Drug resistant tuberculosis is a major opportunistic infection in AIDS patients and resistance to the available drugs is growing. Tuberculosis infection both increases the infectivity of the HIV virus and is a major cause of death in AIDS patients. This project focuses on the role of selected oxidation-reduction processes in the development of Mycobacterium tuberculosis drug resistance and the use of this information for the design of new antitubercular agents. Isoniazid, a first line drug, is activated by the KatG catalase peroxidase to a reactive intermediate. This intermediate, or one of its further reaction products, is responsible for interaction with the cell wall biosynthetic enzymes InhA and KasA-AcpM, the ultimate targets of the drug. Resistance to isoniazid is largely caused by mutations that disable KatG and is associated with the enhanced expression of AhpC and AhpD, two unrelated non-heme alkylhydroperoxidases (peroxiredoxins) that compensate for the loss of KatG . Inhibition of AhpC/AhpD in conjunction with isoniazid treatment is a potentially useful mechanism for counteracting isoniazid resistance. In the expiring grant period, AhpC and AhpD were cloned, expressed, and partially characterized and the crystal structure of AhpD was determined. Building on these results, further structural and mechanistic studies are to be carried out with AhpC/AhpD, and this information is to be used to develop potent and selective inhibitors of these two proteins. Ethionamide is another antitubercular prodrug. It is activated by the enzyme EtaA rather than by KaG but the activated species also appears to act at the level of InhA andor KasA-AcpM. Ethionamide resistance is due to impairment of EtaA activity and is associated with elevations in AhpC/AhpD similar to those observed with isoniazid. We have cloned and expressed EtaA and shown it to be a flavoprotein that catalyzes two sequential step in the activation of ethionamide and other thioamide drugs. We propose to complete characterization of EtaA, including the nature of the reactive intermediate, the mechanisms of its interactions with InhA and KasA-AcpM, and its biological importance. The information is to be used to synthesize agents that inhibit one of these two target proteins and, if warranted, EtaA.
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