Discovery of highly selective inhibitors to probe the physiology of the bacterial cell envelope

Abstract

Traditional high-throughput screening approaches have failed to efficiently discover specific, biologically active inhibitors for use as probes and development into therapeutics. New strategies are therefore required to find small molecules for studying biology and treating disease. This is especially true with regard to antibiotic resistant bacterial infections. Spreading resistance threatens the effectiveness of antibiotics currently in the clinic, so new compounds are needed to facilitate treatment of resistant infections and study of mechanisms of resistance. This thesis describes a pathway-directed screening strategy we have developed to enable efficient discovery of such compounds in the Gram-positive pathogen Staphylococcus aureus. Pathway-directed screening capitalizes on knowledge of genetic relationships to allow rapid identification of inhibitors in diverse cellular pathways. Here, we identified two compounds as the only possible inhibitors of S. aureus lipoteichoic acid (LTA) synthesis from a screen of ~230,000 compounds using only simple growth screens. Both compounds proved to inhibit UgtP, the glycosyltransferase responsible for synthesizing the glycolipid that serves as the membrane anchor for LTA in S. aureus. These compounds restore -lactam sensitivity in a strain of methicillin-resistant S. aureus (MRSA), suggesting inhibitors of this pathway may be useful in a clinical setting. This thesis also describes a second pathway-directed screen in S. aureus and discusses preliminary work in leveraging compounds and knowledge from the UgtP inhibitor discovery process to inform hit prioritization and target ID for this second screen. Together, this work describes an efficient pathway-directed screening platform for inhibitor discovery and biological inquiry in S. aureus, and we believe this strategy can be easily adapted to enable discovery of small molecule inhibitors in other biological systems.