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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

Isoxyl,thiacetazone, and the dehydration step of the FAS-II elongation cycle:(A) structures of ISO and TAC; (B) (3R)-hydroxyacyldehydratases HadAB and HadBC catalyze the (reversible) dehydrationof β-hydroxyacyl-ACP meromycolate precursors to yield trans-Δ2-enoyl-ACP products (the substrates of theenoyl-CoA reductase, InhA) in the FAS-II elongation cycle. The HadABheterodimer is thought to be involved in the early stages of the elongationcycle, whereas the HadBC heterodimer, which displays a greater affinityfor longer fatty acyl substrates than HadAB in vitro, is believedto participate in the late stages of the elongation of the meromycolicacid chain.
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fig1: Isoxyl,thiacetazone, and the dehydration step of the FAS-II elongation cycle:(A) structures of ISO and TAC; (B) (3R)-hydroxyacyldehydratases HadAB and HadBC catalyze the (reversible) dehydrationof β-hydroxyacyl-ACP meromycolate precursors to yield trans-Δ2-enoyl-ACP products (the substrates of theenoyl-CoA reductase, InhA) in the FAS-II elongation cycle. The HadABheterodimer is thought to be involved in the early stages of the elongationcycle, whereas the HadBC heterodimer, which displays a greater affinityfor longer fatty acyl substrates than HadAB in vitro, is believedto participate in the late stages of the elongation of the meromycolicacid chain.

Mentions: The continuingrise of multidrug-resistant tuberculosis (TB) throughout the worldplaces a high priority on the development of new anti-TB drugs withbactericidal mechanisms different from those of the presently availableagents. In this context, elucidating the mechanism of action of drugsformerly deemed efficient in the treatment of TB may prove usefulin identifying validated targets of therapeutic interest and developingnew anti-TB agents with greater potency, improved pharmacokinetics,and reduced toxicity. Two such drugs, isoxyl (ISO) and thiacetazone(TAC), display minimal inhibitory concentrations (MIC) against clinicalisolates of Mycobacterium tuberculosis (Mtb), including multidrug-resistant ones, in theranges of 1–10 and 0.1–0.5 μg/mL, respectively.1,2 Both are thiocarbamide-containing prodrugs (Figure 1A) that require activation of their thiocarbonyl moiety bythe flavin-dependent monooxygenase EthA for bactericidal activity.3−6 Although the oxidation of ISO and TAC by purified EthA in vitrohas led to the identification of a number of metabolites of thosedrugs,4,6 the nature of their reactive intermediate(s)is still unknown. Moreover, while the finding in drug-resistant mutantsof Mtb of missense and frameshift mutations affectingmycolic acid methyltransferases has led to speculations that otherenzymes may be required for the activation of ISO and TAC, biochemicalproof for this assumption has been lacking.2,7,8 Following activation, ISO and TAC inhibitmycolic acid biosynthesis, resulting in bacterial death.1,5,9 ISO and TAC thus target the samecritical pathway as the TB drugs isoniazid (INH) and ethionamide (ETH),albeit through a distinct mechanism independent of the enoyl-CoA reductaseInhA.10 Indeed, recent biochemical andgenetic evidence established that ISO and TAC inhibit the dehydrationstep of the type II fatty acid synthase (FAS-II) elongation cycle2,8,11,12 (Figure 1B). Due to our limited understandingof how the two (3R)-hydroxyacyl-ACP-dehydratase heterodimersof FAS-II, HadAB and HadBC,13 functionin whole cells and of the complexity of FAS-II wherein protein interactionsgovern the activity and substrate specificity of the entire complex,14−16 details of the molecular mechanism of action of ISO and TAC werelacking. In particular, earlier studies failed to establish whetherthe two drugs acted as direct inhibitors of one or the two dehydratasesor rather as molecules perturbing protein–protein interactionswithin FAS-II in a way that indirectly abolished the activity of theseenzymes.8 The present studies were undertakenwith the goal of resolving these issues.


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)

Isoxyl,thiacetazone, and the dehydration step of the FAS-II elongation cycle:(A) structures of ISO and TAC; (B) (3R)-hydroxyacyldehydratases HadAB and HadBC catalyze the (reversible) dehydrationof β-hydroxyacyl-ACP meromycolate precursors to yield trans-Δ2-enoyl-ACP products (the substrates of theenoyl-CoA reductase, InhA) in the FAS-II elongation cycle. The HadABheterodimer is thought to be involved in the early stages of the elongationcycle, whereas the HadBC heterodimer, which displays a greater affinityfor longer fatty acyl substrates than HadAB in vitro, is believedto participate in the late stages of the elongation of the meromycolicacid chain.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Isoxyl,thiacetazone, and the dehydration step of the FAS-II elongation cycle:(A) structures of ISO and TAC; (B) (3R)-hydroxyacyldehydratases HadAB and HadBC catalyze the (reversible) dehydrationof β-hydroxyacyl-ACP meromycolate precursors to yield trans-Δ2-enoyl-ACP products (the substrates of theenoyl-CoA reductase, InhA) in the FAS-II elongation cycle. The HadABheterodimer is thought to be involved in the early stages of the elongationcycle, whereas the HadBC heterodimer, which displays a greater affinityfor longer fatty acyl substrates than HadAB in vitro, is believedto participate in the late stages of the elongation of the meromycolicacid chain.
Mentions: The continuingrise of multidrug-resistant tuberculosis (TB) throughout the worldplaces a high priority on the development of new anti-TB drugs withbactericidal mechanisms different from those of the presently availableagents. In this context, elucidating the mechanism of action of drugsformerly deemed efficient in the treatment of TB may prove usefulin identifying validated targets of therapeutic interest and developingnew anti-TB agents with greater potency, improved pharmacokinetics,and reduced toxicity. Two such drugs, isoxyl (ISO) and thiacetazone(TAC), display minimal inhibitory concentrations (MIC) against clinicalisolates of Mycobacterium tuberculosis (Mtb), including multidrug-resistant ones, in theranges of 1–10 and 0.1–0.5 μg/mL, respectively.1,2 Both are thiocarbamide-containing prodrugs (Figure 1A) that require activation of their thiocarbonyl moiety bythe flavin-dependent monooxygenase EthA for bactericidal activity.3−6 Although the oxidation of ISO and TAC by purified EthA in vitrohas led to the identification of a number of metabolites of thosedrugs,4,6 the nature of their reactive intermediate(s)is still unknown. Moreover, while the finding in drug-resistant mutantsof Mtb of missense and frameshift mutations affectingmycolic acid methyltransferases has led to speculations that otherenzymes may be required for the activation of ISO and TAC, biochemicalproof for this assumption has been lacking.2,7,8 Following activation, ISO and TAC inhibitmycolic acid biosynthesis, resulting in bacterial death.1,5,9 ISO and TAC thus target the samecritical pathway as the TB drugs isoniazid (INH) and ethionamide (ETH),albeit through a distinct mechanism independent of the enoyl-CoA reductaseInhA.10 Indeed, recent biochemical andgenetic evidence established that ISO and TAC inhibit the dehydrationstep of the type II fatty acid synthase (FAS-II) elongation cycle2,8,11,12 (Figure 1B). Due to our limited understandingof how the two (3R)-hydroxyacyl-ACP-dehydratase heterodimersof FAS-II, HadAB and HadBC,13 functionin whole cells and of the complexity of FAS-II wherein protein interactionsgovern the activity and substrate specificity of the entire complex,14−16 details of the molecular mechanism of action of ISO and TAC werelacking. In particular, earlier studies failed to establish whetherthe two drugs acted as direct inhibitors of one or the two dehydratasesor rather as molecules perturbing protein–protein interactionswithin FAS-II in a way that indirectly abolished the activity of theseenzymes.8 The present studies were undertakenwith the goal of resolving these issues.

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