The terpenic diamine GIB24 inhibits the growth of Trypanosoma cruzi epimastigotes and intracellular amastigotes, with proteomic analysis of drug-resistant epimastigotes

https://doi.org/10.1016/j.cbi.2020.109165Get rights and content

Highlights

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    A diamine showed high selective against intracellular amastigotes of T. cruzi.

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    Geraniol derivative reduces the cell size of T. cruzi. epimastigotes forms.

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    Geraniol derivative can cause incidental cell death in T. cruzi.

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    Terpenoid affects the mitochondrial membrane potential in epimastigote T. cruzi.

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    A Geraniol derivative as potential lead compound against T. cruzi.

Abstract

The effect of N-geranyl-ethane-1,2-diamine dihydochloride (GIB24), a synthetic diamine, was assayed against different developmental forms of the parasitic protozoan Trypanosoma cruzi (strain Dm28c). The compound was effective against culture epimastigote forms (IC50/24h = 5.64 μM; SI = 16.4) and intracellular amastigotes (IC50/24h = 12.89 μM; SI = 7.18), as detected by the MTT methodology and by cell counting, respectively. Incubation of epimastigotes for 6h with 6 μM GIB24 (IC50/24h value) resulted in significant dissipation of the mitochondrial membrane potential, prior to permeabilization of the plasma membrane. Rounded epimastigotes with cell size reduction were observed by scanning electron microscopy. These morpho-physiological changes induced by GIB24 suggest an incidental death process. Treatment of infected Vero cells did not prevent the intracellular amastigotes from completing the intracellular cycle. However, there was a decrease in the number of released parasites, increasing the ratio amastigotes/trypomastigotes. Proteomic analysis of 15 μM GIB24 resistant epimastigotes indicated that the compound acts mainly on mitochondrial components involved in the Krebs cycle and in maintaining the oxidative homeostasis of the parasites. Our data suggest that GIB24 is active against the main morphological forms of T. cruzi.

Section snippets

Hallmarks

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    The diamine GIB24 (SI = 7.18) showed high selective activity against intracellular amastigotes of T. cruzi.

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    Flow cytometric analysis showed that GIB-24 induced dissipation of the mitochondrial membrane potential reduced the cell size and led to phosphatidylserine exposure in treated epimastigotes. The data suggest that GIB-24 cause incidental cell death in T. cruzi.

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    Analysis of the proteome of 15 μM GIB-24 resistant epimastigotes indicated that GIB-24 acts preferentially on proteins involved in

Chemistry

All reagents and solvents were reagent grade and were used without prior purification. All reactions were monitored by thin layer chromatography (TLC, Sigma-Aldrich® 60). The Geranyl bromide (compound 2) was purified by liquid-liquid extraction and the geranyldiamine was purified by liquid-liquid extraction followed by flash chromatography on Sigma-Aldrich® silica gel 60 (230–400 mesh) using CH2Cl2:CH3OH:NH4OH (80:18:2) as eluent. The IR spectra were acquired on a Perkin Elmer Spectrum 100 FTIR

Results

The effect of the diamine GIB24 was first evaluated on T. cruzi epimastigotes, by using the MTT methodology. The compound GIB24 was highly effective against these developmental forms (CI50/24h = 5.64 μM), but its selectivity index (SI) was lower than that of benznidazole (Table 1). Similar results were obtained for intracellular amastigotes in Vero cells, with SI = 7.18 (Table 1).

In order to confirm the MTT colorimetric assay, epimastigotes were treated with 3, 6 or 10 μM GIB24 and the number

Discussion

The terpene diamine GIB24 was effective to inhibit the growth of Trypanosoma cruzi culture epimastigotes and intracellular amastigotes, with IC50/24h values of 5.64 μM (SI = 16.4) and 12.89 μM (SI = 7.18), respectively.

Incubation of infected Vero cells with GIB24 led to a reduction in a number of intracellular amastigotes. As a result, there was a significant decrease in the number of released parasites (trypomastigotes + amastigotes) into the extracellular medium. Interestingly, our data

CRediT authorship contribution statement

Camila Maria Azeredo: who worked on the synthesis and characterization of this series of compounds. And the second group. Mauricio Frota Saraiva: The authors contributed equally to the present work, They were divided into two groups. Maristela Ribeiro de Oliveira: who worked on the synthesis and characterization of this series of compounds. Gisele Barbosa: who worked on the synthesis and characterization of this series of compounds. Mauro Vieira de Almeida: who worked on the synthesis and

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by Conselho Nacional de Desenvolvimento Científico (CNPq), Fundação Oswaldo Cruz (FIOCRUZ) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) (Project: APQ-04302-10). The authors thank the Program for technological Development in Tools for Health-PDTIS-FIOCRUZ for providing technical infrastructure (Mass Spectrometry Facility-RPT02H) and professional assistance. This work is also a collaboration research project of members of the Rede Mineira de

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