Differential Gel Electrophoresis (DIGE) Evaluation of Naphthoimidazoles Mode of Action: A Study in <i>Trypanosoma cruzi</i> Bloodstream Trypomastigotes

<div><p>Background</p><p>The obligate intracellular protozoan <i>Trypanosoma cruzi</i> is the causative agent of Chagas disease, a neglected illness affecting millions of people in Latin America that recently entered non-endemic countries through immigration, as a consequence of globalization. The chemotherapy for this disease is based mainly on benznidazole and nifurtimox, which are very efficient nitroderivatives against the acute stage but present limited efficacy during the chronic phase. Our group has been studying the trypanocidal effects of naturally occurring quinones and their derivatives, and naphthoimidazoles derived from β-lapachone N1, N2 and N3 were the most active. To assess the molecular mechanisms of action of these compounds, we applied proteomic techniques to analyze treated bloodstream trypomastigotes, which are the clinically relevant stage of the parasite.</p><p>Methodology/Principal Findings</p><p>The approach consisted of quantification by 2D-DIGE followed by MALDI-TOF/TOF protein identification. A total of 61 differentially abundant protein spots were detected when comparing the control with each N1, N2 or N3 treatment, for 34 identified spots. Among the differentially abundant proteins were activated protein kinase C receptor, tubulin isoforms, asparagine synthetase, arginine kinase, elongation factor 2, enolase, guanine deaminase, heat shock proteins, hypothetical proteins, paraflagellar rod components, RAB GDP dissociation inhibitor, succinyl-CoA ligase, ATP synthase subunit B and methionine sulfoxide reductase.</p><p>Conclusion/Significance</p><p>Our results point to different modes of action for N1, N2 and N3, which indicate a great variety of metabolic pathways involved and allow for novel perspectives on the development of trypanocidal agents.</p></div

Chemical structures of β-lapachone-derived naphthoimidazoles.

<p>(a) N1, (b) N2, and (c) N3.</p

Identifications of <i>T</i>. <i>cruzi</i> trypomastigotes proteins modulated by treatment with naphthoimidazoles.

<p>Identifications of <i>T</i>. <i>cruzi</i> trypomastigotes proteins modulated by treatment with naphthoimidazoles.</p

Differentially abundant protein spots from <i>T</i>. <i>cruzi</i> bloodstream trypomastigotes that were treated with naphthoimidazoles.

<p>The image depicts the preparative 2D-PAGE of a pool of all trypomastigote samples from this study that made up the internal standard. Sample (500 μg of protein) was initially separated on an 18-cm IPG strip (pH 4–7) followed by 12% SDS-PAGE. The spots that were excised from the preparative gel are indicated by the numbered circles.</p

Differentially abundant protein spots from <i>T</i>. <i>cruzi</i> bloodstream trypomastigotes after naphthoimidazole treatment.

<p>Number of spots related to proteins at higher (A) and lower (B) abundance after treatment with each of the three compounds. The numbers of abundant, excised spots from the reference gel are represented by black bars and those that were effectively identified by MALDI TOF/TOF MS are shown in grey.</p

Cellular component classification of proteins that were differentially abundant in <i>T</i>. <i>cruzi</i> bloodstream trypomastigotes treated with naphthoimidazoles.

<p>The total protein number for each classification is represented as a percentage.</p