Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid

Vilchèze, Catherine; Wang, Feng; Arai, Masayoshi; Hazbón, Manzour Hernando; Colangeli, Roberto; Kremer, Laurent; Weisbrod, Torin R.; Alland, David; Sacchettini, James C.; Jacobs, William R.

doi:10.1038/nm1466

Cited by 292 publications

(316 citation statements)

References 14 publications

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“…14 More recently, it was shown by specialized linkage transduction that the introduction of inhA (S94A) point mutation in M. tuberculosis confers clinically relevant levels of resistance to INH killing and inhibition of mycolic acid biosynthesis. 15 INH is a pro-drug that is activated by the mycobacterial KatG-encoded catalase-peroxidase enzyme in the presence of manganese ions, NAD(H), and oxygen. 16 The KatGproduced acylpyridine fragment of isoniazid is covalently attached to the C4 position of NADH, and this isonicotinyl-NAD + adduct forms a binary complex with InhA, 17 which is a slow, tight-binding competitive inhibitor with an overall dissociation constant (K i *) value of 0.75 × 10 -9 mol L -1 .…”

Section: Introductionmentioning

confidence: 99%

An inorganic complex that inhibits Mycobacterium tuberculosis enoyl reductase as a prototype of a new class of chemotherapeutic agents to treat tuberculosis

Basso¹,

Schneider²,

Santos³

et al. 2010

J. Braz. Chem. Soc.

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Descrevemos a atividade inibitória do IQG607, pentaciano(isoniazida)ferrato(II), frente a cepas de Mycobacterium tuberculosis tanto resistentes quanto sensíveis à isoniazida, assim como a toxicidade oral e a adaptação da síntese química do IQG607 para reatores maiores. O IQG607 representa um potencial agente quimioterápico que inibe um alvo molecular definido.Here we describe the inhibitory activity of IQG607, pentacyano(isoniazid)ferrate(II), on isoniazid-sensitive and isoniazid-resistant strains of Mycobacterium tuberculosis, its oral toxicity, and efforts to adapt IQG607 synthesis to large chemical reactors. IQG607 represents a promising chemotherapeutic agent aiming at the inhibition of a validated and druggable molecular target. Keywords: Mycobacterium tuberculosis, enoyl reductase, toxicology, large-scale synthesis, metallodrug IntroductionTuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. 1 In 2007, there were an estimated 9.27 million cases of TB, and 1.37 million (15%) were also HIV-positive patients, who are more likely to develop active TB. 2 Brazil ranks 14 th in terms of the total number of incident cases amongst the high-burden countries. 2 There were an estimated 0.5 million cases of multidrugresistant TB (MDR-TB), which is caused by strains resistant to, at least, isoniazid and rifampicin. 2 The emergence of extensively drug-resistant (XDR) TB cases, which are found in TB-infected patients whose isolates are MDR and also resistant to a fluoroquinolone and, at least, one second-line injectable agent, 2,3 its widespread distribution, 4 and unprecedented fatality rate, 5 raise the prospect of virtually incurable and deadly TB worldwide. The factors that most influence the emergence of drugresistant strains include inappropriate treatment regimens and patient noncompliance in completing the prescribed courses of therapy due to the lengthy standard "shortcourse" treatment (isoniazid, rifampicin, pyrazinamide, and ethambutol or streptomycin for two months, followed by a combination of isoniazid and rifampicin for additional four months) or when the side effects become unbearable. 6 Moreover, no sustainable control of TB epidemic can be reached in any country without properly addressing this global public health problem, including research as a key component. 7 M. tuberculosis has been considered the world's most successful pathogen, and this is largely due to the ability of the bacillum to persist in host tissues, where drugs that are rapidly bactericidal in vitro require prolonged administration to achieve comparable in vivo effects. 8 Hence, more effective and less toxic anti-tubercular agents are immediately needed to shorten the duration of current treatment, improve the treatment of drug-resistant TB, and to provide effective treatment of latent TB infection.The modern approach in the development of new chemical entities (NCEs) against TB is based on the use of defined molecular targets. This involves (i) the search and identification ...

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Section: Introductionmentioning

confidence: 99%

An inorganic complex that inhibits Mycobacterium tuberculosis enoyl reductase as a prototype of a new class of chemotherapeutic agents to treat tuberculosis

Basso¹,

Schneider²,

Santos³

et al. 2010

J. Braz. Chem. Soc.

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“…Two enzymes of the mycobacterial FASII system are already targets of antimycobacterial drugs: the ␤-keto-acyl-ACP reductase KasA, targeted by thiolactomycin (34), and the enoyl-ACP reductase InhA, which is inhibited by INH (40). INH is a prodrug that is activated by the catalase-peroxidase KatG (13) to form an isonicotinoyl radical which reacts with NAD to produce an 31,42), which inhibits InhA (17,23,29,40,42). A large majority of INH-resistant (INH R ) M. tuberculosis clinical isolates have mutations in the activator of INH, KatG, not the target of INH, InhA (38).…”

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Novel Inhibitors of InhA Efficiently Kill Mycobacterium tuberculosis under Aerobic and Anaerobic Conditions

Vilchèze

Baughn

Tufariello

et al. 2011

Antimicrob Agents Chemother

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Drug resistance in Mycobacterium tuberculosis has become a serious global health threat, which is now complicated by the emergence of extensively drug-resistant strains. New drugs that are active against drugresistant tuberculosis (TB) are needed. We chose to search for new inhibitors of the enoyl-acyl carrier protein (ACP) reductase InhA, the target of the first-line TB drug isoniazid (also known as isonicotinoic acid hydrazide [INH]). A subset of a chemical library, composed of 300 compounds inhibiting Plasmodium falciparum enoyl reductase, was tested against M. tuberculosis. Four compounds were found to inhibit M. tuberculosis growth with MICs ranging from 1 M to 10 M. Testing of these compounds against M. tuberculosis in vitro revealed that only two compounds (CD39 and CD117) were bactericidal against drug-susceptible and drug-resistant M. tuberculosis. These two compounds were also bactericidal against M. tuberculosis incubated under anaerobic conditions. Furthermore, CD39 and CD117 exhibited increased bactericidal activity when used in combination with INH or rifampin, but CD39 was shown to be toxic to eukaryotic cells. The compounds inhibit InhA as well the fatty acid synthase type I, and CD117 was found to also inhibit tuberculostearic acid synthesis. This study provides the TB drug development community with two chemical scaffolds that are suitable for structureactivity relationship study to improve on their cytotoxicities and bactericidal activities in vitro and in vivo.The fight against tuberculosis (TB), a disease caused by the bacillus Mycobacterium tuberculosis, is facing new challenges with the surge of multidrug-resistant (MDR) TB and the recent emergence of extensively drug-resistant (XDR) TB (8). TB strains resistant to the first-line anti-TB drugs (FLDs) isoniazid (also known as isonicotinoic acid hydrazide [INH]) and rifampin (RIF) are classified as MDR-TB, while XDR-TB is defined as MDR-TB resistant to any fluoroquinolone and one or more of the three injectable drugs (6). The need for novel TB drugs has spurred a new interest in TB drug development, and several new TB drugs are currently being tested in clinical trials (18). Nevertheless, expanding the pharmacopeia of active TB drugs remains an important goal, as M. tuberculosis has proven to be more than adept at acquiring drug resistance. One strategy to develop new drugs effective against MDR-and XDR-TB is to target essential functions that are not aims of the current anti-TB drug regimen. An alternative is to develop new drugs with novel requirements for inhibition of a bona fide target, with the goal of circumventing extant drug resistance.TB is resistant to most commonly used antibacterial agents due in part to its unusual cell wall structure. The mycobacterial cell wall contains unique long-chain (C 70 to C 90 ), ␣-alkyl, ␤-hydroxy fatty acids called mycolic acids. The synthesis of these fatty acids requires the coenzyme A (CoA)-dependent fatty acid synthase type I (FASI) and the acyl carrier protein (ACP)-dependent multienzyme fatty ...

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“…During the second step of the elongation cycle, the resulting ␤-ketoacyl-ACP product is reduced by MabA, the NADPH-dependent ␤-ketoacyl reductase of M. tuberculosis (19,20). The resulting ␤-hydroxyacyl-ACP is then dehydrated by a set of dehydratases, HadABC (21,22), and finally reduced by the enoyl-ACP reductase, InhA, the primary target of isoniazid (23). The succeeding steps of condensation of the elongating chain with malonyl-ACP units are performed by the ␤-ketoacyl-ACP synthases KasA and KasB (8,24,25), leading to very long-chain meromycolyl-ACPs (up to C 56 ), which are the direct precursors of mycolic acids.…”

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Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

Veyron‐Churlet

Zanella-Cléon

Cohen‐Gonsaud

et al. 2010

Journal of Biological Chemistry

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Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential ␤-ketoacylacyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr 191 being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr 191 alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

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Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid

Cited by 292 publications

References 14 publications

An inorganic complex that inhibits Mycobacterium tuberculosis enoyl reductase as a prototype of a new class of chemotherapeutic agents to treat tuberculosis

An inorganic complex that inhibits Mycobacterium tuberculosis enoyl reductase as a prototype of a new class of chemotherapeutic agents to treat tuberculosis

Novel Inhibitors of InhA Efficiently Kill Mycobacterium tuberculosis under Aerobic and Anaerobic Conditions

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

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