Inhibitors of trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease.

Chagas disease remains one of the most neglected diseases in the world despite being the most important parasitic disease in Latin America. The characteristic chronic manifestation of chagasic cardiomyopathy is the region's leading cause of heart-related illness, causing significant mortality and morbidity. Due to the limited available therapeutic options, new drugs are urgently needed to control the disease. Sirtuins, also called Silent information regulator 2 (Sir2) proteins have long been suggested as interesting targets to treat different diseases, including parasitic infections. Recent studies on Trypanosoma cruzi sirtuins have hinted at the possibility to exploit these enzymes as a possible drug targets. In the present work, the T. cruzi Sir2 related protein 1 (TcSir2rp1) is genetically validated as a drug target and biochemically characterized for its NAD+-dependent deacetylase activity and its inhibition by the classic sirtuin inhibitor nicotinamide, as well as by bisnaphthalimidopropyl (BNIP) derivatives, a class of parasite sirtuin inhibitors. BNIPs ability to inhibit TcSir2rp1, and anti-parasitic activity against T. cruzi amastigotes in vitro were investigated. The compound BNIP Spermidine (BNIPSpd) (9), was found to be the most potent inhibitor of TcSir2rp1. Moreover, this compound showed altered trypanocidal activity against TcSir2rp1 overexpressing epimastigotes and anti-parasitic activity similar to the reference drug benznidazole against the medically important amastigotes, while having the highest selectivity index amongst the compounds tested. Unfortunately, BNIPSpd failed to treat a mouse model of Chagas disease, possibly due to its pharmacokinetic profile. Medicinal chemistry modifications of the compound, as well as alternative formulations may improve activity and pharmacokinetics in the future. Additionally, an initial TcSIR2rp1 model in complex with p53 peptide substrate was obtained from low resolution X-ray data (3.5 Å) to gain insight into the potential specificity of the interaction with the BNIP compounds. In conclusion, the search for TcSir2rp1 specific inhibitors may represent a valuable strategy for drug discovery against T. cruzi

The expression of sirtuins is developmentally regulated.

<p>Equal amounts of parasite lysate from epimastigotes (E), amastigotes (A) and trypomastigotes (T) were loaded on SDS-PAGE followed by (A) Coomassie staining or (B) western blot analysis using the following antibodies: anti-<i>Tc</i>SIR2RP1, anti-<i>Tc</i>SIR2RP3 and anti-Tubulin as load control.</p

Sirtuins inducible expression throughout <i>T</i>. <i>cruzi</i> life cycle.

<p>(A) Western blot analysis. Equal amounts of parasite lysate from epimastigotes (E), amastigotes (A) and trypomastigotes (T) in the absence (-) or presence (+) of 0.25 μg/ml Tetracycline for 24 hours were loaded on SDS-PAGE followed by western blot analysis using rat anti-HA monoclonal antibodies. (B) Immunofluorescence confocal microscopy of induced (0.25 μg/ml Tetracycline, 24 hours) parasites using rat anti-HA, FITC-conjugated anti-rat antibodies (green), and DAPI (blue). Scale bar: 5 μm.</p

IC₅₀^* values of sirtuin inhibitors on <i>T</i>. <i>cruzi</i> Dm28<i>c</i> epimastigotes.

<p>*IC₅₀ was calculated after 72 hs culture.</p><p>ND: not determined.</p><p>IC₅₀^{<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003725#t001fn001" target="_blank">*</a>} values of sirtuin inhibitors on <i>T</i>. <i>cruzi</i> Dm28<i>c</i> epimastigotes.</p

Overexpression of sirtuins changes the acetylation pattern in epimastigotes.

<p>Equal amounts of parasite lysate from each line in the absence (-, white bars) or presence (+, grey bars) of 0.25 μg/ml Tetracycline 24 hours post induction, were loaded on SDS-PAGE (A) followed by Western blot analysis using rabbit anti-Acetyl-lysine (B) and mouse anti-Tubulin and anti-Acetylated Tubulin antibodies (C). The deacetylated proteins in the anti-Acetyl-lysine western blot are depicted with black arrowsheads. The intensity of the acetylated-tubulin bands was quantified from n = 3 independent experiments and normalized to α-tubulin intensity. The bar graph represents the mean ± SEM; * p<0.05, ** p<0.005 (unpaired, two-tailed Student t test).</p

Overexpression of sirtuins protects epimastigotes from the growth defect caused by sirtuin inhibitors.

<p>Dm28<i>c</i> wild type (white bars) and uninduced (grey bars) and induced (black bars) epimastigotes of both transfected lines (Dm28<i>c</i> p<i>Tc</i>INDEXGW-<i>Tc</i>SIR2RP1HA and <i>Tc</i>SIR2RP3HA) were treated with three sirtuin inhibitors with concentrations above their IC₅₀ values: 75 μM Nicotinamide, 100 μM Cambinol and 250 μM Ex-527. The experiment was performed in triplicates and cell growth was determined after culture for 72 hours by counting viable forms. The values obtained were normalized to the wild type growth without inhibitors. The growth rate of each transfected line with inhibitors was compared to the corresponding untreated one. We also compared the viability of the uninduced and induced overexpressing lines for each sirtuin inhibitor. The bar graph represents the mean ± SEM; * p<0.05, ** p<0.005, *** p<0.001 (unpaired, two-tailed Student t test).</p

Inducible expression of sirtuins in epimastigotes.

<p>Equal amounts of parasite total lysate from each line (p<i>Tc</i>INDEXGW-SIR2RP1HA and p<i>Tc</i>INDEXGW-SIR2RP3HA) in the absence (-) or presence (+) of 0.25 μg/ml Tetracycline for 24 hours, were loaded on SDS-PAGE and stained with Coomassie (left panel), followed by western blot analysis using rat anti-HA monoclonal antibodies (A), or mouse anti-Tubulin and specific rabbit polyclonal antibodies against <i>Tc</i>SIR2RP1 and <i>Tc</i>SIR2RP3 (B). The degree of overexpression observed with the specific antibodies were quantified and normalized to α-tubulin intensity. (C) Immunofluorescence microscopy of uninduced and induced (0.25 μg/ml Tetracycline, 24 hours) parasites using rat anti-HA and FITC-conjugated anti-rat antibodies (green). DNA was stained with DAPI (blue).</p

<i>Tc</i>SIR2RP1 localizes in the cytoplasm and <i>Tc</i>SIR2RP3 in the mitochondrion.

<p>(A) Immunofluorescence assay of Dm28<i>c</i> wild type epimastigotes using rabbit antibodies against <i>Tc</i>SIR2RP1 and <i>Tc</i>SIR2RP3 and cytosolic and mitochondrial markers (mouse anti-TAT and MitoTracker, respectively). Cy3-conjugated anti-rabbit (red) and FITC-conjugated anti-mouse and anti-rabbit (green) were used as secondary antibodies, and DNA was counterstained with DAPI (blue). Scale bar: 5 μm. (B) Immunofluorescence assay of Dm28<i>c</i> expressing <i>Tc</i>SIR2RP1HA and <i>Tc</i>SIR2RP3HA (0.25 μg/ml Tetracycline) using rat anti-HA and cytosolic and mitochondrial markers (rabbit anti-TAT and anti-MDHm). Cy3-conjugated anti-rabbit (red) and FITC-conjugated anti-rat (green) were used as secondary antibodies, and DNA was counterstained with DAPI (blue). Scale bar: 5 μm. (C) Western blot analysis of the subcellular fractionation of the induced lines obtained by differential centrifugation. Equal amounts of each fraction were loaded on SDS-PAGE (left panel) followed by western blot with anti-TAT (cytosolic marker), anti-MDHm (mitochondrial marker), anti-BDF2 (nuclear marker) and anti-HA. N, nucleus; LG, large granules; SG, small granules; M, microsomes; S, final supernatant.</p

Overexpression of <i>Tc</i>SIR2RP1HA affects <i>in vitro</i> metacyclogenesis rate.

<p><i>In vitro</i> metacyclogenesis using TAU medium of lines harboring transgenes encoding <i>Tc</i>SIR2RP1HA and <i>Tc</i>SIR2RP3HA uninduced (- Tet) or induced (+ Tet) with 0.5 μg/ml Tetracycline. The bar graph represents the mean ± SEM from three independent experiments; **** p<0.0001 (unpaired, two-tailed Student t test).</p