Arylidene ketones with Potent Trypanosomicidal Activity that Causes Late Apoptosis / Necrosis Like Nifurtimox

1 Grupo de Química Medicinal, Facultad de Ciencias Universidad de la República, Iguá. 4225, Montevideo, 11400, Uruguay. 2 Laboratorio de Interacciones Moleculares, Facultad de Ciencias, Universidad de la República, Iguá, 4225, Montevideo. 11400, Uruguay. 3 Área de Radiofarmacia, Centro de Investigaciones Nucleares, Universidad de la República, Mataojo S/N, Montevideo, 11400, Uruguay. 4 Laboratorio de Moléculas Bioactivas, Centro Universitario Regional Litoral Norte, Universidad de la República, Rute 3 km 363, Paysandú, 60000. Uruguay.

• Chagas disease remains the major parasite disease in Latin America • Migration of infected people has spread the disease to non-endemic areas, presenting a new worldwide challenge.• Approximately 6-8 million of people is infected and more than 70 million is at risk of getting the disease.

Current pharmacotherapy
Both drugs are: -Toxic -They are not good in eliminating the amastigote form of the parasite -Mutagenic

Background of the present work
Trypanosomicidal structures used for the design of new and simpler symmetric diarylidene ketone from dibenzalketone (Prototype I) and furylthiazolidines (Prototype II).

Apoptosis/ Necrosis
Annexin V acts by binding to phosphatidylserine exposed in apoptotic cells.Propidium iodide is used to identify necrotic cells as it binds to the DNA or RNA of cells as the integrity of the membrane disappears.For Chagas disease or any disease, it is preferable that the parasite dies by apoptosis than by necrosis because it is less invasive and cause less inflammation events.

Materials and methods
 1x10 6 of T. cruzi strain Tulahuen 2 were incubated with the compounds to be evaluated on a 12-well plate.
 The compounds were incubated at 28 ° C for 24 h.Compound 1 at a concentration of 100 µM; 2 at a concentration of 12µ; 3 at a concentration of 0,8 µM (20X IC 50 ).Nfx and Bnz were as control reference drugs, at concentrations 160µM and 140µM respectively (20X IC 50 ).Untreated parasites were incubated with the solvent (in this case dimethylsulfoxide) in a concentration that never exceeding 1%.
• Finally the Cell death mechanism was analyzed using the Dead Cell Apoptosis Kit (Thermo Fisher Scientific) following the manufacturer's instructions.Briefly, untreated and drug treated parasites were harvested by centrifugation, washed with 1X PBS and incubated for 15 min at room temperature (RT) with 5 mg/mL Alexa Fluor® 488 annexin V (AV) and 10 mg/mL propidium iodide (PI) diluted in annexin-V binding buffer containing Ca2+.Cells were immediately analyzed on a flow cytometer.Dual-parameter flow cytometric analysis was performed on an Accuri C6 (BD Bioscience) flow cytometer, using a 533/30 nm signal detector (FL1-H) for AV and a 670 nm long pass PI emission signal detector (FL3-H).Fluorescence intensity was acquired for 10,000 events and the data were analyzed using BD CSampler software (BD Bioscience).
All graphs shown correspond to the evaluation of compounds at 20X IC 50 Parasites death by Late apoptosis/Necrosis Parasites that did not die by apoptosis or necrosis Apoptosis Aguilera, E., Varela J., Birriel, E., Serna E., Yaluff G., Vera de Bilbao ,N., Aguirre-López, B., Cabrera, N., Díaz Mazariegos, S., Truena de Gómez-Puyou, M. Gómez-Puyou, A., Perez-Montfort, R., Minini, L., Merlino, A., Cerecetto, H., González, M., Alvarez, G. ChemMedChem, 2016, 11, 1328-1338.Compound 2 produced more than 70 % of Late Apoptosis/Necrosis at 24 h post incubation.It was evaluated at 20X IC 50 .Apoptosis was not significantly observed Parasites death by Late apoptosis/Necrosis Apoptosis Parasites that did not die by apoptosis or necrosis Compound 3 did at 20X IC 50 not produced Apoptosis nor Necrosis at 24 h post incubation.The cells were alive or suffer different types of cellular death like autophagia in this condition.Late apoptosis/Necrosis Apoptosis Parasites that did not die by apoptosis or necrosis produced more than 75 % of Late Apoptosis/Necrosis at 24 h post incubation evaluated at a concentration of 20X IC 50 .The same effect on Necrosis of NFx was observed previously using TUNNEL microscopy and 1 H RMN Benitez, D., Pezaroglo, H., Martínez, V., Casanova, G., Cabrera, G., Galanti, N., González, M., Cerecetto, H. Parasitology.2012, 139(4),506-515 Parasites death by Late apoptosis/Necrosis Apoptosis Parasites that did not die by apoptosis or necrosis not die by apoptosis or necrosis