Structure of Ddn, the Deazaflavin-Dependent Nitroreductase from Mycobacterium tuberculosis Involved in Bioreductive Activation of PA-824

Cellitti, Susan E.; Shaffer, Jennifer; Jones, David H.; Mukherjee, Tathagata; Gurumurthy, Meera; Bursulaya, Badry; Boshoff, Helena I.; Choi, Inhee; Nayyar, Amit; Lee, Yong Sok; Cherian, Joseph; Niyomrattanakit, Pornwaratt; Dick, Thomas; Manjunatha, Ujjini H.; Barry, Clifton E.; Spraggon, Glen; Geierstanger, Bernhard H.

doi:10.1016/j.str.2011.11.001

Cited by 91 publications

(135 citation statements)

References 58 publications

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“…In Mycobacterium tuberculosis metabolism of ( S )-PA-824 is catalyzed by an unusual deazaflavin-dependent nitroreductase (Ddn) [23–25], an enzyme which is absent in Leishmania spp . Incubation of ( S )-PA-824 with recombinant M .…”

Section: Discussionmentioning

confidence: 99%

Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania

et al. 2016

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Drug discovery pipelines for the “neglected diseases” are now heavily populated with nitroheterocyclic compounds. Recently, the bicyclic nitro-compounds (R)-PA-824, DNDI-VL-2098 and delamanid have been identified as potential candidates for the treatment of visceral leishmaniasis. Using a combination of quantitative proteomics and whole genome sequencing of susceptible and drug-resistant parasites we identified a putative NAD(P)H oxidase as the activating nitroreductase (NTR2). Whole genome sequencing revealed that deletion of a single cytosine in the gene for NTR2 that is likely to result in the expression of a non-functional truncated protein. Susceptibility of leishmania was restored by reintroduction of the wild-type gene into the resistant line, which was accompanied by the ability to metabolise these compounds. Overexpression of NTR2 in wild-type parasites rendered cells hyper-sensitive to bicyclic nitro-compounds, but only marginally to the monocyclic nitro-drugs, nifurtimox and fexinidazole sulfone, known to be activated by a mitochondrial oxygen-insensitive nitroreductase (NTR1). Conversely, a double knockout NTR2 null cell line was completely resistant to bicyclic nitro-compounds and only marginally resistant to nifurtimox. Sensitivity was fully restored on expression of NTR2 in the null background. Thus, NTR2 is necessary and sufficient for activation of these bicyclic nitro-drugs. Recombinant NTR2 was capable of reducing bicyclic nitro-compounds in the same rank order as drug sensitivity in vitro. These findings may aid the future development of better, novel anti-leishmanial drugs. Moreover, the discovery of anti-leishmanial nitro-drugs with independent modes of activation and independent mechanisms of resistance alleviates many of the concerns over the continued development of these compound series.

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Section: Discussionmentioning

confidence: 99%

Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania

et al. 2016

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“…F 420 was docked into the cofactor-binding pockets based on the cofactor-bound structures of Rv3547 (PDB: 3R5R; Cellitti et al, 2012) and Rv2074 (PDB: 5JAB; Ahmed et al, 2016) respectively. AutoDock Vina was used to computationally dock the substrates into their corresponding enzymes, with enzymes and ligands prepared using AutoDockTools operating with default settings (Morris et al, 2009).…”

Section: Methodsmentioning

confidence: 99%

Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism

et al. 2017

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An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H-) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,β-unsaturated compounds.

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“…Ddn (Rv3547) converts the prodrugs into three primary metabolites, a des-nitroimidazole and two unstable byproducts (4). Ddn is likely a membrane-bound protein (5) that is involved in a protective mechanism under oxidative stress (6). The major mechanism of action of nitroimidazole in active disease under aerobic conditions is to hinder the formation of mycolic acids, and under anaerobic conditions, the mechanism involves the induction of respiratory poisoning (4,7).…”

mentioning

confidence: 99%

“…After the reduction of F 420 to the active protonated cofactor, F 420 -H 2 , Ddn catalyzes the reverse reaction to oxidize it back to F 420 . It has been hypothesized that Ddn orients PA-824 so that hydride transfer from F 420 can occur and stabilize the transition state during this biochemical reaction (5). In F 420 biosynthesis, the FbiC gene encodes a 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) synthase, which transfers the hydroxybenzyl group from 4-hydroxy-phenylpyruvate to pyrimidinedione (12).…”

mentioning

confidence: 99%

Mutations in Genes for the F ₄₂₀ Biosynthetic Pathway and a Nitroreductase Enzyme Are the Primary Resistance Determinants in Spontaneous In Vitro -Selected PA-824-Resistant Mutants of Mycobacterium tuberculosis

Haver

Chua

Ghode

et al. 2015

Antimicrob Agents Chemother

127

113

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Alleviating the burden of tuberculosis (TB) requires an understanding of the genetic basis that determines the emergence of drug-resistant mutants. PA-824 (pretomanid) is a bicyclic nitroimidazole class compound presently undergoing the phase III STAND clinical trial, despite lacking identifiable genetic markers for drug-specific resistant Mycobacterium tuberculosis. In the present study, we aimed to characterize the genetic polymorphisms of spontaneously generated PA-824-resistant mutant strains by surveying drug metabolism genes for potential mutations. Of the 183 independently selected PA-824-resistant M. tuberculosis mutants, 83% harbored a single mutation in one of five nonessential genes associated with either PA-824 prodrug activation (ddn, 29%; fgd1, 7%) or the tangential F 420 biosynthetic pathway (fbiA, 19%; fbiB, 2%; fbiC, 26%). Crystal structure analysis indicated that identified mutations were specifically located within the protein catalytic domain that would hinder the activity of the enzymes required for prodrug activation. This systematic analysis conducted of genotypes resistant to PA-824 may contribute to future efforts in monitoring clinical strain susceptibility with this new drug therapy.T uberculosis (TB) remains a major global health concern, with Ͼ8 million new cases and 1.8 million deaths occurring annually (WHO). This pandemic is exacerbated by the pervasive spread of multidrug-resistant (MDR)-TB that challenges clinicians to fight a disease with a limited arsenal of resources. The bicyclic 4-nitroimidazole chemotype has yielded two promising candidates, delamanid (OPC67683) and pretomanid (PA-824), which actively inhibit both nonreplicating and rapidly growing bacilli under aerobic and anaerobic conditions (1). Both drugs are undergoing clinical evaluation and FDA approval is pending for the treatment of MDR-TB. In 2013, delamanid received conditional marketing authorization by the European Medicines Agency (EMA) for use in adult patients deprived of other treatment options (2). PA-824 is in the phase III STAND clinical trial, and at this stage of the development pipeline, it would be beneficial to monitor the genetic basis of resistant clinical strains as they emerge in the wake of future implementation into a treatment protocol.Bicyclic 4-nitroimidazoles are prodrugs that require metabolic activation by a deazaflavin (cofactor F 420 )-dependent nitroreductase (Ddn) (3). Ddn (Rv3547) converts the prodrugs into three primary metabolites, a des-nitroimidazole and two unstable byproducts (4). Ddn is likely a membrane-bound protein (5) that is involved in a protective mechanism under oxidative stress (6). The major mechanism of action of nitroimidazole in active disease under aerobic conditions is to hinder the formation of mycolic acids, and under anaerobic conditions, the mechanism involves the induction of respiratory poisoning (4, 7). By inhibiting the formation of ketomycolates, a class of mycolic acids, nitroimidazole interferes with Mycobacterium tuberculosis cell wall formation, ...

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Structure of Ddn, the Deazaflavin-Dependent Nitroreductase from Mycobacterium tuberculosis Involved in Bioreductive Activation of PA-824

Cited by 91 publications

References 58 publications

Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania

Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania

Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism

Mutations in Genes for the F ₄₂₀ Biosynthetic Pathway and a Nitroreductase Enzyme Are the Primary Resistance Determinants in Spontaneous In Vitro -Selected PA-824-Resistant Mutants of Mycobacterium tuberculosis

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Structure of Ddn, the Deazaflavin-Dependent Nitroreductase from Mycobacterium tuberculosis Involved in Bioreductive Activation of PA-824

Cited by 91 publications

References 58 publications

Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania

Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania

Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism

Mutations in Genes for the F 420 Biosynthetic Pathway and a Nitroreductase Enzyme Are the Primary Resistance Determinants in Spontaneous In Vitro -Selected PA-824-Resistant Mutants of Mycobacterium tuberculosis

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Mutations in Genes for the F ₄₂₀ Biosynthetic Pathway and a Nitroreductase Enzyme Are the Primary Resistance Determinants in Spontaneous In Vitro -Selected PA-824-Resistant Mutants of Mycobacterium tuberculosis