fibrosis airway disease to persist during chronic infection, metabolize nutrients, and upregulate virulence factors. Studies have demonstrated that isocitrate lyase (ICL), the first enzyme in the glyoxylate pathway is constitutively upregulated in P. aeruginosa isolates from the lungs of CF patients, but not in isolates from acute infections. These findings illustrate the importance of ICL for chronic infection of P. aeruginosa and combined with the lack of ICL orthologs in humans, make it an attractive target for antimicrobials.

Methods: A small molecule compound library was screened in-silico via a docking model to ICL. The top 40 candidates were selected by their docking score, commercial availability, and feasibility of synthesis. These small molecules were screened for their ability to inhibit P. aeruginosa growth (OD₆₀₀) when grown in minimal media acetate, which requires ICL activity, and not inhibit the bacterium when it was grown in minimal media containing glucose or succinate. Strains PAO1 (acute wound isolate) and FRD1 (chronic CF isolate) were used in this study. Small molecules that inhibited P. aeruginosa on acetate, but not glucose and succinate, were further characterized for their ability to disrupt alginate, pyocyanin, pyoverdine formation, and biofilm development, which are virulence factors that require optimal ICL activity. Additionally, we tested the ability of candidate compounds to rescue Drosophila melanogaster from killing by P. aeruginosa.

Results: Five candidate compounds were able to inhibit P. aeruginosa growth on acetate, in addition to glucose and succinate. However, one small molecule candidate designated 37A, reduced growth on acetate, but not when P. aeruginosa was grown on glucose and succinate, indicating that 37A may selectively inhibit ICL activity, which is required for growth on acetate. Small molecule 37A also decreased production of biofilm, pyoverdine, and alginate production in the FRD1 background. Further, 37A increased the survival of Drosophila melanogaster that were infected with P. aeruginosa compared to flies that were not exposed to 37A.

Conclusions: In conclusion, our data indicate that 37A inhibits P. aeruginosa growth on acetate, pyocyanin, pyoverdine, and biofilm production. Further, 37A protected D. melanogaster from killing by P. aeruginosa. Future experiments will explore the efficacy of 37A to directly bind to ICL to inhibit enzymatic activity, in addition to determine whether 37A reduces P. aeruginosa pathogenesis in a mammalian in vivo model. Overall, interference of the glyoxylate pathway using a small molecule strategy may be an effective strategy for targeting P. aeruginosa during chronic infections.

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