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Nitro/nitrosyl-ruthenium complexes are potent and selective anti-Trypanosoma cruzi agents causing autophagy and necrotic parasite death.

ABSTRACT: cis-[RuCl(NO2)(dppb)(5,5'-mebipy)] (complex 1), cis-[Ru(NO2)2(dppb)(5,5'-mebipy)] (complex 2), ct-[RuCl(NO)(dppb)(5,5'-mebipy)](PF6)2 (complex 3), and cc-[RuCl(NO)(dppb)(5,5'-mebipy)](PF6)2 (complex 4), where 5,5'-mebipy is 5,5'-dimethyl-2,2'-bipyridine and dppb is 1,4-bis(diphenylphosphino)butane, were synthesized and characterized. The structure of complex 2 was determined by X-ray crystallography. These complexes exhibited a higher anti-Trypanosoma cruzi activity than benznidazole, the current antiparasitic drug. Complex 3 was the most potent, displaying a 50% effective concentration (EC50) of 2.1 ± 0.6 μM against trypomastigotes and a 50% inhibitory concentration (IC50) of 1.3 ± 0.2 μM against amastigotes, while it displayed a 50% cytotoxic concentration (CC50) of 51.4 ± 0.2 μM in macrophages. It was observed that the nitrosyl complex 3, but not its analog lacking the nitrosyl group, releases nitric oxide into parasite cells. This release has a diminished effect on the trypanosomal protease cruzain but induces substantial parasite autophagy, which is followed by a series of irreversible morphological impairments to the parasites and finally results in cell death by necrosis. In infected mice, orally administered complex 3 (five times at a dose of 75 μmol/kg of body weight) reduced blood parasitemia and increased the survival rate of the mice. Combination index analysis of complex 3 indicated that its in vitro activity against trypomastigotes is synergic with benznidazole. In addition, drug combination enhanced efficacy in infected mice, suggesting that ruthenium-nitrosyl complexes are potential constituents for drug combinations.

SUBMITTER: Bastos TM

PROVIDER: S-EPMC4187982 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Nitro/nitrosyl-ruthenium complexes are potent and selective anti-Trypanosoma cruzi agents causing autophagy and necrotic parasite death.

Bastos Tanira M TM Barbosa Marília I F MI da Silva Monize M MM da C Júnior José W JW Meira Cássio S CS Guimaraes Elisalva T ET Ellena Javier J Moreira Diogo R M DR Batista Alzir A AA Soares Milena B P MB

Antimicrobial agents and chemotherapy 20140804 10

cis-[RuCl(NO2)(dppb)(5,5'-mebipy)] (complex 1), cis-[Ru(NO2)2(dppb)(5,5'-mebipy)] (complex 2), ct-[RuCl(NO)(dppb)(5,5'-mebipy)](PF6)2 (complex 3), and cc-[RuCl(NO)(dppb)(5,5'-mebipy)](PF6)2 (complex 4), where 5,5'-mebipy is 5,5'-dimethyl-2,2'-bipyridine and dppb is 1,4-bis(diphenylphosphino)butane, were synthesized and characterized. The structure of complex 2 was determined by X-ray crystallography. These complexes exhibited a higher anti-Trypanosoma cruzi activity than benznidazole, the curren ...[more]

PMID: 25092707

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Project description:BackgroundTrypanosomatids are a group of protozoan parasites that includes the etiologic agents of important human illnesses as Chagas disease, sleeping sickness and leishmaniasis. These parasites have a significant distinction from other eukaryotes concerning mRNA structure, since all mature mRNAs have an identical species-specific sequence of 39 nucleotides at the 5' extremity, named spliced leader (SL). Considering this peculiar aspect of trypanosomatid mRNA, the aim of the present work was to develop a Trypanosoma cruzi specific in vitro transcription (IVT) linear mRNA amplification method in order to improve parasite transcriptomics analyses.MethodsWe designed an oligonucleotide complementary to the last 21 bases of T. cruzi SL sequence, bearing an upstream T7 promoter (T7SL primer), which was used to direct the synthesis of second-strand cDNA. Original mRNA was then amplified by IVT using T7 RNA polymerase. T7SL-amplified RNA from two distinct T. cruzi stages (epimastigotes and trypomastigotes) were deep sequenced in SOLiD platform. Usual poly(A) + RNA and and T7-oligo(dT) amplified RNA (Eberwine method) were also sequenced. RNA-Seq reads were aligned to our new and improved T. cruzi Dm28c genome assembly (PacBio technology) and resulting transcriptome pattern from these three RNA preparation methods were compared, mainly concerning the conservation of mRNA transcritional levels and DEGs detection between epimastigotes and trypomastigotes.ResultsT7SL IVT method detected more potential differentially expressed genes in comparison to either poly(A) + RNA or T7dT IVT, and was also able to produce reliable quantifications of the parasite transcriptome down to 3 ng of total RNA. Furthermore, amplification of parasite mRNA in HeLa/epimastigote RNA mixtures showed that T7SL IVT generates transcriptome quantification with similar detection of differentially expressed genes when parasite RNA mass was only 0.1% of the total mixture (R = 0.78 when compared to poly(A) + RNA).ConclusionsThe T7SL IVT amplification method presented here allows the detection of more potential parasite differentially expressed genes (in comparison to poly(A) + RNA) in host-parasite mixtures or samples with low amount of RNA. This method is especially useful for trypanosomatid transcriptomics because it produces less bias than PCR-based mRNA amplification. Additionally, by simply changing the complementary region of the T7SL primer, the present method can be applied to any trypanosomatid species.

Dataset Information

Nitro/nitrosyl-ruthenium complexes are potent and selective anti-Trypanosoma cruzi agents causing autophagy and necrotic parasite death.

Publications

Nitro/nitrosyl-ruthenium complexes are potent and selective anti-Trypanosoma cruzi agents causing autophagy and necrotic parasite death.

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