The long-term goal of this project is to develop a co-drug that will increase the effectiveness of sulfonamides and para-aminosalicylic acid (PAS) in the treatment of tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (Mtb). A continued increase in the number of multiple drug resistant (MDR) TB cases calls for the development of new TB drugs, which is a challenging endeavor. A viable alternative or parallel solution is to increase the effectiveness of FDA-approved TB-drugs that have become less attractive. Sulfonamides were used as TB drugs until the early 1950s, but, due to poor effectiveness and toxicity of their early forms, they were discontinued for the treatment of TB. Sulfamethoxazole (SMX), a sulfonamide that was approved in 1961 for the treatment of bacterial infections in humans and animals, is also well-tolerated by Mtb. PAS was first used as a TB drug in 1944. However, it is less effective than newer drugs and is required to be administered in a high dose. For these reasons PAS is no longer a first-line TB-drug but, instead, is a second- line drug used for MDR TB. Thus, an improvement in the effectiveness of SMX and PAS would bring major help in combating TB, especially the drug resistant forms of the disease. The proposed project will leverage one of our discoveries for making sulfonamides and PAS more effective in killing Mtb. Both of these compounds are anti-folates. Some of the folate synthesis enzymes activate these compounds, which in turn inhibit the folate biosynthesis system; sulfonamides inhibit even in their unmodified form. Our preliminary results suggest that Mtb use F420-gammaglutamyl-ligase (FbiB), a protein that is unrelated to folate biosynthesis, to counter the actions of SMX and PAS; consequently, a co-drug that inhibits FbiB will make SMX and PAS more effective TB-drugs. This protein, encoded by the fbiB gene, catalyzes the synthesis of the polyglutamate side chain of coenzyme F420. Both F420 and FbiB are found in all methanogenic archaea and certain bacteria including all mycobacteria, but are rarely found in eukarya and are absent in humans. A deletion of fbiB makes Mycolicibacterium smegmatis (Msmeg), a relative of Mtb, hypersensitive to SMX and PAS, and a complementation with the MtbfbiB gene restores the ability to tolerate high levels of these compounds. Based on preliminary analysis, we have developed two hypotheses: i) FbiB provides two alternate folate biosynthesizing enzymatic activities that do not activate PAS and are not sensitive to sulfonamides, PAS, and their activated forms. (ii) FbiB transforms or degrades these drugs into non-inhibitory compounds. In the proposed exploratory project, we will test these hypotheses through an investigation with the following specific aims. 1. To functionally and structurally characterize two folate biosynthesizing enzymatic activities of MtbFbiB and the effects of sulfonamides, PAS, and their activated forms on these activities. 2. To ch...