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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

Proposed mechanism for generation of mycolic acid sub-types by the action of CMAS enzymes (A) and inhibition by TAC/SRI-224 (B).The generation of α- and the oxygenated mycolic acids is considered to follow to two independent pathways. A common, di-unsaturated precursor, Y, is envisaged for the two pathways. Y is subsequently transformed into α-mycolic acids by the action of the MmaA2 and PcaA that modify the distal or proximal double bond, respectively. Action of MmaA4 commits Y to the pathway for the oxygenated mycolic acids, by producing the precursor X. MmaA3, which is required for generation of methoxy-mycolic acids in M. tb is inactive in M. bovis BCG Pasteur due to the presence of a point mutation [50]. The proximal double bond is modified by the CmaA2 (and MmaA2) or PcaA to generate trans- or cis-cyclopropanated derivatives, respectively. In the presence of TAC, all the CMASs mentioned above are inhibited, except for MmaA4. Due to inhibition of MmaA2, excess of Y is diverted to MmaA4 leading to generation of X, which accumulates due to lack of activities of CmaA2 and MmaA2. SRI-224 appears to affect MmaA4 to a certain degree, leading to accumulation Y in addition to X. (For simplicity, only the meromycolyl moiety of mycolates has been depicted).
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pone-0001343-g008: Proposed mechanism for generation of mycolic acid sub-types by the action of CMAS enzymes (A) and inhibition by TAC/SRI-224 (B).The generation of α- and the oxygenated mycolic acids is considered to follow to two independent pathways. A common, di-unsaturated precursor, Y, is envisaged for the two pathways. Y is subsequently transformed into α-mycolic acids by the action of the MmaA2 and PcaA that modify the distal or proximal double bond, respectively. Action of MmaA4 commits Y to the pathway for the oxygenated mycolic acids, by producing the precursor X. MmaA3, which is required for generation of methoxy-mycolic acids in M. tb is inactive in M. bovis BCG Pasteur due to the presence of a point mutation [50]. The proximal double bond is modified by the CmaA2 (and MmaA2) or PcaA to generate trans- or cis-cyclopropanated derivatives, respectively. In the presence of TAC, all the CMASs mentioned above are inhibited, except for MmaA4. Due to inhibition of MmaA2, excess of Y is diverted to MmaA4 leading to generation of X, which accumulates due to lack of activities of CmaA2 and MmaA2. SRI-224 appears to affect MmaA4 to a certain degree, leading to accumulation Y in addition to X. (For simplicity, only the meromycolyl moiety of mycolates has been depicted).

Mentions: Collation of current knowledge of CMAS enzymology and the data presented here on the structures of mycolic acids synthesized in the presence of TAC or its analogues, leads us to propose a model for the action of these drugs. A simplified schematic shown in Figure 8A illustrates generation of the mycolic acid sub-types in M. bovis BCG as a result of action of different CMAS enzymes. Lipid Y, which is unsaturated at the distal and proximal positions, can be regarded as a common precursor for generation of both α- and keto-mycolates, and could serve as a substrate for both MmaA2 and MmaA4. MmaA2 would introduce a distal cis-cyclopropane and thus commit Y to the α-mycolic acid branch of synthesis, while action of MmaA4 would initiate synthesis of the oxygenated lipid X, committing it to keto-mycolate synthesis [23], [46]. Next, PcaA or CmaA2 would introduce the proximal cyclopropane rings [12], [41], generating either the α- or keto- mycolates, respectively. From the structural analysis of the lipids X and Y, it appears that the CMASs are differentially inhibited by TAC or SRI-224. According to our model (Figure 8B), MmaA2 is most strongly inhibited by TAC, while MmaA4 is largely unaffected. We speculate that in TAC-treated cells, owing to the inhibition of MmaA2 activity, the initial excess of common precursor Y would be shunted to serve as a substrate for MmaA4 resulting in synthesis of X, which accumulates due to lack of activity of CmaA2 and MmaA2. Thus, treatment with TAC led to a more significant reduction in the content of α-mycolates compared to the keto-mycolates with concomitant build-up of X. A similar but opposite situation has been reported in a MmaA4 knock-out mutant [46], wherein the Y is shunted to serve as a substrate for MmaA2 instead, leading to lack of oxygenated mycolates with concomitant accumulation of precursors of α-mycolates. Treatment with SRI-224 appears to inhibit MmaA4 as well as the other CMASs, leading to accumulation of the precursor Y. Thus, despite their high degree of structural homology [21], [47], the CMASs show differential sensitivity to TAC and SRI-224, resulting in abnormal ratios of mycolates.


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)

Proposed mechanism for generation of mycolic acid sub-types by the action of CMAS enzymes (A) and inhibition by TAC/SRI-224 (B).The generation of α- and the oxygenated mycolic acids is considered to follow to two independent pathways. A common, di-unsaturated precursor, Y, is envisaged for the two pathways. Y is subsequently transformed into α-mycolic acids by the action of the MmaA2 and PcaA that modify the distal or proximal double bond, respectively. Action of MmaA4 commits Y to the pathway for the oxygenated mycolic acids, by producing the precursor X. MmaA3, which is required for generation of methoxy-mycolic acids in M. tb is inactive in M. bovis BCG Pasteur due to the presence of a point mutation [50]. The proximal double bond is modified by the CmaA2 (and MmaA2) or PcaA to generate trans- or cis-cyclopropanated derivatives, respectively. In the presence of TAC, all the CMASs mentioned above are inhibited, except for MmaA4. Due to inhibition of MmaA2, excess of Y is diverted to MmaA4 leading to generation of X, which accumulates due to lack of activities of CmaA2 and MmaA2. SRI-224 appears to affect MmaA4 to a certain degree, leading to accumulation Y in addition to X. (For simplicity, only the meromycolyl moiety of mycolates has been depicted).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001343-g008: Proposed mechanism for generation of mycolic acid sub-types by the action of CMAS enzymes (A) and inhibition by TAC/SRI-224 (B).The generation of α- and the oxygenated mycolic acids is considered to follow to two independent pathways. A common, di-unsaturated precursor, Y, is envisaged for the two pathways. Y is subsequently transformed into α-mycolic acids by the action of the MmaA2 and PcaA that modify the distal or proximal double bond, respectively. Action of MmaA4 commits Y to the pathway for the oxygenated mycolic acids, by producing the precursor X. MmaA3, which is required for generation of methoxy-mycolic acids in M. tb is inactive in M. bovis BCG Pasteur due to the presence of a point mutation [50]. The proximal double bond is modified by the CmaA2 (and MmaA2) or PcaA to generate trans- or cis-cyclopropanated derivatives, respectively. In the presence of TAC, all the CMASs mentioned above are inhibited, except for MmaA4. Due to inhibition of MmaA2, excess of Y is diverted to MmaA4 leading to generation of X, which accumulates due to lack of activities of CmaA2 and MmaA2. SRI-224 appears to affect MmaA4 to a certain degree, leading to accumulation Y in addition to X. (For simplicity, only the meromycolyl moiety of mycolates has been depicted).
Mentions: Collation of current knowledge of CMAS enzymology and the data presented here on the structures of mycolic acids synthesized in the presence of TAC or its analogues, leads us to propose a model for the action of these drugs. A simplified schematic shown in Figure 8A illustrates generation of the mycolic acid sub-types in M. bovis BCG as a result of action of different CMAS enzymes. Lipid Y, which is unsaturated at the distal and proximal positions, can be regarded as a common precursor for generation of both α- and keto-mycolates, and could serve as a substrate for both MmaA2 and MmaA4. MmaA2 would introduce a distal cis-cyclopropane and thus commit Y to the α-mycolic acid branch of synthesis, while action of MmaA4 would initiate synthesis of the oxygenated lipid X, committing it to keto-mycolate synthesis [23], [46]. Next, PcaA or CmaA2 would introduce the proximal cyclopropane rings [12], [41], generating either the α- or keto- mycolates, respectively. From the structural analysis of the lipids X and Y, it appears that the CMASs are differentially inhibited by TAC or SRI-224. According to our model (Figure 8B), MmaA2 is most strongly inhibited by TAC, while MmaA4 is largely unaffected. We speculate that in TAC-treated cells, owing to the inhibition of MmaA2 activity, the initial excess of common precursor Y would be shunted to serve as a substrate for MmaA4 resulting in synthesis of X, which accumulates due to lack of activity of CmaA2 and MmaA2. Thus, treatment with TAC led to a more significant reduction in the content of α-mycolates compared to the keto-mycolates with concomitant build-up of X. A similar but opposite situation has been reported in a MmaA4 knock-out mutant [46], wherein the Y is shunted to serve as a substrate for MmaA2 instead, leading to lack of oxygenated mycolates with concomitant accumulation of precursors of α-mycolates. Treatment with SRI-224 appears to inhibit MmaA4 as well as the other CMASs, leading to accumulation of the precursor Y. Thus, despite their high degree of structural homology [21], [47], the CMASs show differential sensitivity to TAC and SRI-224, resulting in abnormal ratios of mycolates.

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