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Covalent modification of the Mycobacterium tuberculosis FAS-II dehydratase by Isoxyl and Thiacetazone.

Grzegorzewicz AE, Eynard N, Quémard A, North EJ, Margolis A, Lindenberger JJ, Jones V, Korduláková J, Brennan PJ, Lee RE, Ronning DR, McNeil MR, Jackson M - ACS Infect Dis (2015)

Bottom Line: We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity.Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs.Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

View Article: PubMed Central - PubMed

Affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.

ABSTRACT

Isoxyl and Thiacetazone are two antitubercular prodrugs formerly used in the clinical treatment of tuberculosis. Although both prodrugs have recently been shown to kill Mycobacterium tuberculosis through the inhibition of the dehydration step of the type II fatty acid synthase pathway, their detailed mechanism of inhibition, the precise number of enzymes involved in their activation and the nature of their activated forms remained unknown. We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity. Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs. Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

No MeSH data available.


Related in: MedlinePlus

Covalent binding of ISO and TAC to HadAC105A-His in whole M. bovis BCG cells. (A) HadAC105A-His was partially purified from recombinant M. bovis BCG cells that were either untreated or treated with ISO or TACas in Figure 2. The samples annotated as ISO-DTTand TAC-DTT were incubated with 500 mM DTT for 5 min at 70 °Cprior to SDS-PAGE. (B) LC-MS analysis of the intact HadAC105A-His protein devoid of drug (untreated) and in covalent complex withTAC. The ESI mass spectra show the mass of HadAC105A-Hisin the untreated control and both the masses of HadAC105A-His and HadAC105A-His in covalent complex with TAC (massexpected from attachment to Cys61 via an S–S bond) in the TAC-treatedsample. Calculated masses: HadAC105A-His = 18381.45; HadAC105A-His-TAC = 18,615.15; (∗) an unidentified proteinwas observed in the TAC-treated sample. (C) HadAC105A-Hispurified from TAC-treated BCG cells was in-gel trypsin digested, andthe peptides were analyzed by LC-MS/MS. Cys61-containing tryptic peptidein complex with TAC at charge states (+2) and (+3) = 938.48 (M + 2H)/2and 625.99 (M + 3H)/3, respectively. (D) Structure of the proposedHadAC105-TAC adduct purified from M. bovis BCG. (E) Activity of the HadAC105A-His proteins purifiedfrom drug-treated and untreated M. bovis BCG cellsshown in (A). Kinetic assays in the presence of C12:1-CoA were performedas described in the SI and initial velocitiesdetermined by linear fitting. Shown are the average initial velocities± standard deviations of at least three independent measurements.
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fig3: Covalent binding of ISO and TAC to HadAC105A-His in whole M. bovis BCG cells. (A) HadAC105A-His was partially purified from recombinant M. bovis BCG cells that were either untreated or treated with ISO or TACas in Figure 2. The samples annotated as ISO-DTTand TAC-DTT were incubated with 500 mM DTT for 5 min at 70 °Cprior to SDS-PAGE. (B) LC-MS analysis of the intact HadAC105A-His protein devoid of drug (untreated) and in covalent complex withTAC. The ESI mass spectra show the mass of HadAC105A-Hisin the untreated control and both the masses of HadAC105A-His and HadAC105A-His in covalent complex with TAC (massexpected from attachment to Cys61 via an S–S bond) in the TAC-treatedsample. Calculated masses: HadAC105A-His = 18381.45; HadAC105A-His-TAC = 18,615.15; (∗) an unidentified proteinwas observed in the TAC-treated sample. (C) HadAC105A-Hispurified from TAC-treated BCG cells was in-gel trypsin digested, andthe peptides were analyzed by LC-MS/MS. Cys61-containing tryptic peptidein complex with TAC at charge states (+2) and (+3) = 938.48 (M + 2H)/2and 625.99 (M + 3H)/3, respectively. (D) Structure of the proposedHadAC105-TAC adduct purified from M. bovis BCG. (E) Activity of the HadAC105A-His proteins purifiedfrom drug-treated and untreated M. bovis BCG cellsshown in (A). Kinetic assays in the presence of C12:1-CoA were performedas described in the SI and initial velocitiesdetermined by linear fitting. Shown are the average initial velocities± standard deviations of at least three independent measurements.

Mentions: C61G and C61S point mutationsin HadA are the most common amino acid changes associated with high-levelISO and TAC resistance in the FAS-II dehydratases of Mtb and Mycobacterium bovis BCG and resultin >20–80-fold increases in MIC, respectively.2,8,11,12 This observation raised the possibility that the inhibition of thedehydratase activity of FAS-II by the prodrugs resulted from the formationof a covalent complex between residue Cys61 and the thiocarbonyl moietyof ISO and TAC. To test this hypothesis, M. bovis BCG expressing a C-terminal His-tagged recombinant form of HadA(HadAWT-His) was incubated for 15 h with ISO (10 μg/mL;4 × MIC; the solubility limit of ISO in this culture medium)or TAC (10 μg/mL; 20 × MIC) at 37 °C with shakingand subsequently lysed in Tris-HCl buffer [see the Supporting Information (SI)]. These experiments were performedin situ using whole mycobacterial cells rather than in vitro usingpurified HadAB to ensure the proper activation of the prodrugs. Previousattempts to incubate purified HadAB with ISO and TAC in a cell-freeassay had indeed failed to reveal any inhibition of the dehydrataseby the drugs.8 HadAWT-His fromdrug-treated and untreated cells was partially purified by affinitychromatography and analyzed by SDS-PAGE. As expected, HadAWT-His copurified with untagged HadB13 (Figure 2). Changes affecting the migration of HadAWT-His were clearly visible in the ISO- and TAC-treated cells. AlthoughHadAWT-His from untreated cells yielded a single proteinband of the expected size (18.4 kDa), two forms of HadAWT-His were detected in the drug-treated samples. One of them migratedsimilarly to HadAWT-His from untreated BCG, and the othermigrated with an apparent slightly lower molecular weight. Consistentwith the likely reactivity of the drugs with Cys61, this migrationshift was not observed when a C61S mutant of HadA (HadAC61S-His), similarly expressed and purified from M. bovis BCG, was used (see the SI) (Figure 2), and high-level resistance to ISO and TAC wasnoted for this recombinant strain (MIC of both drugs >25 μg/mLcompared to MICs of 2.5 μg/mL for ISO and 0.5 μg/mL forTAC against WT M. bovis BCG or BCG expressing HadAWT-His). Subsequent purification of the treated and untreatedforms of HadAWT-His from the gel and analysis of the full-sizeproteins and corresponding derived tryptic peptides by liquid chromatographytandem mass spectrometry (LC-MS/MS) under denaturing conditions (seethe SI) unexpectedly revealed that drugtreatment had caused the oxidation of the two cysteines in HadA toform a disulfide bond as demonstrated by the presence in the drug-treatedsamples of a protein approximately 2 amus lower in mass than the reducedform of HadAWT (Figure S1A-C). Consistent with this finding, the SDS-PAGE migration shift ofHadAWT-His in the drug-treated samples was reversed upontreatment with dithiothreitol (DTT) (Figure 2), and only the reduced form of HadAWT-His was presentby LC-MS analysis (Figure S1A). Suspectingthat the only other cysteine in HadA (Cys105) reacted with the Cys61drug complexes to form the oxidized protein, we next sought to repeatthese experiments in the absence of thiols by mutating Cys105 to analanine. That Cys105 is not required for the drugs to inhibit HadABwas supported by the fact that the MICs of ISO and TAC against BCGexpressing HadAC105A-His are similar to those against BCGexpressing HadAWT-His or wild-type M. bovis BCG (2.5 μg/mL for ISO; 0.5 μg/mL for TAC). WhereasHadAC105A-His from untreated BCG cells yielded a singleprotein band of the expected size (18.3 kDa), HadAC105A-His from the drug-treated samples migrated as two more diffuse bands(Figure 3A). Reduction of the samples withDTT prior to SDS-PAGE reversed the migration shift of HadAC105A-His in the ISO- and TAC-treated samples (Figure 3A). MS analysis of the treated and untreated forms of HadAC105A-His revealed that the full-size protein and derived Cys61-containingpeptides exhibited a shift in mass of 234 amu in the TAC-treated sample(Figure 3B,C) which matched that expected forthe covalent HadAC105A-His-TAC complex presented in Figure 3D. This shows that a single site on the Cys61-containingpeptides was covalently modified with one TAC metabolite. Whetherdue to the insufficient amount or degree of purity of the materialpurified from the ISO-treated BCG cells or otherwise, MS analysisfailed to reveal the nature of the HadAC105A-His-ISO complex.


Covalent modification of the Mycobacterium tuberculosis FAS-II dehydratase by Isoxyl and Thiacetazone.

Grzegorzewicz AE, Eynard N, Quémard A, North EJ, Margolis A, Lindenberger JJ, Jones V, Korduláková J, Brennan PJ, Lee RE, Ronning DR, McNeil MR, Jackson M - ACS Infect Dis (2015)

Covalent binding of ISO and TAC to HadAC105A-His in whole M. bovis BCG cells. (A) HadAC105A-His was partially purified from recombinant M. bovis BCG cells that were either untreated or treated with ISO or TACas in Figure 2. The samples annotated as ISO-DTTand TAC-DTT were incubated with 500 mM DTT for 5 min at 70 °Cprior to SDS-PAGE. (B) LC-MS analysis of the intact HadAC105A-His protein devoid of drug (untreated) and in covalent complex withTAC. The ESI mass spectra show the mass of HadAC105A-Hisin the untreated control and both the masses of HadAC105A-His and HadAC105A-His in covalent complex with TAC (massexpected from attachment to Cys61 via an S–S bond) in the TAC-treatedsample. Calculated masses: HadAC105A-His = 18381.45; HadAC105A-His-TAC = 18,615.15; (∗) an unidentified proteinwas observed in the TAC-treated sample. (C) HadAC105A-Hispurified from TAC-treated BCG cells was in-gel trypsin digested, andthe peptides were analyzed by LC-MS/MS. Cys61-containing tryptic peptidein complex with TAC at charge states (+2) and (+3) = 938.48 (M + 2H)/2and 625.99 (M + 3H)/3, respectively. (D) Structure of the proposedHadAC105-TAC adduct purified from M. bovis BCG. (E) Activity of the HadAC105A-His proteins purifiedfrom drug-treated and untreated M. bovis BCG cellsshown in (A). Kinetic assays in the presence of C12:1-CoA were performedas described in the SI and initial velocitiesdetermined by linear fitting. Shown are the average initial velocities± standard deviations of at least three independent measurements.
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fig3: Covalent binding of ISO and TAC to HadAC105A-His in whole M. bovis BCG cells. (A) HadAC105A-His was partially purified from recombinant M. bovis BCG cells that were either untreated or treated with ISO or TACas in Figure 2. The samples annotated as ISO-DTTand TAC-DTT were incubated with 500 mM DTT for 5 min at 70 °Cprior to SDS-PAGE. (B) LC-MS analysis of the intact HadAC105A-His protein devoid of drug (untreated) and in covalent complex withTAC. The ESI mass spectra show the mass of HadAC105A-Hisin the untreated control and both the masses of HadAC105A-His and HadAC105A-His in covalent complex with TAC (massexpected from attachment to Cys61 via an S–S bond) in the TAC-treatedsample. Calculated masses: HadAC105A-His = 18381.45; HadAC105A-His-TAC = 18,615.15; (∗) an unidentified proteinwas observed in the TAC-treated sample. (C) HadAC105A-Hispurified from TAC-treated BCG cells was in-gel trypsin digested, andthe peptides were analyzed by LC-MS/MS. Cys61-containing tryptic peptidein complex with TAC at charge states (+2) and (+3) = 938.48 (M + 2H)/2and 625.99 (M + 3H)/3, respectively. (D) Structure of the proposedHadAC105-TAC adduct purified from M. bovis BCG. (E) Activity of the HadAC105A-His proteins purifiedfrom drug-treated and untreated M. bovis BCG cellsshown in (A). Kinetic assays in the presence of C12:1-CoA were performedas described in the SI and initial velocitiesdetermined by linear fitting. Shown are the average initial velocities± standard deviations of at least three independent measurements.
Mentions: C61G and C61S point mutationsin HadA are the most common amino acid changes associated with high-levelISO and TAC resistance in the FAS-II dehydratases of Mtb and Mycobacterium bovis BCG and resultin >20–80-fold increases in MIC, respectively.2,8,11,12 This observation raised the possibility that the inhibition of thedehydratase activity of FAS-II by the prodrugs resulted from the formationof a covalent complex between residue Cys61 and the thiocarbonyl moietyof ISO and TAC. To test this hypothesis, M. bovis BCG expressing a C-terminal His-tagged recombinant form of HadA(HadAWT-His) was incubated for 15 h with ISO (10 μg/mL;4 × MIC; the solubility limit of ISO in this culture medium)or TAC (10 μg/mL; 20 × MIC) at 37 °C with shakingand subsequently lysed in Tris-HCl buffer [see the Supporting Information (SI)]. These experiments were performedin situ using whole mycobacterial cells rather than in vitro usingpurified HadAB to ensure the proper activation of the prodrugs. Previousattempts to incubate purified HadAB with ISO and TAC in a cell-freeassay had indeed failed to reveal any inhibition of the dehydrataseby the drugs.8 HadAWT-His fromdrug-treated and untreated cells was partially purified by affinitychromatography and analyzed by SDS-PAGE. As expected, HadAWT-His copurified with untagged HadB13 (Figure 2). Changes affecting the migration of HadAWT-His were clearly visible in the ISO- and TAC-treated cells. AlthoughHadAWT-His from untreated cells yielded a single proteinband of the expected size (18.4 kDa), two forms of HadAWT-His were detected in the drug-treated samples. One of them migratedsimilarly to HadAWT-His from untreated BCG, and the othermigrated with an apparent slightly lower molecular weight. Consistentwith the likely reactivity of the drugs with Cys61, this migrationshift was not observed when a C61S mutant of HadA (HadAC61S-His), similarly expressed and purified from M. bovis BCG, was used (see the SI) (Figure 2), and high-level resistance to ISO and TAC wasnoted for this recombinant strain (MIC of both drugs >25 μg/mLcompared to MICs of 2.5 μg/mL for ISO and 0.5 μg/mL forTAC against WT M. bovis BCG or BCG expressing HadAWT-His). Subsequent purification of the treated and untreatedforms of HadAWT-His from the gel and analysis of the full-sizeproteins and corresponding derived tryptic peptides by liquid chromatographytandem mass spectrometry (LC-MS/MS) under denaturing conditions (seethe SI) unexpectedly revealed that drugtreatment had caused the oxidation of the two cysteines in HadA toform a disulfide bond as demonstrated by the presence in the drug-treatedsamples of a protein approximately 2 amus lower in mass than the reducedform of HadAWT (Figure S1A-C). Consistent with this finding, the SDS-PAGE migration shift ofHadAWT-His in the drug-treated samples was reversed upontreatment with dithiothreitol (DTT) (Figure 2), and only the reduced form of HadAWT-His was presentby LC-MS analysis (Figure S1A). Suspectingthat the only other cysteine in HadA (Cys105) reacted with the Cys61drug complexes to form the oxidized protein, we next sought to repeatthese experiments in the absence of thiols by mutating Cys105 to analanine. That Cys105 is not required for the drugs to inhibit HadABwas supported by the fact that the MICs of ISO and TAC against BCGexpressing HadAC105A-His are similar to those against BCGexpressing HadAWT-His or wild-type M. bovis BCG (2.5 μg/mL for ISO; 0.5 μg/mL for TAC). WhereasHadAC105A-His from untreated BCG cells yielded a singleprotein band of the expected size (18.3 kDa), HadAC105A-His from the drug-treated samples migrated as two more diffuse bands(Figure 3A). Reduction of the samples withDTT prior to SDS-PAGE reversed the migration shift of HadAC105A-His in the ISO- and TAC-treated samples (Figure 3A). MS analysis of the treated and untreated forms of HadAC105A-His revealed that the full-size protein and derived Cys61-containingpeptides exhibited a shift in mass of 234 amu in the TAC-treated sample(Figure 3B,C) which matched that expected forthe covalent HadAC105A-His-TAC complex presented in Figure 3D. This shows that a single site on the Cys61-containingpeptides was covalently modified with one TAC metabolite. Whetherdue to the insufficient amount or degree of purity of the materialpurified from the ISO-treated BCG cells or otherwise, MS analysisfailed to reveal the nature of the HadAC105A-His-ISO complex.

Bottom Line: We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity.Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs.Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

View Article: PubMed Central - PubMed

Affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.

ABSTRACT

Isoxyl and Thiacetazone are two antitubercular prodrugs formerly used in the clinical treatment of tuberculosis. Although both prodrugs have recently been shown to kill Mycobacterium tuberculosis through the inhibition of the dehydration step of the type II fatty acid synthase pathway, their detailed mechanism of inhibition, the precise number of enzymes involved in their activation and the nature of their activated forms remained unknown. We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity. Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs. Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

No MeSH data available.


Related in: MedlinePlus