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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

Covalentbinding of ISO to HadAC105A-His in the E. coli activation system. (A) LC-MS analysis of the intact HadAC105A-His protein devoid of drug (untreated; top panel) and in covalentcomplex with ISO (bottom panel). The HadA-ISO complex exhibited alonger retention time on the HPLC column and, as expected, was notpresent in the untreated control (data not shown). Calculated masses:HadAC105A-His = 18268.24; HadAC105A-His-ISOwhere ISO is attached via an S–S bond = 18666.08. (B) Structureof the proposed HadAC105-ISO adduct purified from E. coli.
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fig4: Covalentbinding of ISO to HadAC105A-His in the E. coli activation system. (A) LC-MS analysis of the intact HadAC105A-His protein devoid of drug (untreated; top panel) and in covalentcomplex with ISO (bottom panel). The HadA-ISO complex exhibited alonger retention time on the HPLC column and, as expected, was notpresent in the untreated control (data not shown). Calculated masses:HadAC105A-His = 18268.24; HadAC105A-His-ISOwhere ISO is attached via an S–S bond = 18666.08. (B) Structureof the proposed HadAC105-ISO adduct purified from E. coli.

Mentions: To overcome this problem, we followeda similar Escherichia coli-based approachas that used previously to study the mechanism of inhibition of theenoyl-CoA reductase InhA by ETH.17 To thisend, ethA and hadAC105ABC (allowing for the expression of an N-terminalhexahistidine-tagged form of HadAC105A) were coexpressedin E. coli BL21(DE3), and HadAC105AB purifiedfrom treated and untreated E. coli cells was analyzedby LC-MS for covalent modification of HadAC105A by thedrugs. MS analyses of the HadAC105A-His-TAC adduct confirmedthe data previously obtained in BCG, whereas that of the HadAC105A-His-ISO complex revealed for the first time the natureof the active form of ISO in covalent linkage with its target; thefull-size protein exhibited a shift in mass of 398 amu in the ISO-treatedsample (Figure 4A) that matched the mass expectedfor the covalent HadAC105A-His-ISO adduct presented inFigure 4B. The results of these E.coli-based experiments further indicated that the coexpressionof ethA potentiated the formation of HadAC105A-His-TAC and -ISO complexes in the cells, although the presence ofthis gene is not an absolute requirement for the drugs to react withthe dehydratase. We believe this to be due either to the spontaneousoxidation of ISO and TAC inside the cells or to the existence of E. coli monooxygenase(s) capable of activating the two prodrugs.


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)

Covalentbinding of ISO to HadAC105A-His in the E. coli activation system. (A) LC-MS analysis of the intact HadAC105A-His protein devoid of drug (untreated; top panel) and in covalentcomplex with ISO (bottom panel). The HadA-ISO complex exhibited alonger retention time on the HPLC column and, as expected, was notpresent in the untreated control (data not shown). Calculated masses:HadAC105A-His = 18268.24; HadAC105A-His-ISOwhere ISO is attached via an S–S bond = 18666.08. (B) Structureof the proposed HadAC105-ISO adduct purified from E. coli.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401429&req=5

fig4: Covalentbinding of ISO to HadAC105A-His in the E. coli activation system. (A) LC-MS analysis of the intact HadAC105A-His protein devoid of drug (untreated; top panel) and in covalentcomplex with ISO (bottom panel). The HadA-ISO complex exhibited alonger retention time on the HPLC column and, as expected, was notpresent in the untreated control (data not shown). Calculated masses:HadAC105A-His = 18268.24; HadAC105A-His-ISOwhere ISO is attached via an S–S bond = 18666.08. (B) Structureof the proposed HadAC105-ISO adduct purified from E. coli.
Mentions: To overcome this problem, we followeda similar Escherichia coli-based approachas that used previously to study the mechanism of inhibition of theenoyl-CoA reductase InhA by ETH.17 To thisend, ethA and hadAC105ABC (allowing for the expression of an N-terminalhexahistidine-tagged form of HadAC105A) were coexpressedin E. coli BL21(DE3), and HadAC105AB purifiedfrom treated and untreated E. coli cells was analyzedby LC-MS for covalent modification of HadAC105A by thedrugs. MS analyses of the HadAC105A-His-TAC adduct confirmedthe data previously obtained in BCG, whereas that of the HadAC105A-His-ISO complex revealed for the first time the natureof the active form of ISO in covalent linkage with its target; thefull-size protein exhibited a shift in mass of 398 amu in the ISO-treatedsample (Figure 4A) that matched the mass expectedfor the covalent HadAC105A-His-ISO adduct presented inFigure 4B. The results of these E.coli-based experiments further indicated that the coexpressionof ethA potentiated the formation of HadAC105A-His-TAC and -ISO complexes in the cells, although the presence ofthis gene is not an absolute requirement for the drugs to react withthe dehydratase. We believe this to be due either to the spontaneousoxidation of ISO and TAC inside the cells or to the existence of E. coli monooxygenase(s) capable of activating the two prodrugs.

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