Thursday, November 21, 2024
11/21/2024
Penicillin-binding proteins (PBPs) are proven ß-lactam drug targets yet resistance to ß-lactam antibiotics, such as carbapenems and cephalosporins, has resulted in a global health problem. In particular, extended- spectrum ß-lactamase (ESBL) producing or carbapenem-resistant Enterobacteriaceae, which includes Escherichia coli, are serious threats and are often linked to hospital-acquired infections. Bloodstream infections caused by these pathogens have a high mortality rate. ß-lactam antimicrobial resistance mechanism in E. coli are multiple and include, for example, the expression of ß-lactamases that can degrade ß-lactams, deletion of porins, and the overexpression of efflux pumps. Resistance is developing even against new ß-lactam/ß-lactamase inhibitor combinations. This alarming resistance spurs the need to develop different mechanisms of PBP inhibition to break this resistance cycle. PBP1b, one of the key PBPs in E. coli, has two peptidoglycan (PG)-related catalytic activities: a transglycosylase activity and a transpeptidase activity. Both activities build the PG mesh that provides critical mechanical strength and shape for bacteria. PBP1b is activated by LpoB binding to PBP1b, leading to a conformational change that stimulates both activities of PBP1b. Our goal is to develop a novel approach to inhibiting PBP1b, by targeting the activation of PBP1b by LpoB. Deletion of LpoB or mutations in LpoB that disrupt PBP1b binding leads to hypersensitivity to certain ß-lactam antibiotics. Aim 1: We propose to develop inhibitors of PBP1b activation by screening and developing compounds that bind to the PBP1b-recognition site on activator LpoB via a fragment-based structural approach. We will use thermal shift and split luciferase complementation assays to screen fragment library compounds. Hits from these orthogonal assays are further probed using dose-response measurements, biophysical tools, and a TG activity assay testing for a decrease of LpoB-mediated activation of PBP1b. Aim 2: Fragment hits will be targeted for crystallographic analysis in complex with LpoB. The combined structural information, affinity, activity, and thermal shift data will be used to design novel LpoB-directed inhibitors in an iterative fashion. Top lead compounds will advance to microbiological testing. The successful completion of our comprehensive high-risk/high-reward PPI targeting approach will lead to a new strategy of re-sensitizing PBP-targeting antibiotics, which is urgently needed in light of the current antibiotic resistance problem. The LpoB:PBP1b system is conserved in Enterobacteriaceae so our results could extend to other pathogens. Furthermore, the successful outcome of this proposal could lead to a paradigm shift in antibiotic development, re-focusing efforts on targeting PPIs in bacterial pathogens.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
