Identification by High Throughput Screening of Inhibitors of the Mycobacterium tuberculosis ESX-1 and ESX-5 Type VII Secretion Systems – critical virulence determinants and novel drug targets

PROJECT SUMMARY/ABSTRACT Tuberculosis (TB) is a serious global health problem, causing ~10.6 million active cases and 1.3 million deaths/year. Better drugs are urgently needed to shorten the burdensomely long treatment course and to combat the global emergence of drug resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent. Attractive and novel targets not previously exploited for new drug development are the newly identified Type 7 Secretion Systems (T7SSs), designated ESX-1 to ESX-5, that transport factors through the Mtb hydrophobic cell wall that are essential to Mtb viability and virulence. Here, in a collaborative study involving researchers in a laboratory with years of experience studying the cell biology and pathogenesis of Mtb and developing novel drug regimens to combat TB and the Director of the state-of-the-art Molecular Screening Shared Resources (MSSR) facility at UCLA, we propose to identify small molecule inhibitors of the Mtb T7SS. In preliminary work, we have developed split-luciferase based high-throughput assays targeting a critical step in the function of the ESX-1 and ESX-5 secretion systems. We now propose to use these assays to screen the extensive small compound molecule libraries (> 300,000 compounds) at the UCLA MSSR to identify small molecule inhibitors of Mtb ESX-1 and ESX-5. Hit compounds will be retested in orthogonal assays to confirm their capacity to block Mtb T7SS secretion and Mtb growth in human macrophages in cell culture; ESX-5 inhibitors will additionally be evaluated for capacity to block Mtb growth in broth culture. We anticipate that validated hit compounds will fall into several series of structurally related compounds. We shall choose 5 - 10 of our most potent structural series for analoging (at least 20 analogs per series). We shall test the potency of the analogs in dose response in our original and orthogonal assays and assess their performance in in vitro ADMET assays. These studies will identify the most promising lead compounds with the highest therapeutic ratio for further development. Such compounds will serve as vital tools for additional studies of the role of the T7SS in Mtb pathogenesis as well as lead compounds for development of a new class of antibiotics to treat TB.