DRUG RESISTANT TUBERCULOSIS

Worldwide, tuberculosis is a leading cause of mortality: 2-3 million people die of the disease each year, 8-10 million new cases are reported, and the total number of infected individuals is ~1700 million. The development of multiple drug resistant M. tuberculosis strains is a major problem for immunocompromised patients, such as those with AIDS, and poses a long-term threat to the general population unless new therapeutic approaches are developed. Resistance to isoniazid, the first line drug, is due in many instances to mutations that inactivate the M. tuberculosis catalase-peroxidase, the enzyme responsible for activating isoniazid to its active (reactive) form. Inactivation of this protein poses a survival dilemma, as the catalase peroxidase contributes to the antioxidant defense that allows M. tuberculosis to spend much of its natural life pends in the oxidative environment of the macrophage.

Isoniazid is a one of the leading drugs used in the treatment of tuberculosis, but widespread resistance to this drug has developed. Loss of KatG catalase-peroxidase activity in isoniazid-resistant M. tuberculosis is associated with the elevated expression of a few genes, among which AhpC is the only one relevant to antioxidant defense. AhpD codes for a non-heme dependent alkylhydroperoxidase is a member of the peroxiredoxin class of enzymes. AhpD, which follows immediately after AhpC in the M. tuberculosis genome, has no sequence identity with AhpD. We have clones and characterized both the M. tuberculosis AhpC and AhpD and shown that, despite their lack of sequence identity, both proteins function as alkylhydroperoxidases. AhpC and AhpD are therefore potential targets for antituberculosis drugs, as their inhibition might compromise the ability of the isoniazid-resistant mycobacteria to survive in the host macrophage environment.

The mechanisms of the two proteins have not yet been fully elucidated, but like other AhpC proteins probably involve reaction of a sulfhydryl group with the peroxide to give a sulfenic acid (-S-OH) intermediate (Figure 1). Intramolecular reduction of this sulfenic acid by a second cysteine residue then yields a disulfide, which can be recycled to the resting enzyme by reaction with a reductive partner. We have confirmed the identities of the critical sulfhydryl groups in both AhpC and AhpD by site specific mutagenesis. In AhpC, Cys-61 probably reacts with the peroxide and is then reduced by either Cys-174 or Cys-176. In AhpD, Cys-130 appears to be the group that reacts with the peroxide, and Cys-133 the moiety that reduces the sulfenic acid intermediate. Crystallization of AphD in collaboration with a group in London has provided the first structure of an AhpD protein. This structure will facilitate mechanistic and inhibitor development studies.

Figure 1. General mechanism for the alkylhydroperoxidases. The reductive partner (Red) can vary and has not actually been identified in the case of M. tuberculosis. The tuberculosis enzyme can be turned over, however, with surrogate electron donor partners.

In a parallel effort, the gene coding for the protein that activates ethionamide to its active antitubercular form has been expressed and the protein has been partially characterized. The protein is a flavoprotein that oxidizes ethionamide, first to the sulfoxide product and finally to a doubly oxidized (sulfinic) species that is presumably responsible for the antimycobacterial activity. Resistance to ethionamide appears to be tightly linked to loss of the activity of the flavoprotein. Interestingly, resistance to ethionamide also results in elevation of AhpC. Mechanistic, structural, and drug design studies based on the ethionamide model are ongoing.

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