Dec 8: Garrett Booher (VanNieuwenhze lab) "Fluorescent D-amino acids as tools for studying peptidoglycan synthase activity and developing a high-throughput screening assay to identify novel antibiotics" at 4:00 pm
Peptidoglycan is a unique structure to bacteria, which allows this structure to be an important target for antibiotics due to its orthogonality to human cell biology. One target of such antibiotics is the bacterial transpeptidases, mainly penicillin binding proteins (PBPs), that have been widely studied since the discovery of penicillin in the early 1900's. However, tools to study the dynamics of these enzymes in vivo and in vitro have been lacking. Recently, the VanNieuwenhze lab discovered that transpeptidases can utilize fluorescent D-amino acids (FDAAs) to incorporate a fluorescent tag into peptidoglycan. Thus, resulting in the labeling of peptidoglycan to study the growth modes of various different bacterial species. These FDAAs have been applied to a wide range of studies, including dynamics of peptidoglycan synthesis, peptidoglycan-enzyme interactions, and peptidoglycan metabolism. Herein, I report additional applications of FDAAs to further the understanding of the enzymes involved in peptidoglycan processes. Specifically, the development of several methods for studying the enzymatic reactions of various transpeptidases. Furthermore, an assay developed to study PBPs in vitro in real-time is reported. This assay has been optimized to the point that a robust screening of natural products was performed, and several hits were identified. Though the positive hits did not meet the threshold for clinical drugs, chemical modification of the compounds could enhance activity. Specifically, these chemical compounds can be modified to incorporate a D-amino acid backbone. The hypothesis being that since the enzymes involved in peptidoglycan synthesis can use FDAAs, substituting the fluorophore with a reactive compound could lead to a novel class of antibiotics. This work demonstrates the value of such an assay to screen for compounds. These can be further optimized to allow new and potent antibiotics to be discovered and implemented as therapeutics.