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 M were identified; four of these showed activities against whole M. tuberculosis cells, with MICs from 128 to 16 g/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 g/ml.The necessity for new drugs against Mycobacterium tuberculosis due to increasing resistance to the present chemotherapeutic agents is well documented (6,12,21,33,41,44). An attractive target for such new agents is the mycobacterial cell wall (2,3,29), since the wall is necessary for viability and several known drugs such as isoniazid (52) and ethambutol (11, 49) inhibit cell wall synthesis. The mycobacterial cell wall core consists of three interconnected macromolecules (Fig. 1). The outermost, the mycolic acids, are 70-to 90-carbon-containing, branched fatty acids which form an outer lipid layer in some ways similar to the classical outer membrane of gram-negative bacteria (5). The mycolic acids are esterified to the middle component, arabinogalactan (AG), a polymer composed primarily of D-galactofuranosyl and D-arabinofuranosyl residues. AG is connected via a linker disaccharide, ␣-L-rhamnosyl-(133)-␣-D-N-acetyl-glucosaminosyl-1-phosphate, to the 6 position of a muramic acid residue in the peptidoglycan. The peptidoglycan is the innermost of the three cell wall core macromolecules.This structural arrangement shows why AG is necessary for mycobacterial viability, as it tethers the lipid layer to the peptidoglycan layer. Moreover, a rhamnosyl residue, a sugar not found in humans, plays a crucial structural role in the attachment of AG to peptidoglycan (Fig. 1). (L-Rhamnosyl residues are found in other bacteria as components of O anti...

Two novel alkaloids, named manadomanzamines A (1) and B (2), were isolated from an Indonesian sponge Acanthostrongylophora sp. (Haplosclerida: Petrosiidae). Their structures were elucidated and shown to be a novel organic skeleton related to the manzamine type alkaloids. Their absolute configuration and conformation were determined by CD, NOESY, and molecular modeling analysis. The microbial community analysis for the sponge that produces these unprecedented alkaloids has also been completed. Manadomanzamines A (1) and B (2) exhibited strong activity against Mycobacterium tuberculosis (Mtb) with MIC values of 1.9 and 1.5 µg/mL, respectively. Manadomanzamines A and B also exhibit activities against human immunodeficiency virus (HIV-1) and AIDS opportunistic fungal infections.