Michael S. Scherman

Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.

Decaprenylphosphoryl Arabinofuranose, the Donor of the d-Arabinofuranosyl Residues of Mycobacterial Arabinan, Is Formed via a Two-Step Epimerization of Decaprenylphosphoryl Ribose

The major cell wall polysaccharide of mycobacteria is a branched-chain arabinogalactan in which arabinan chains are attached to the 5 carbon of some of the 6-linked galactofuranose residues; these arabinan chains are composed exclusively of D-arabinofuranose (Araf) residues. The immediate precursor of the polymerized Araf is decaprenylphosphoryl-D-Araf, which is derived from 5-phosphoribose 1-diphosphate (pRpp) in an undefined manner. On the basis of time course, feedback, and chemical reduction experiment results we propose that decaprenylphosphoryl-Araf is synthesized by the following sequence of events. (i) pRpp is transferred to a decaprenyl-phosphate molecule to form decaprenylphosphoryl-beta-D-5-phosphoribose. (ii) Decaprenylphosphoryl-beta-D-5-phosphoribose is dephosphorylated to form decaprenylphosphoryl-beta-D-ribose. (iii) The hydroxyl group at the 2 position of the ribose is oxidized and is likely to form decaprenylphosphoryl-2-keto-beta-D-erythro-pentofuranose. (iv) Decaprenylphosphoryl-2-keto-beta-D-erythro-pentofuranose is reduced to form decaprenylphosphoryl-beta-D-Araf. Thus, the epimerization of the ribosyl to an arabinosyl residue occurs at the lipid-linked level; this is the first report of an epimerase that utilizes a lipid-linked sugar as a substrate. On the basis of similarity to proteins implicated in the arabinosylation of the Azorhizobium caulidans nodulation factor, two genes were cloned from the Mycobacterium tuberculosis genome and expressed in a heterologous host, and the protein was purified. Together, these proteins (Rv3790 and Rv3791) are able to catalyze the epimerization, although neither protein individually is sufficient to support the activity.

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 inEscherichia 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 (OD340). Inhibitor candidates were monitored for their ability to lower the rate of OD340change. To test the robustness and practicality of the assay, a chemical library of 8,000 compounds was screened. Eleven inhibitors active at 10 μM were identified; four of these showed activities against whole M. tuberculosis cells, with MICs from 128 to 16 μg/ml. A rhodanine structural motif was present in three of the enzyme inhibitors, and two of these showed activity against wholeM. tuberculosis cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth ofM. tuberculosis in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 μg/ml.

Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope.

Ethambutol is known to rapidly inhibit biosynthesis of the arabinan component of the mycobacterial cell wall core polymer, arabinogalactan (K. Takayama and J. O. Kilburn, Antimicrob. Agents Chemother. 33:1493-1499, 1989). This effect was confirmed, and it was also shown that ethambutol inhibits biosynthesis of the arabinan of lipoarabinomannan, a lipopolysaccharide noncovalently associated with the cell wall core. In contrast to cell wall core arabinan, which is completely inhibited by ethambutol, synthesis of the arabinan of lipoarabinomannan was only partially affected, demonstrating a differential effect on arabinan synthesis in the two locales. Further studies of the effect of ethambutol on cell wall biosynthesis revealed that the synthesis of galactan in the cell wall core is strongly inhibited by the drug. In addition, ethambutol treatment resulted in the cleavage of arabinosyl residues present in the mycobacterial cell wall; more than 50% of the arabinan in the cell wall core was removed from the wall 1 h after addition of the drug to growing mycobacterial cultures. In contrast, galactan was not released from the cell wall during ethambutol treatment. The natural function of the arabinosyl-releasing enzyme remains unknown, but its action in combination with inhibition of synthesis during ethambutol treatment results in severe disruption of the mycobacterial cell wall. Accordingly, ethambutol-induced damage to the cell wall provides a ready molecular explanation for the known synergetic effects of ethambutol with other chemotherapeutic agents. Nevertheless, the initial direct effect of ethambutol remains to be elucidated.

Polyprenylphosphate-pentoses in Mycobacteria Are Synthesized from 5-Phosphoribose Pyrophosphate

Polyprenylphosphate-arabinose (in which the polyprenyl unit is found both as decaprenyl and octahydroheptaprenyl) is a donor of mycobacterial cell wall arabinosyl residues. Because of this important role, its biosynthetic pathway, and that of the related lipid, polyprenylphosphate-D-ribose, was investigated. Surprisingly, phosphoribose pyrophosphate was shown to be a key intermediate on the pathway to both polyprenylphosphate-D-pentoses. Thus, incubation of 5-phospho-D-[14C]ribose pyrophosphate with membranes prepared from Mycobacterium smegmatis resulted in the presence of organic-soluble radioactivity that was shown to be, in part, polyprenylphosphate-[14C]arabinose and polyprenylphosphate-[14C]ribose. Two additional intermediates, polyprenylphosphate-5-phospho[14C]ribose and polyprenylphosphate-5-phospho[14C]arabinose, were identified. Further experiments showed that the mature polyprenylphosphate-ribose is formed from phosphoribose pyrophosphate via a two-step pathway involving a transferase to form polyprenylphosphate-5-phosphoribose and then a phosphatase to form the final polyprenylphosphateribose. Polyprenylphosphate-arabinose is formed by a similar pathway with an additional step being the epimerization at C-2 of the ribosyl residue. This epimerization occurs at either the level of phosphoribose pyrophosphate or at the level of polyprenylphosphate-5-phosphoribose.

Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues

SETTING Mycobacterial galactofuran is essential to the linking of the peptidoglycan and mycolic acid cell wall layers. Galactofuran biosynthesis should thus be essential for viability. OBJECTIVE The objective was to determine the pathway of galactofuranosyl biosynthesis and to clone a gene encoding an essential enzyme necessary for its formation. DESIGN Specific enzymatic conversions involved in formation of galactopyranose and galactofuranose residues in other bacteria were tested for in Mycobacterium smegmatis. M. tuberculosis deoxyribonucleic acid (DNA) was identified by homology. RESULTS It was shown that the de novo synthesis of the galactose carbon skeleton occurred in M. smegmatis by the transformation of UDP-glucopyranose to UDP-galactopyranose via the enzyme UDP-glucose 4-epimerase (E.C. 5.1.3.2). The N-terminal sequence of this enzyme was obtained after purification. The galactose salvage pathway enzyme, UDP-glucose-galactose-1-phosphate uridylyltransferase (E.C. 2.7.7.12), was also shown to be present. The critical biosynthetic transformation of the galactopyranose to galactofuranose ring form was shown to occur at the sugar nucleotide level via the enzyme UDP-galactopyranose mutase (E.C. 5.4.99.9). The M. tuberculosis DNA encoding this enzyme was sequenced, the gene expressed in Escherichia coli, and the expected enzymatic activity demonstrated. CONCLUSION Galactofuranose biosynthesis can now be pursued as a potential drug target in M. tuberculosis.

Spectinamides: a new class of semisynthetic antituberculosis agents that overcome native drug efflux

Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, we generated a new semisynthetic series of spectinomycin analogs with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross resistance with existing tuberculosis therapeutics, activity against multidrug-resistant (MDR) and extensively drug-resistant tuberculosis and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage-induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target-based tuberculosis drug discovery.

A microbiological assessment of novel nitrofuranylamides as anti-tuberculosis agents

OBJECTIVES Nitrofuranylamides (NFAs) are nitroaromatic compounds that have recently been discovered and have potent anti-tuberculosis (TB) activity. A foundational study was performed to evaluate whether this class of agents possesses microbiological properties suitable for future antimycobacterial therapy. METHODS Five representative compounds of the NFA series were evaluated by standard microbiological assays to determine MICs, MBCs, activity against anaerobic non-replicating persistent Mycobacterium tuberculosis, post-antibiotic effects (PAEs), antibiotic synergy and the basis for resistance. RESULTS The antimicrobial activity of these compounds was restricted to bacteria of the M. tuberculosis complex, and all compounds were highly active against drug-susceptible and -resistant strains of M. tuberculosis, with MICs 0.0004-0.05 mg/L. Moreover, no antagonism was observed with front-line anti-TB drugs. Activity was also retained against dormant bacilli in two in vitro low-oxygen models for M. tuberculosis persistence. A long PAE was observed, which was comparable to that of rifampicin, but superior to isoniazid and ethambutol. Spontaneous NFA-resistant mutants arose at a frequency of 10(-5)-10(-7), comparable to that for isoniazid (10(-5)-10(-6)). Some of these mutants exhibited cross-resistance to one or both of the nitroimidazoles PA-824 and OPC-67683. Cross-resistance was associated with inactivation of the reduced F(420)-deazaflavin cofactor pathway and not with inactivation of the Rv3547, the nitroreductase for PA-824 and OPC-67683. CONCLUSIONS Based on these studies, NFAs have many useful antimycobacterial properties applicable to TB chemotherapy and probably possess a unique mode of action that results in good activity against active and dormant M. tuberculosis. Therefore, the further development of lead compounds in this series is warranted.

Inactivation of the Mycobacterial Rhamnosyltransferase, Which Is Needed for the Formation of the Arabinogalactan-Peptidoglycan Linker, Leads to Irreversible Loss of Viability

Temperature-sensitive mutant 2-20/32 of Mycobacterium smegmatis mc2155 was isolated and genetically complemented with a Mycobacterium tuberculosis H37Rv DNA fragment that contained a single open reading frame. This open reading frame is designated Rv3265c in the M. tuberculosis H37Rv genome. Rv3265c shows homology to the Escherichia coli gene wbbL, which encodes a dTDP-Rha:α-d-GlcNAc-pyrophosphate polyprenol, α-3-l-rhamnosyltransferase. In E. coli this enzyme is involved in O-antigen synthesis, but in mycobacteria it is required for the rhamnosyl-containing linker unit responsible for the attachment of the cell wall polymer mycolyl-arabinogalactan to the peptidoglycan. The M. tuberculosis wbbL homologue, encoded by Rv3265c, was shown to be capable of restoring an E. coli K12 strain containing an insertionally inactivated wbbL to O-antigen positive. Likewise, the E. coli wbbL gene allowed 2-20/32 to grow at higher non-permissive temperatures. The rhamnosyltransferase activity of M. tuberculosis WbbL was demonstrated in 2-20/32 as was the loss of this transferase activity in 2-20/32 at elevated temperatures. The wbbL of the temperature-sensitive mutant contained a single-base change that converted what was a proline in mc2155 to a serine residue. Exposure of 2-20/32 to higher non-permissive temperatures resulted in bacteria that could not be recovered at the lower permissive temperatures.

Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Development of a Microtiter Plate-Based Screen for UDP-Galactopyranose Mutase and Identification of an Inhibitor from a Uridine-Based Library

ABSTRACT A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.