Through the NIAID-sponsored Tuberculosis Anti-microbial Acquisition and Coordinating Facility (TAACF), we have identified numerous anti-Mycobacterium tuberculosis lead compounds, which are structurally similar to purine bases and nucleosides. In the current grant period (1999 to 2003) we have (1) synthesized many new compounds and evaluated them for activity against M. tb, (2) characterized the biochemical pharmacology of one of the lead compounds (2-methyladenosine), and (3) begun the characterization of the enzymes involved in purine salvage in M. tb. In the new grant proposal we plan to continue these studies to further our understanding of the enzymes involved in the purine salvage pathway in M. tb and to design and synthesize new agents with selective activity against M. tb. Much is known about the substrate requirements of human enzymes involved in purine salvage, because of the considerable effort to develop nucleoside analogs for the treatment of cancer and viral diseases. However, very little is currently known about the substrate characteristics of these enzymes in M. tb. We have identified 3 purine salvage enzymes that are expressed in M. tb that could be exploited in the development of new selective anti-/W. tb agents. Two of these enzymes (adenosine cleavage and guanosine kinase) are not expressed in human cells and we have recently shown that the third enzyme (adenosine kinase) has unique characteristics that could also be used for selective activation of nucleoside analogs. The biochemical and genetic characterization of these enzymes should provide valuable information that will be useful in the rational design and development of new agents based on metabolic differences in purine metabolism between humans and M. tb. The proposed specific aims to accomplish the goals of this grant proposal are: (1) identification, cloning, expression, and purification of M. tb adenosine cleavage and guanosine kinase activities; (2) biochemical characterization of M. tb adenosine kinase, adenosine cleavage, and guanosine kinase activities; (3) metabolic studies with new agents that have potent and selective anti-M. tb activity; and (4) design and synthesis of purine and purine nucleoside analogs with selective activity against M. tb.
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