DOI: 10.57715/unr/axngvk
title: Supplementary data for: Amino Acids metabolism as drug target for anti-trypanosomatids drug discovery

description: Description: Experimental data on the antiparasitic drug discovery project. New compounds characterization data (NMR, UV-Vis, chemoinformatics data) T. cruzi growing curves (IC50, washout experiments) Cell death experiments (flow cytometry) The present dataset is focused in the obtaining of different proline analogues and the biological validation as T. cruzi epimastigotes growth inhibitors, as well as the determination/validation of their mode of action. The datasets contain: Supporting Information 01 (file "Ballari_et_al_Supporting_Information_01_T_cruzi_growing_curves.pdf"): Growth curves, dose response curves, and IC50s obtained for treatments of different strains of T. cruzi epimastigotes with variable concentrations of proline analogues, as well as growth curves for washout experiments on T. cruzi epimastigotes. Exponentially proliferating T. cruzi epimastigotes (2.5x106 cells/mL) were seeded in 96-well plates and further treated with different concentrations of each analogue (concentration range from 25 to 250 µM) or untreated (negative control). As a positive control, a combination of rotenone (60 µM) and antimycin (0.5 µM) was used. The plates were incubated at 28 °C for 8 to 10 days. Cell proliferation was spectrophotometrically measured by recording the absorbance at 620 nm every 24 h for 8 to 10 days. The absorbance was transformed into cell density values (cells/mL) using a linear regression equation that was previously obtained under the same conditions. Data from the mid-exponential growth phase were used to determine the concentration of the analogues that inhibited 50% of the parasite proliferation (IC50). For this, we explored classic dose-response sigmoid functions to find the best fit to the obtained data. Each experiment was made in triplicates, and the results presented correspond to the average of three independent experiments. Supporting Information 02 (file "Ballari_et_al_Supporting_Information_02_Cell_death_experiments.pdf"): Analysis of cell death and cell cycle arrest mechanisms on T. cruzi epimastigotes caused by treatment with proline analogues. To analyse cell death mechanism, T. cruzi, strain CL14 epimastigotes (2.5x106 cells/mL) were cultured in LIT medium at 28 °C and treated with compounds 1, 2 and 3 at their respective IC50 or IC80 concentration. After 24, 48 and 72 h of incubation, treated cells and controls (1x106 cells/mL) were washed once with annexin buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl, pH 7.4) and resuspended in 50 µL of the same buffer. The parasites were incubated in ice (protected from light), for 15 min, in the presence of annexin-V FITC (Invitrogen, Eugene, Oregon, USA), according to the manufacturer's instructions, and 1 µg/mL propidium iodide. Then, 450 µL of annexin buffer was added, and the parasites were analysed by flow cytometry (AccuriTM C6 Plus cytometer, BD Biosciences), with 10,000 events collected and analysed using Flowjo_v10.8.1 software. On the other hand, to explore cell cycle arrest mechanism, epimastigote forms were cultivated in LIT medium and treated with IC50 or IC80 of the compounds 1, 2 and 3 during 24, 48 and 72 h. To analyse the DNA content, the parasites were washed once in PBS and resuspended in lysis buffer (phosphate buffer Na2HPO4 7.7 mM, KH2PO4 2.3 mM, pH 7.4) and digitonin 64 μM. Then, the parasites were incubated in ice for 30 min, and propidium iodide 20 μg/mL was added. The parasites were analysed by flow cytometry (AccuriTM C6 Plus cytometer, BD Biosciences), with 10,000 events collected and analysed using Flowjo_v10.8.1 software. Supporting Information 03 (file "Ballari_et_al_Supporting_Information_03_Compounds_Characterization.pdf"): UV-vis characterization of the fluorescent proline analogue 1-NBD, as well as NMR spectral characterization of all final compounds. Prediction of physicochemical properties of fluorescent analogues was performed using molinspiration platform (http://www.molinspiration.com). UV-visible absorption spectra were obtained using a Shimadzu MultiSpec 1501 UV-vis spectrophotometer. Emision and excitation spectra were obtained using a Varian Cary Eclipse spectroluminometer. 1H and 13C NMR spectra were acquired on a Bruker Avance II 300 MHz (75.13 MHz) using CDCl3 as solvent. Chemical shifts (δ) were reported in ppm downﬁeld from tetramethylsilane and coupling constants are in hertz (Hz). NMR spectra were obtained at 298 K unless otherwise stated and samples run as a dilute solution of the stated solvent. All NMR spectra were referenced to the residual undeuterated solvent as an internal reference. Value of the data: The general significance of this dataset revolves around the generation of new ways of treatment of Chagas Disease, a Neglected Tropical Disease which treatment nowadays relays only on two drugs that are partially effective and present multiple side effects.