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Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria.

Alahari A, Trivelli X, Guérardel Y, Dover LG, Besra GS, Sacchettini JC, Reynolds RC, Coxon GD, Kremer L - PLoS ONE (2007)

Bottom Line: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation.Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope.The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier II et I, Centre National de Recherche Scientifique (CNRS), UMR 5235, Montpellier, France.

ABSTRACT

Background: Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.

Methodology/principle findings: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the alpha- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs.

Conclusions/significance: This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

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Related in: MedlinePlus

Structures of chemical analogues of thiacetazone and their corresponding minimum inhibitory concentrations (MICs) in M. tb H37Rv.MICs were determined by BACTEC 460 radiometric assay. *Data from MABA assay.
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pone-0001343-g002: Structures of chemical analogues of thiacetazone and their corresponding minimum inhibitory concentrations (MICs) in M. tb H37Rv.MICs were determined by BACTEC 460 radiometric assay. *Data from MABA assay.

Mentions: TAC was purchased from Sigma (Saint Louis, MO), while SRI-224 and other chemical analogues of TAC were synthesized at the Southern Research Institute (Birmingham, AL). The chemical synthesis of the analogues will be documented separately. A primary screen was conducted by the BACTEC 460 radiometric assay at an initial screening concentration of 12.5 µg/ml against M. tb H37Rv (ATCC 27294) in BACTEC 12B medium as reported [30]. Two samples (entries 11 and 16, Figure 2) were screened against M. tb H37Rv in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA) [30]. Drug stocks were prepared in DMSO for dilution, and the stocks were filter sterilized and stored at –70°C until usage. Compounds demonstrating at least 90% inhibition in the primary screen were tested again at lower concentrations in the BACTEC 460 system against M. tb H37Rv to determine the minimum inhibitory concentration (MIC). The MIC is defined as the lowest concentration effecting a reduction in fluorescence of 99% relative to controls. For entry 11 in Figure 2, MIC90 was derived using the MABA assay. It has been reported that the MABA MIC90 correlates well with a MIC99 for the BACTEC 460 assay [30].


Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria.

Alahari A, Trivelli X, Guérardel Y, Dover LG, Besra GS, Sacchettini JC, Reynolds RC, Coxon GD, Kremer L - PLoS ONE (2007)

Structures of chemical analogues of thiacetazone and their corresponding minimum inhibitory concentrations (MICs) in M. tb H37Rv.MICs were determined by BACTEC 460 radiometric assay. *Data from MABA assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2147073&req=5

pone-0001343-g002: Structures of chemical analogues of thiacetazone and their corresponding minimum inhibitory concentrations (MICs) in M. tb H37Rv.MICs were determined by BACTEC 460 radiometric assay. *Data from MABA assay.
Mentions: TAC was purchased from Sigma (Saint Louis, MO), while SRI-224 and other chemical analogues of TAC were synthesized at the Southern Research Institute (Birmingham, AL). The chemical synthesis of the analogues will be documented separately. A primary screen was conducted by the BACTEC 460 radiometric assay at an initial screening concentration of 12.5 µg/ml against M. tb H37Rv (ATCC 27294) in BACTEC 12B medium as reported [30]. Two samples (entries 11 and 16, Figure 2) were screened against M. tb H37Rv in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA) [30]. Drug stocks were prepared in DMSO for dilution, and the stocks were filter sterilized and stored at –70°C until usage. Compounds demonstrating at least 90% inhibition in the primary screen were tested again at lower concentrations in the BACTEC 460 system against M. tb H37Rv to determine the minimum inhibitory concentration (MIC). The MIC is defined as the lowest concentration effecting a reduction in fluorescence of 99% relative to controls. For entry 11 in Figure 2, MIC90 was derived using the MABA assay. It has been reported that the MABA MIC90 correlates well with a MIC99 for the BACTEC 460 assay [30].

Bottom Line: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation.Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope.The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier II et I, Centre National de Recherche Scientifique (CNRS), UMR 5235, Montpellier, France.

ABSTRACT

Background: Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.

Methodology/principle findings: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the alpha- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs.

Conclusions/significance: This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

Show MeSH
Related in: MedlinePlus