Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania.
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.
Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.
Susan
http://orcid.org/0000-0001-8810-5605
S
Wyllie
Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.
Adam J
http://orcid.org/0000-0002-6834-549X
AJ
Roberts
Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.
Suzanne
http://orcid.org/0000-0002-9186-1994
S
Norval
Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.
Stephen
S
Patterson
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
Bernardo J
http://orcid.org/0000-0001-8374-5590
BJ
Foth
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
Matthew
http://orcid.org/0000-0002-9581-0377
M
Berriman
Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.
Kevin D
KD
Read
Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.
Alan H
http://orcid.org/0000-0001-5134-0329
AH
Fairlamb
D000818:Animals; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D007898:Leishmaniasis, Visceral / Q000469:parasitology*; D013058:Mass Spectrometry; D058069:Neglected Diseases / Q000469:parasitology; D009601:Nitroreductases / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D021261:Parasitic Sensitivity Tests; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000378:metabolism*; D014344:Trypanocidal Agents / Q000494:pharmacology*
D015800:Protozoan Proteins; D014344:Trypanocidal Agents; D009601:Nitroreductases