An Essential Type I Nitroreductase from Leishmania major Can Be Used to Activate Leishmanicidal Prodrugs

Voak, Andrew A.; Gobalakrishnapillai, Vithurshaa; Seifert, Karlheinz; Balczo, Edina; Hall, Belinda S.; Wilkinson, Shane R.

doi:10.1074/jbc.m113.494781

Cited by 30 publications

(31 citation statements)

References 50 publications

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“…To demonstrate that LmNTR played a role in prodrug activiation in the parasite itself, the susceptibility of LmNTR ϩ/Ϫ heterozygous promastigotes to CB1954, NH10, and NH11 was evaluated; attempts (16 independent transformations) to generate L. major LmNTR Ϫ/Ϫ null mutants failed to produce recombinant cells, leading us to speculate that this activity is essential in insectstage parasites (48). This screening demonstrated that cells expressing lower levels of the oxidoreductase were between 2-and 4-fold more resistant to the ANB than controls; wild-type L. major had IC 50 …”

Section: Resultsmentioning

confidence: 99%

Evaluating Aziridinyl Nitrobenzamide Compounds as Leishmanicidal Prodrugs

Voak

Seifert

Helsby

et al. 2014

Antimicrob Agents Chemother

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cMany of the nitroaromatic agents used in medicine function as prodrugs and must undergo activation before exerting their toxic effects. In most cases, this is catalyzed by flavin mononucleotide (FMN)-dependent type I nitroreductases (NTRs), a class of enzyme absent from higher eukaryotes but expressed by bacteria and several eukaryotic microbes, including trypanosomes and Leishmania. Here, we utilize this difference to evaluate whether members of a library of aziridinyl nitrobenzamides have activity against Leishmania major. Biochemical screens using purified L. major NTR (LmNTR) revealed that compounds containing an aziridinyl-2,4-dinitrobenzyl core were effective substrates for the enzyme and showed that the 4-nitro group was important for this activity. To facilitate drug screening against intracellular amastigote parasites, we generated leishmanial cells that expressed the luciferase reporter gene and optimized a mammalian infection model in a 96-well plate format. A subset of aziridinyl-2,4-dinitrobenzyl compounds possessing a 5-amide substituent displayed significant growth-inhibitory properties against the parasite, with the most potent agents generating 50% inhibitory concentrations of <100 nM for the intracellular form. This antimicrobial activity was shown to be LmNTR specific since L. major NTR ؉/؊ heterozygote parasites were slightly resistance to most aziridinyl dinitrobenzyl agents tested. When the most potent leishmanicidal agents were screened against the mammalian cells in which the amastigote parasites were propagated, no growth-inhibitory effect was observed at concentrations of up to 100 M. We conclude that the aziridinyl nitrobenzamides represent a new lead structure that may have the potential to treat leishmanial infections.

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Section: Resultsmentioning

confidence: 99%

Evaluating Aziridinyl Nitrobenzamide Compounds as Leishmanicidal Prodrugs

Voak

Seifert

Helsby

et al. 2014

Antimicrob Agents Chemother

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“…This could explain why knock-down approaches have failed, so far in our hands, and why a nitroreductase is essential for Leishmania donovani ( Voak et al., 2013 ). From an evolutionary point of view, the reduction of nitro compounds could be a side effect without negative selective pressure until a nitro compound yielding toxic intermediates after reduction comes into play.…”

Section: Discussionmentioning

confidence: 99%

Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds

Müller

Samuel

Leitsch

et al. 2015

International Journal for Parasitology: Drugs and Drug Resistan

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“…It is worth mentioning that nitroreductase-encoding genes are not only commonly present in enterobacteria but also found in other bacterial species such as Staphylococcus aureus, Bacillus subtilis, Vibrio fischeri and parasites (e.g. Trypanosoma brucei, Leishmania major) [21][22][23][24]. Nitroreductase enzymes play different physiological roles in different species; in E. coli, multiple functions have been proposed for NfsA and NfsB, including dihydropteridine reductase, chromate reductase, quinone-dependent azo reductase, and part of the oxidative stress response [21].…”

Section: Introductionmentioning

confidence: 99%

In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria

Olivera

Spagnuolo

et al. 2020

BMC Microbiol

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Background: Antimicrobial combinations have been proven as a promising approach in the confrontation with multi-drug resistant bacterial pathogens. In the present study, we identify and characterize a synergistic interaction of broad-spectrum nitroreductase-activated prodrugs 5-nitrofurans, with a secondary bile salt, Sodium Deoxycholate (DOC) in growth inhibition and killing of enterobacteria. Results: Using checkerboard assay, we show that combination of nitrofuran furazolidone (FZ) and DOC generates a profound synergistic effect on growth inhibition in several enterobacterial species including Escherichia coli, Salmonella enterica, Citrobacter gillenii and Klebsiella pneumoniae. The Fractional Inhibitory Concentration Index (FICI) for DOC-FZ synergy ranges from 0.125 to 0.35 that remains unchanged in an ampicillin-resistant E. coli strain containing a β-lactamase-producing plasmid. Findings from the time-kill assay further highlight the synergy with respect to bacterial killing in E. coli and Salmonella. We further characterize the mechanism of synergy in E. coli K12, showing that disruption of the tolC or acrA genes that encode components of multidrug efflux pumps causes, respectively, a complete or partial loss, of the DOC-FZ synergy. This finding indicates the key role of TolC-associated efflux pumps in the DOC-FZ synergy. Overexpression of Nitric Oxide-detoxifying enzyme Hmp results in a threefold increase in FICI for DOC-FZ interaction, suggesting a role of nitric oxide in the synergy. We further demonstrate that DOC-FZ synergy is largely independent of NfsA and NfsB, the two major activation enzymes of the nitrofuran prodrugs. Conclusions: This study is to our knowledge the first report of nitrofuran-deoxycholate synergy against Gramnegative bacteria, offering potential applications in antimicrobial therapeutics. The mechanism of DOC-FZ synergy involves FZ-mediated inhibition of TolC-associated efflux pumps that normally remove DOC from bacterial cells. One possible route contributing to that effect is via FZ-mediated nitric oxide production.

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An Essential Type I Nitroreductase from Leishmania major Can Be Used to Activate Leishmanicidal Prodrugs

Cited by 30 publications

References 50 publications

Evaluating Aziridinyl Nitrobenzamide Compounds as Leishmanicidal Prodrugs

Evaluating Aziridinyl Nitrobenzamide Compounds as Leishmanicidal Prodrugs

Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds

In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria

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