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

Enzymatic inhibition of TcSir2rp1 by BNIP derivatives.

<p><b>A-B)</b> The percentage of inhibition of the NAD⁺-dependent deacetylase activity of TcSir2rp1 by BNIP derivatives is represented in the y axis. Positive (no drug) and negative (NAM, nicotinamide at 2 mM) controls are represented. Bars represent the average + standard deviation of at least two independent experiments. Differences between the experimental groups were considered significant as follows: * p<0.05, *** p<0.001 and **** p<0.0001.</p

<i>In vitro</i> activity of BNIP derivatives.

<p><b>A)</b> Primary screening of BNIP derivatives by an <i>in vitro</i> assay for intracellular <i>T</i>. <i>cruzi</i> amastigotes at a single dose of 2.5 μM. Values for both anti-parasitic activity and cell ratio are represented in the dot plot graph. The blue square represents the zone of selection for active, low toxicity hits to be evaluated by dose-response curve analysis. The green square represents the zone of selection for compounds to be tested at a higher dose. <b>B)</b> Primary screening of the compounds selected for testing at a higher dose of 10 μM. Values for both anti-parasitic activity and cell ratio are represented in the dot plot graph. The blue square represents the zone of selection for active, low toxicity hits to be evaluated by dose-response curve analysis. <b>C)</b> Representation of the selectivity indexes of the compounds analysed by dose-response curve analysis, with the determined anti-parasitic activity (EC₅₀) in the y axis, and the cell ratio (CC₅₀) in the x axis. Dots represent the average of three independent experiments.</p

<i>In vitro</i> hepatocytes and neurons injury scores for BNIPSpd (9) by HCS.

<p>The score was calculated as the sum of individual scores obtained from a panel of <i>in vitro</i> cytotoxicity assays that include: mitochondrial dysfunction measured by TMRM probe dynamics in cells; membrane integrity assayed by lactate dehydrogenase quantification; DNA damage by imaging with H2AX antibody; apoptosis by caspase 3/7 activation; and neurite outgrowth as imaged with an anti-tubulin III antibody. Nimesulide (400 μM), an approved drug with a mild toxicological profile, was included as a toxicity control.</p

TcSir2rp1 structural model and docking with compound 9.

<p><b>A</b>) The 3.5 Å structural model shows a large Rossmann-fold domain (composed of 6 parallel β-strands, sandwiched between 2 layers of α-helices), and a small zinc binding domain. The substrate acetylated peptide p53 is bound to the cleft between the small and the large domains. <b>B-C</b>) The substrate p53 peptide was removed from the structure and docking studies conducted with compound <b>9</b>. Several conformations (only 2 shown here) of compound <b>9</b> were possible in a putative ligand binding site close to the NAD binding site.</p

Chemical structures of the bisnaphthalimidopropyl derivatives used in the present study.

<p>R = H in all compounds except for compound 13.</p

EC₅₀ values (μM) of BNIP derivatives and nifurtimox against <i>T</i>. <i>cruzi</i> epimastigotes, wild-type (WT) and cells overexpressing TcSir2rp1 (OE) with the pTcINDEX.

<p>EC₅₀ values (μM) of BNIP derivatives and nifurtimox against <i>T</i>. <i>cruzi</i> epimastigotes, wild-type (WT) and cells overexpressing TcSir2rp1 (OE) with the pTcINDEX.</p

Anti-parasitic activity, cytotoxicity and selectivity index of BNIP derivatives selected for dose-response curve analysis.

<p>Anti-parasitic activity, cytotoxicity and selectivity index of BNIP derivatives selected for dose-response curve analysis.</p

Cytotoxicity and selectivity of BNIPSpd (9).

<p>Cytotoxicity and selectivity of BNIPSpd (9).</p