Two mycobacteria pose a major threat to individuals infected with the human immunodeficiency virus (HIV), Mycobacterium avium and Mycobacterium tuberculosis. Development of new drugs for M. avium and M. tuberculosis is an urgent necessity if efforts to control their effect on the outcome of acquired immunodeficiency syndrome (AIDS) are to be successful. The purpose of this project is to evaluate the antimycobacterial effectiveness of a unique class of drugs that inhibit mycobacterial dihydrofolate reductase (DHFR), a key enzyme in the biosynthetic pathway of folates that are necessary for RNA, DNA and protein synthesis. This enzyme represents a strategic target because of the ability to develop selective inhibitors that can be several thousand times more active against bacterial DHFR than mammalian DHFR. Through the efforts of the NIAID sponsored Tuberculosis Antimicrobial Acquisition Coordinating Facility (TAACF), several lipophilic DHFR inhibitors have been identified from a collection of 2,4-diamino-5-methyl-5-deazapteridine derivatives synthesized at Southern Research Institute (SRI). In the TAACF screening, these novel lipophilic antifolates exhibited low minimal inhibitory concentrations (MICs) against M. tuberculosis H37Rv, and low toxicity to Vero cells. At SRI, the derivatives have shown selective inhibition in a cell-free DHFR assay from M. avium serovar 4, a common serovar found in AIDS patients. Evaluation of the antimycobacterial activity of these novel lipophilic DHFR inhibitors will be conducted at three levels against M. avium and M. tuberculosis: 1) screening of 2,4-diamino-5-methyl-5-deazapterdine derivatives will be performed with an in vitro broth microdilution assay and cell-free mycobacterial DHFR assays and compared with toxicity to human cell lines and activity against recombinant human DHFR, 2) intracellular activity of selected compounds will be evaluated in murine and human monocytic cell line models and, 3) in vivo activity of candidate compounds will be determined in infected mouse models. M. avium and M. tuberculosis H37Ra DHFR will be purified and sequenced for comparison with, and cloning of, recombinant DHFR. The DHFR gene will be cloned from M. avium and M. tuberculosis H37Ra. Recombinant DHFR will be expressed and purified in order to obtain sufficient quantities for specific inhibition studies to be used to develop derivatives with improved selectivity. With information from this study, a comparative molecular field analysis (CoMFA) will be used to predict more effective and selective DHFR inhibitors. These studies will eventually lead to the development of site directed lipophilic DHFR inhibitors using X-ray crystallographic and molecular graphic techniques.
