Mechanistic details of key integral membrane enzymes for antimicrobial discovery

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Project Summary Title: Mechanistic details of key integral-membrane enzymes for antimicrobial discovery The increasing number of antibiotic resistant strains of bacteria represents a significant threat to human health, making the development of novel therapeutic strategies critical. The major component of the bacterial cell wall is the peptidoglycan layer that is a unique meshwork providing essential structural support; therefore, identifying ways to weaken this layer is an ideal antibiotic strategy. Currently, numerous therapeutics target the peptidoglycan synthesis pathway and their use has been extremely successful in medicine. The enzymes involved in the pathway have been extensively characterized except in the case of the membrane components. Most notable are MraY and MurG, essential proteins that catalyze the membrane steps of peptidoglycan biosynthesis. There are a few known inhibitors of MraY, such as tunicamycin, demonstrating its potential as an antibiotic target; however, none of them has found usefulness in the clinic. Our group has developed efficient total synthesis schemes for two of the most promising natural products, capuramycin and muraymycin, and in the last funding period we have leveraged this to create novel compounds with improved therapeutic potential. In this proposal, we describe our plans to use our functional MraY homologs to solve structures in a lipid environment with various inhibitors and substrate analogs by EM and X-ray crystallography. We have developed a new assay for MurG that allowed us to identify novel inhibitors. We will further screen additional compounds and solve their structures with MurG. We will further explore the MurG interaction with the lipid bilayer and MraY. Our novel inhibitors, APPB and CPPB, have broad efficacy against bacterial pathogens and show potential as anti-cancer therapeutics. We will leverage the structural work to design the next round of compound libraries. The breadth of effectiveness leads us to pursue structures of other phosphotransferases, bacterial WecA and human DPAGT1, in complex with our compounds. This will allow for more targeted small molecule development. The aims are to 1) perform structural and mechanistic studies of MraY and the development of inhibitors, 2) carry out mechanistic and structural studies of MurG, and 3) develop novel and improved phosphotransferase inhibitors. Our combined team of structural biologists and synthetic chemists provides an innovative approach to achieve these important goals.