The novel antimicrobial compound M789-2225 partially inhibits two glycolytic enzymes pyruvate kinase and pyruvate dehydrogenase

Abstract

Since the discovery of penicillin in 1928, antibiotics have greatly contributed to the increase in human lifespan across the globe, and its modern applications in medicine, research and agriculture are invaluable. However, increased rates of antibiotic resistance combined with reduced identification of novel antibiotics in the past decades has motivated the use of novel screening techniques. A novel screening technique employed by the Gitai Laboratory has identified a group of antimicrobial compounds with potent and unusual mechanisms of action (MoA). Compound M789-2225 (2225) identified by the screen showed differential media activity in minimal media (MOPS) and rich media (TB), but not standard growth media (LB) which suggests a novel mechanism of action. To investigate the MoA of 2225, I implemented metabolomics assays, resistance mutant screenings and RNA sequencing to identify significantly altered cellular pathways upon 2225 treatment. Carbon metabolism pathways glycolysis and the citric acid cycle were significantly affected, and a S. aureus resistance mutant mapped to the pyruvate kinase (PK) gene. Enzymatic activity assays were performed to investigate the effect of 2225 on glycolysis enzymes, showing inhibition of both PK and pyruvate dehydrogenase complex PDH. The inhibitory effects of 2225 on PK and PDH were confirmed in two ways. First, overexpression of the PK subunit pykF rescued 2225 treated cells. Secondly, addition of Acetyl-CoA which is the direct downstream product of PDH fully rescued 2225 treated E. coli ΔtolC. In addition, 2225 loses its media specific activity under anaerobic conditions likely due to restraints of amino acid catabolism. A modified metabolomics screen and thermal proteome profiling could highlight differences in metabolite levels and potential drug interaction partners under both aerobic and anaerobic conditions. Antibiotics affecting several targets have previously been described for their activity in multi antibiotic resistant pathogens and their ability to avoid resistance. PK and PDH inhibitors are a powerful novel class of antibiotics. The centrality of PK and PDH to cellular metabolism and high conservation across different bacterial species suggests potential low resistance. Promising experiments with 2225 were performed in the Gitai Laboratory with human cell lines, showing no toxicity in HEPG2 cells likely due to low homology of human PK with bacterial PK. However, 2225 showed activity in HCT116 human cells where glycolysis if upregulated. These results suggest further exploration of differential 2225 effects in human cells in the context of cancer and obesity treatments.