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

Abstract

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...

Synthesis and Evaluation of l-Rhamnose 1C-Phosphonates as Nucleotidylyltransferase Inhibitors

“…6 Cell wall biosynthesis is a commonly pursued target for a variety of bacterial enzyme inhibitors as cell wall assembly is essential for bacterial survival and virulence. L-Rhamnose (6-deoxy-L-mannose) serves as the linking unit between arabinogalactan and peptidoglycan 7,8 and is a necessary constituent of the bacterial cell wall in many bacterial species. 9 The biosynthesis of L-rhamnose begins with nucleotidylyltransferase enzymes (Cps2L/RmlA) responsible for generating activated sugars in the form of glycosylated nucleoside diphosphates (NDPs).…”

Metabolism of Plasma Membrane Lipids in Mycobacteria and Corynebacteria

“…A PIM biosynthetic membrane, enriched in the early steps, has been purified by sucrose gradient fractionation as a membrane subdomain termed PMf, which is distinct from the bulk plasma membrane [102]. Mannosyltransferases performing the early steps utilize the water-soluble mannose donor, GDP-Man, which can be produced from exogenously acquired mannose or via de novo synthesis from the glycolytic pathway when fructose-6-phosphate is transformed by the enzymes ManA (Rv3255c), ManB (Rv3257c) and ManC (Rv3264c) [108][109][110][111], with a degree of redundancy reported at the ManC (NCgl0710) step in C. glutamicum [112]. The first step of PIM synthesis involves mannosylation of the C2-position of the inositol ring of PI by the enzyme PimA (Rv2610c) to form PIM1 [113,114].…”

Activated Sugar Precursors: Biosynthetic Pathways and Biological Roles of an Important Class of Intermediate Metabolites in Bacteria

“…This component is connected, via the linker disaccharide -L-rhamnosyl-(1→3)--D-N-acetylglucosaminosyl-1-phosphate, to the 6 position of a muramic acid residue of the inner component peptidoglycan. Presently, it is known that M. tuberculosis strains have increased resistance to the antimicrobials agents in use, and therefore new antituberculosis drugs are necessary (Ma et al, 2001). In this context, the four enzyme activities (RmlA to RmlD) involved in the dTDP-L-rhamnose biosynthetic pathway have been studied as attractive targets for the development of new antimicrobials.…”

“…For example, in Shigella and Salmonella species, the O-antigen repeating unit is mainly constituted by L-rhamnose (Van den Bosch et al, 1997). The L-rhamnosyl residue has also an essential structural role in the cell wall of Mycobacterium tuberculosis (Ma et al, 2001). The donor of the L-rhamnose moiety found in bacterial structures is deoxythymidinediphosphate (dTDP)-L-rhamnose.…”

“…The described multi-step pathway does not exist in humans, being these four enzyme activities potential targets for the design of new therapeutic agents, as is the case of the development of new antimycobacterial agents, an area of intensive research (Ma et al, 2001). …”