TB DRUGS VIA INHIBITORS OF CELL WALL SYNTHETIC ENZYMES

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The thrust of the PO-1 is the development of new TB drugs against the cell wall of M. tuberculosis. Projects 1 & 2 focus on inhibiting mycolic acid synthesis and project 3 on the synthesis of the lipid polyisoprene carriers. This Project 4 rounds out the P01 by targeting the polysaccharide arabinogalactan (AG), which anchors the waxy mycolic acid layer to the inner peptidoglycan. AG is a proven drug target as it is the target of ethambutol. Also, we have a TS mutant of M. smegmatis that is complemented by the M. tuberculosis gene encoding the AG biosynthetic target enzyme, rhamnosyl transferase (WbbL). The non-complemented TS mutant is no longer viable after treatment at the non-permissive temperature. Earlier research has developed AG as a drug target by defining its structure and biosynthetic pathway, by cloning and expressing five essential biosynthetic enzymes, and by developing assays amenable to microtiter plate screening. Herein we propose to identify inhibitors of AG biosynthesis by screening potential inhibitors in AG biosynthetic enzyme assays. The assays include: the three enzymes (RmIB-D) required to form dTDP-rha, the enzyme (Gif) required to form UDP-Galf; the rhamnosyl transferase (WbbL); the enzymes which form decaprenylphosphoryl-D-arabinose; and, in a system requiring some development, the arabinosyl and galactofuranosyl transferases. Many complementary sources of potential inhibitors will be used. These include: 1) compounds known to inhibit the growth of M. tuberculosis where the mode of action is unknown, 2) compounds designed by a collaborating organic chemist based on knowledge of the crystal structures of some of the enzymes, 3) natural product mixtures which are known to inhibit growth of M. tuberculosis, and whole bacteria will be further pursued. Compound rights will usually remain with the original owner. The mission is to see that the screens, basic science, and collaborations with X-ray crystallographers and organic chemists are used to bring compounds as far forward as possible. Finally we will provide both the screening expertise and the actual compounds to other components of the PO1 including the development of inhibitors of deoxyxylulose-5 phosphate synthetase and the sterol demethylase (Project 3), the mycolyl transferase (Project 1) and mycolyl condensation enzyme (Project 2).