With increasing incidence of antibiotic resistance, development of new therapies against bacterial pathogens is essential for global public health. The bacterial type III secretion system (T3SS) represents an excellent drug target because it is externally accessible to small molecules and enables virulence of Pseudomonas, Salmonella, Chlamydia, and numerous other important pathogens. We have developed a high throughput screening pipeline to discover T3SS inhibitors and have shown the robustness of our approach through pilot screens identifying three classes of compounds active against the T3SS. We now propose to broaden our scope and screen three unique libraries comprising ~58,000 natural product fractions developed by members of our consortium as well as two commercial synthetic chemical libraries. According to the CDC, every year over 50,000 healthcare-associated Pseudomonas aeruginosa infections occur in the U.S., >6,000 of which are caused by multidrug resistant strains. To identify Pseudomonas T3SS inhibitors and potential future therapeutics, we will carry out the following aims.
In Aim 1, we will implement our primary screen and counterscreens to identify natural product fractions and synthetic compounds with specific T3SS inhibitory activity.
In Aim 2, we will validate hits identified in Aim 1, using three orthogonal distinct approaches. The identity and structure of bioactive natural products will be determined and initial structure activity analysis performed on identified synthetic scaffolds. Prioritized compounds will be purified or synthesized and evaluated for off target activity, if any, as well as breadth of activity against T3SSs in multiple relevant pathogens.
In Aim 3, mode of action will be determined, using parallel genetic and biochemical approaches. This rigorous strategy will provide ~10 T3SS inhibitor chemical probes with identified molecular targets active against P. aeruginosa and potentially other pathogens.
Antibiotic resistance is on the rise, necessitating development of new drugs to treat bacterial infections. As evidence mounts on the importance of a healthy microbiota to human health, future therapeutics should serve to eliminate pathogens while minimizing microbiota disruption. This project implements a primary screen and downstream experimental pipeline to identify and validate chemical probes that target a virulence-associated structure used by dozens of Gram negative pathogens.
