Purine derivatives as potential agents against Chagas disease: Ex vivo, in vitro, in silico evaluation, and identification of targets in Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi, affects millions of people worldwide and remains inadequately treated because the currently available drugs, benznidazole (Bzn) and nifurtimox (Nfx), show variable efficacy and significant toxicity across different disease stages. Given that purine nucleoside analogues have demonstrated antitrypanosomal activity and may target essential parasite proteins such as cruzipain (TcCZP) and bromodomain factor 2 (TcBDF2), this study aimed to determine whether a set of 20 purine derivatives possessed antiparasitic properties in relation to these targets using ex vivo, in vitro and in silico approaches. The results indicated that, in a screening assay against circulating trypomastigotes from two Mexican strains of T. cruzi (NINOA and INC-5), most of these purines outperformed the reference drugs Nfx and Bzn, according to the half-maximal lethal concentration (LC50) values (which ranged from 6.28 to 144 µM for the NINOA strain and from 18.42 to 166.87 µM for the INC-5 strain), with the most active purines being the most active purines 9e, 9i, 9j, 9m, 9n, and 9t. Structure-activity relationship (SAR) analyses explained the influence of some fragments in the purine scaffold related to the trypanocidal effect. Subsequently, selected purines were tested against intracellular Tulahuen-2 amastigotes, where four demonstrated activities at sub-10 µM concentrations, and 9m achieved approximately 1.5-fold greater potency than Bzn. Due to the low inhibitory activity of the purines tested against TcCZP (the best compound, 9j, had an IC₅₀ of 87.1 µM), this target was ruled out as the source of the trypanocidal activity. However, when the compounds were evaluated on TcBDF2, the results showed that 9i and 9t were the two best compounds with binding capacities to TcBDF2 (Kd = 13.5 and 19.6 µM, respectively), which was more closely aligned with trypanocidal activity. In addition, 9i and 9t did not bind to TcBDF3, indicating selectivity among these targets. Molecular docking and 200 ns molecular dynamics simulations supported the stable binding of purine 9i to the TcBDF2 bromodomain pocket. In addition, purines 9i, 9j, 9m, and 9t were predicted to have good pharmacokinetic profiles for oral administration of the drug. Overall, trisubstituted purines emerged as promising antichagasic leads, with TcBDF2 engagement appearing more consistent with the observed trypanocidal activity, thereby guiding future optimisation towards a balance of potency, selectivity, and cytotoxicity.