Tuberculosis (TB) is a lasting global epidemic that claims ~1.5 million human lives annually. Mycobacterium tuberculosis (Mtb) is the causative agent of TB and this bacterium establishes long term infections by surviving within macrophages and manipulating the host immune response. Within lung tissue Mtb can persist for decades sequestered away from the pressures of the host immune response. During this persistent infection Mtb solely utilizes host-derived nutrients for survival. Mtb has the ability to utilize host-derived cholesterol during an infection to supply energy producing and/or biosynthetic pathways. Therefore, inhibition of cholesterol catabolism may be a weakness that can be exploited for discovery of new anti-Mtb drugs. For this project we will develop a cell-based assay suitable for HTS that can identify inhibitors of cholesterol catabolism in Mtb. Aim 1: we will develop a robust chemical-suppressor HTS assay 384-well format using the Mtb ΔIcl1 strain. Aim 2: we will complete a pilot HTS and validate hits from the screen. Aim 3: will focus on identifying the enzyme targets of the HTS hits. These studies will yield a HTS ready assay and provide “proof of principle” that our approach can identify selective inhibitors of cholesterol catabolism in Mtb.