Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis

Larsen, Michelle H.; Vilchèze, Catherine; Kremer, Laurent; Besra, Gurdyal S.; Parsons, Linda M.; Salfinger, Max; Heifets, Leonid; Hazbón, Manzour Hernando; Alland, David; Sacchettini, James C.; Jacobs, William R.

doi:10.1046/j.1365-2958.2002.03162.x

Cited by 187 publications

(154 citation statements)

References 61 publications

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“…In addition, a single mutation in the inhA gene confers resistance to both INH and ETH (12). As shown in Table III, overexpression of inhA in M. smegmatis leads to a 20-fold increase in resistance to ETH, in agreement with a recent study (28) demonstrating that overexpression of inhA in M. smegmatis, M. bovis BCG, and M. tuberculosis increases resistance against both INH and ETH. These observations suggest that the mode of action of both drugs is identical, although their modes of activation differ.…”

Section: Induction Of the Kasa-containing Complex By Inha-inhibitory supporting

confidence: 90%

“…To date, no one has performed a gene transfer experiment demonstrating that any of these mutations confer INH resistance to susceptible strains of mycobacteria. Furthermore, the failure of overexpression of kasA in M. bovis BCG (27), M. tuberculosis, and M. smegmatis (28) to confer resistance to INH is inconsistent with a previous report (29) which raises doubts as to whether KasA plays a relevant role in INH action. Clearly, additional in vivo and in vitro studies are required in order to determine the potential role of KasA and the participation of the proposed ternary AcpMKasA-INH complex in INH and ETH resistance.…”

Section: Isoniazid (Inh)mentioning

confidence: 62%

“…Due to conflicting reports, the mode of action of INH and the structurally related drug ETH has recently been re-examined. A recent study (28) showed that overexpression of inhA, but not of kasA, confers resistance to INH and ETH in M. smegmatis, M. bovis BCG, and M. tuberculosis, and it was therefore concluded that InhA represents the primary target of action of INH and ETH in all three species. On the other hand, KasA was found to be covalently associated with AcpM and a low molecular weight molecule, which was postulated to be a reactive intermediate of INH, to form an 80-kDa complex in M. tuberculosis, leading to the proposal that KasA represents the primary target of INH (13).…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Kremer

Dover

Morbidoni

et al. 2003

Journal of Biological Chemistry

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(6) further demonstrated that the inhibition of mycolic acid biosynthesis mediated by INH leads to the accumulation of a saturated C 26 fatty acid within 1 h of INH treatment. By using scanning electron microscopy, it was established that M. tuberculosis treated with INH undergoes a defined order of molecular events that leads to lysis 24 h after initiation of treatment, thus after approximately one cell generation (7). The events that lead from the inhibition of mycolic acid biosynthesis and the accumulation of a saturated C 26 fatty acid to cell lysis are still poorly understood. Moreover, there exists controversy as to which enzymes of the mycolic acid biosynthetic pathway are the actual targets of INH.The mode of action of INH appears to be rather complex and requires activation by the mycobacterial catalase-peroxidase KatG (8,9). Activation of the pro-drug leads to reactive radicals that exert a toxic effect on the tubercle bacillus (9 -11). Presumably activated INH inhibits one or more targets of the mycolic acid biosynthetic pathway which eventually leads to cell death. At least two enzymes have been identified as potential targets of activated INH, the enoyl-ACP reductase InhA (12) and the ␤-ketoacyl-ACP synthase KasA (13). The inhA gene was first identified by conferring co-resistance to INH and ethionamide (ETH) after overexpression in mycobacteria (12). In addition, mutations in inhA confer resistance to both INH and ETH in drug-resistant M. tuberculosis isolates (14 -17).

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Section: Induction Of the Kasa-containing Complex By Inha-inhibitory supporting

confidence: 90%

Section: Isoniazid (Inh)mentioning

confidence: 62%

Section: Discussionmentioning

confidence: 99%

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Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Kremer

Dover

Morbidoni

et al. 2003

Journal of Biological Chemistry

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“…However, although there is convincing evidence that InhA is inhibited by INH, Barry and coworkers (18) have also proposed that KasA, one of three ketoacyl synthases in the FASII pathway (Scheme 2), is a target for INH in vivo. Subsequent experiments involving the effect of drugs on gene (19,20) and protein expression (21), and studying the effect of InhA and KasA expression on drug resistance (20,22), have highlighted the apparent complexity in the mode of action of INH.To better understand the molecular basis for INH resistance in clinical isolates carrying InhA mutations, we purified and isolated the INH-NAD adduct and quantified its affinity toward WT and drug-resistant mutants. Our studies show that INH-NAD is a slow, tight-binding competitive inhibitor of InhA that binds with an overall dissociation constant of K i ϭ 0.75 Ϯ 0.08 nM.…”

mentioning

confidence: 99%

The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: Adduct affinity and drug resistance

Rawat

Whitty²,

Tonge³

2003

Proc. Natl. Acad. Sci. U.S.A.

288

254

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Isoniazid (INH), a frontline antitubercular drug, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. Here, we report that the INH-NAD adduct is a slow, tight-binding competitive inhibitor of InhA. Demonstration that the adduct binds to WT InhA by a two-step enzyme inhibition mechanism, with initial, weak binding (K ؊1 ‫؍‬ 16 ؎ 11 nM) followed by slow conversion to a final inhibited complex (EI*) with overall K i ‫؍‬ 0. 75 A ttempts to treat tuberculosis, a disease that kills more than two million people every year, are hindered by the spread of multidrug-resistant strains of Mycobacterium tuberculosis (MDRTB), the causative agent, and by the increased susceptibility of HIV-positive individuals to this disease (1-4). Although isoniazid (INH; Scheme 1) has been the most effective and widely used drug for the treatment of tuberculosis since the 1950s, the mode of action of this compound is still not completely understood. INH is a prodrug that is activated by the mycobacterial catalase-peroxidase enzyme KatG (Scheme 1) (5-8) and a substantial fraction of all clinical isolates that are resistant to INH result from KatG mutations (2, 9-11). Consequently, compounds that inhibit the ultimate molecular target(s) of INH, but that do not require activation by KatG, have tremendous promise as novel drugs for combating MDRTB.Two enzymes, InhA and KasA, have been proposed as targets for INH. Both are members of the type II dissociated fatty acid biosynthesis pathway (FASII) in M. tuberculosis (Scheme 2), consistent with the observation that INH interferes with the biosynthesis of mycolic acids, very long chain fatty acid components of the mycobacterial cell wall. InhA, an enoyl reductase that catalyzes the NADH-dependent reduction of long chain trans-2-enoyl-acyl carrier proteins (ACPs), was first identified as a target by Jacobs and coworkers (6, 12) who observed mutations in the inhA gene in INH-resistant clinical isolates and identified a point mutant (S94A) that conferred resistance to INH and ethionamide in Mycobacterium smegmatis and in Mycobacterium bovis. Subsequently, Blanchard, Sacchettini, and coworkers (13-15) demonstrated that InhA was inhibited in vitro by a covalent adduct formed between activated INH and the nicotinamide head group of NAD (Scheme 1). InhA mutations observed in INH-resistant clinical isolates were found to be localized to the cofactor binding site and were shown to result in decreased affinity of the purified enzyme for NADH (12,16), consistent with the hypothesis that binding of NADH to the enzyme precedes adduct formation. In addition, Vilcheze et al. (17) used a temperature-sensitive mutation in the inhA gene to show that the phenotypic response to InhA inactivation in M. smegmatis was identical to that caused by treatment with INH, thereby validating InhA as a target for drug discovery. However, although there is convincing evidence that InhA is inhibited by INH, Barry and coworkers (18) have also proposed that KasA, on...

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“…Consistent with InhA as the major target of INH mode of action, inactivation of the M. tuberculosis inhAencoded enoyl reductase and INH treatment resulted in similar morphological changes to the mycobacterial cell wall leading to cell lysis (Vilchèze et al 2000). Overexpression of inhA has been shown to confer resistance to INH and ethionamide (ETH) in M. smegmatis, M. bovis BCG, and M. tuberculosis (Larsen et al 2002). Further biochemical and genetic evidence has been given likewise showing that InhA is the primary target of INH The arabinogalactan is a branched-chain polysaccharide consisting of a proximal galactose chain linked to a distal arabinose chain.…”

Section: Biosynthesis Of Mycolic Acids (Fas-ii System)mentioning

confidence: 92%

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

Basso

Silva

Fett-Neto

et al. 2005

Mem. Inst. Oswaldo Cruz

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Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis

Cited by 187 publications

References 61 publications

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: Adduct affinity and drug resistance

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

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