Project Summary Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major public health problem worldwide and the most common presenting illness among people living with HIV accounting for one in four HIV/AIDS-associated deaths. The continuing rise of multidrug-resistant strains of Mtb makes the development of new effective anti-TB drugs a high priority. In this context, re-examining the molecular mechanism of action of established antitubercular drugs that target the same well-validated biosynthetic pathway as front-line anti- TB agents but at a distinct catalytic step, thereby bypassing widespread resistance mechanisms, could have a major impact on the treatment of drug-resistant TB. Two such prodrugs previously used in the clinical treatment of TB, Isoxyl (ISO) and Thiacetazone (TAC), have been the object of our investigations. We identified the essential FAS-II dehydratase, HadAB, as the enzyme targeted by both ISO and TAC to inhibit mycolic acid biosynthesis and elucidated some of the most common mechanisms of resistance associated with the inhibition of this enzyme in Mtb. Recent collaborative studies between Colorado State University and the University of Cape Town Drug Discovery and Development Centre (H3D) (South Africa), have led to the discovery of a new generation of HadAB inhibitors, structurally unrelated to ISO and TAC, that displays potent activity against whole Mtb bacilli. Preliminary mode of action studies indicate that these compounds: (i) unlike ISO and TAC, do not require activation by the monooxygenase EthA; (ii) interact with HadAB differently from the former drugs; and (iii) that a subset of them have the ability to inhibit the second dehydratase of FAS-II, HadBC, which accounts for some of the resistance mechanisms associated with HadAB inhibition in Mtb. As a result, this new compound series has the potential to bypass some of the most common resistance mechanisms associated with the chemical inhibition of Mtb HadAB. This proposal aims to develop novel effective therapeutics based on H3D’s compounds with which to treat Mtb infections. This goal will be achieved by optimizing existing hits through chemical analoging (Aim 1), biological profiling (Aim 2) and in vivo efficacy testing of emerging leads (Aim 3).