SUMMARY Tuberculosis (TB) remains a significant global health problem causing 10 million new cases and 1.2 million deaths just last year according to the World Health Organization (WHO). The single greatest impediment to TB control is its ability to form persisters, the subpopulation of Mycobacterium tuberculosis (Mtb) cells that are phenotypically resistant to killing by bactericidal drugs or immune effectors. We have discovered that arginine starvation, induced by inoculating mutants of Mtb cells into arginine-free media or mice, mediates rapid sterilization that kills both actively growing Mtb cells and Mtb persister cells both in vitro and in vivo. Moreover, we have successfully determined the structure of the Mtb ArgB enzyme and used Fragment-Based Drug Discovery (FBDD) to discover two scaffold compounds that bind and inhibit the enzymatic activity of ArgB. The absence of the de novo arginine biosynthesis pathway enzymes Arg A, B, C, D, and J in humans and our demonstration that Mtb cannot bypass argB or argF deletions make Arginine Biosynthetic Enzymes attractive drug targets for TB drug development. Transcriptomic, metabolomics and flow-cytometric analyses on the arginine starving cells have provided new insights into the sterilization process. By comparing arginine starvation to three other sterilizing regimens, we identified a set of six genes that provide a sterilization signature. We plan to exploit these genes for improved drug discovery, by identifying novel drugs to inhibit the activity of Mtb ArgB using fragment-based drug design, confirm genetically any additional targets of the arginine biosynthetic pathway to expand the drug target space, and determining the correlative or causative role the common sterilizing signature genes play in Mtb sterilization. Together these studies will make available a new way to rapidly sterilize cultures of Mtb and provide new drug possibilities and new insights to shorten TB chemotherapy and treat drug-resistant TB.

PROJECT NARRATIVE Tuberculosis (TB) remains a global health problem with an estimated 10 million new infections and 1.2 million deaths per year despite existing effective chemotherapies that are long due to the presence of Mycobacterium tuberculosis (Mtb) subpopulation called “persisters”. We have discovered that arginine starvation leads to rapid sterilization of Mtb in vitro as well as in mice and, by using fragment-based drug design, we have identified two scaffold molecules that bind and inhibit the enzymatic activity of ArgB. This proposal seeks to optimize scaffold molecules to novel drugs that rapidly sterilize cultures of Mtb and expanding the drug target space by further validating other enzymes in the pathway as potential drug targets providing new drug possibilities and new insights to shorten TB chemotherapy and to treat Multidrug-resistant Mtb strains.