Activation of bicyclic nitro-drugs by a novel nitroreductase (NTR2) in Leishmania

Susan Wyllie (Lead / Corresponding author), Adam J. Roberts, Suzanne Norval, Stephen Patterson, Bernardo J. Foth, Matthew Berriman, Kevin D. Read, Alan H. Fairlamb (Lead / Corresponding author)

Research output: Contribution to journalArticle

25 Citations (Scopus)
76 Downloads (Pure)

Abstract

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.
Original languageEnglish
Article numbere1005971
Pages (from-to)1-22
Number of pages22
JournalPLoS Pathogens
Volume12
Issue number11
DOIs
Publication statusPublished - 3 Nov 2016

Fingerprint

Nitroreductases
Leishmania
Nitro Compounds
Nifurtimox
Pharmaceutical Preparations
Parasites
Genome
Neglected Diseases
Null Lymphocytes
Sulfones
Visceral Leishmaniasis
NADPH Oxidase
Cytosine
Drug Discovery
Proteomics
Genes
Oxygen
Cell Line

Keywords

  • PA-824
  • DNDI-VL-2098
  • Delamanid Nitroimidazo-oxazines
  • Leishmania
  • bio-activation

Cite this

Wyllie, Susan ; Roberts, Adam J. ; Norval, Suzanne ; Patterson, Stephen ; Foth, Bernardo J. ; Berriman, Matthew ; Read, Kevin D. ; Fairlamb, Alan H. / Activation of bicyclic nitro-drugs by a novel nitroreductase (NTR2) in Leishmania. In: PLoS Pathogens. 2016 ; Vol. 12, No. 11. pp. 1-22.
@article{0fa3d8e75e874d3ab53c6e73eb560dbc,
title = "Activation of bicyclic nitro-drugs by a novel nitroreductase (NTR2) in Leishmania",
abstract = "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.",
keywords = "PA-824, DNDI-VL-2098, Delamanid Nitroimidazo-oxazines, Leishmania, bio-activation",
author = "Susan Wyllie and Roberts, {Adam J.} and Suzanne Norval and Stephen Patterson and Foth, {Bernardo J.} and Matthew Berriman and Read, {Kevin D.} and Fairlamb, {Alan H.}",
note = "This work was funded by grants from the Wellcome Trust (079838 and 100476 to AHF and 098051 to the Wellcome Trust Sanger Institute). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.",
year = "2016",
month = "11",
day = "3",
doi = "10.1371/journal.ppat.1005971",
language = "English",
volume = "12",
pages = "1--22",
journal = "PLoS Pathogens",
issn = "1553-7366",
publisher = "Public Library of Science",
number = "11",

}

Activation of bicyclic nitro-drugs by a novel nitroreductase (NTR2) in Leishmania. / Wyllie, Susan (Lead / Corresponding author); Roberts, Adam J.; Norval, Suzanne; Patterson, Stephen; Foth, Bernardo J.; Berriman, Matthew; Read, Kevin D.; Fairlamb, Alan H. (Lead / Corresponding author).

In: PLoS Pathogens, Vol. 12, No. 11, e1005971, 03.11.2016, p. 1-22.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Activation of bicyclic nitro-drugs by a novel nitroreductase (NTR2) in Leishmania

AU - Wyllie, Susan

AU - Roberts, Adam J.

AU - Norval, Suzanne

AU - Patterson, Stephen

AU - Foth, Bernardo J.

AU - Berriman, Matthew

AU - Read, Kevin D.

AU - Fairlamb, Alan H.

N1 - This work was funded by grants from the Wellcome Trust (079838 and 100476 to AHF and 098051 to the Wellcome Trust Sanger Institute). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

PY - 2016/11/3

Y1 - 2016/11/3

N2 - 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.

AB - 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.

KW - PA-824

KW - DNDI-VL-2098

KW - Delamanid Nitroimidazo-oxazines

KW - Leishmania

KW - bio-activation

U2 - 10.1371/journal.ppat.1005971

DO - 10.1371/journal.ppat.1005971

M3 - Article

C2 - 27812217

VL - 12

SP - 1

EP - 22

JO - PLoS Pathogens

JF - PLoS Pathogens

SN - 1553-7366

IS - 11

M1 - e1005971

ER -