Cholesterol metabolism in Mtb is an attractive target for basic research and new drug development efforts. The Mtb cholesterol metabolic pathway is specific to the bacteria and mutants defective in this pathway are attenuated in various infection models. This suggests that chemically inhibiting this pathway will also attenuate Mtb virulence in vivo. We recently discovered a compound series that blocks cholesterol uptake in Mtb and stimulates cAMP overproduction in the bacteria. This is important since cAMP regulates mycobacterial central metabolism, transcription, pathogenicity, dormancy, and stress responses. Thus, chemically stimulating cAMP production in Mtb will perturb multiple different aspects of bacterial physiology in addition to cholesterol utilization. Granulomas are a major pathologic barrier that limits immune cell recruitment and antibiotic diffusion. Within a granuloma Mtb is sequestered in a cholesterol rich and hypoxic microenvironment that is thought to favor bacterial persistence. During infection, MΦ’s produce TNF-α which drives TB tissue pathology and is required to form and maintain granulomas. It is thought that TNF-α depletion can disturb the granuloma architecture and promote enhanced antibiotic availability or disrupt the granuloma microenvironment making TB antibiotics more effective. It has long been known that TNF-α production by MΦ’s can be down regulated in response to high levels of cytosolic cAMP and it is also known that Mtb-derived cAMP down regulates TNF-α production in MΦ’s. Our compound series stimulates enough Mtb derived cAMP to down regulate TNF-α production at the infected cell level. Thus, stimulating cAMP overproduction in Mtb could be a novel strategy to reduce TNF-α levels specifically in infected MΦ’s to enhance the activity of current TB drugs. Here we propose to characterize the molecular mechanisms of how these probes block cholesterol uptake and stimulate the overproduction of cAMP in Mtb (Aim 1). We also will determine how Mtb-derived cAMP modulates: (i) the host immune response, (ii) MΦ function, and (iii) evaluate the efficacy of V-59 alone and incombination with known TB antibiotics in vivo with a goal of evaluating the therapeutic potential of V-59 (Aim 2). Because we already have a compound that has excellent potency and demonstrates in vivo efficacy via the oral route, we are well positioned to make progress with a potential TB drug candidate and novel treatment strategy.