Drug targeting Mycobacterium tuberculosis cell wall synthesis: genetics of dTDP-rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-rhamnose.

An L-rhamnosyl residue plays an essential structural role in the cell wall of Mycobacterium tuberculosis. Therefore, the four enzymes (RmlA to RmlD) that form dTDP-rhamnose from dTTP and glucose-1-phosphate are important targets for the development of new tuberculosis therapeutics. M. tuberculosis genes encoding RmlA, RmlC, and RmlD have been identified and expressed in Escherichia coli. It is shown here that genes for only one isotype each of RmlA to RmlD are present in the M. tuberculosis genome. The gene for RmlB is Rv3464. Rv3264c was shown to encode ManB, not a second isotype of RmlA. Using recombinant RmlB, -C, and -D enzymes, a microtiter plate assay was developed to screen for inhibitors of the formation of dTDP-rhamnose. The three enzymes were incubated with dTDP-glucose and NADPH to form dTDP-rhamnose and NADP(+) with a concomitant decrease in optical density at 340 nm (OD(340)). Inhibitor candidates were monitored for their ability to lower the rate of OD(340) change. To test the robustness and practicality of the assay, a chemical library of 8,000 compounds was screened. Eleven inhibitors active at 10 microM were identified; four of these showed activities against whole M. tuberculosis cells, with MICs from 128 to 16 microg/ml. A rhodanine structural motif was present in three of the enzyme inhibitors, and two of these showed activity against whole M. tuberculosis cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth of M. tuberculosis in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 microg/ml.

Recognition of the lipid intermediate for arabinogalactan/arabinomannan biosynthesis and its relation to the mode of action of ethambutol on mycobacteria.

Despite major advances in our understanding of the structure of mycobacterial cell walls, little is known of their biogenesis, and yet they are the site of action of many anti-tuberculosis drugs and implicated in much of the pathology of tuberculosis and leprosy. A family of monoglycosyl polyprenylphosphates was isolated from Mycobacterium smegmatis, containing arabinose, ribose, and mannose. The isoprenoid nature of the lipid components was established by 1H NMR, and fast atom bombardment mass spectroscopy (FAB-MS) demonstrated the presence of C50 decaprenyl-P derivatives and smaller amounts of the C35 octahydroheptaprenyl-P products. The configuration of the mycobacterial decaprenol was established as mono-trans, octa-cis, pointing to carriers of unusual structure. Combined gas chromatography (GC)/MS, FAB-MS/MS, and 1H NMR allowed characterization of one of the primary components as beta-D-arabinofuranosyl-1-monophosphodecaprenol. Pulse-chase metabolic labeling of cells with D-[14C]glucose indicated that the decaprenyl-P-arabinose is an active intermediate in the biosynthesis of the arabinan of cell wall arabinogalactan and arabinomannan. The identification of polyprenyl-P-ribose suggests the existence of ribose-containing polysaccharides in the cell walls of M. smegmatis or/and of a novel epimerase in the D-arabinose biosynthetic pathway. Ethambutol, a powerful anti-tuberculosis drug known to inhibit arabinogalactan and arabinomannan biosynthesis, results in the rapid accumulation of decaprenyl-P-arabinose, indicating that the drug interferes with either the transfer of arabinose from the donor or, alternatively, the synthesis of the arabinose acceptor itself.