RESUMEN
Chagas disease, also known as American trypanosomiasis, is a neglected tropical disease caused by the protozoa Trypanosoma cruzi, affecting nearly 7 million people only in the Americas. Polyamines are essential compounds for parasite growth, survival, and differentiation. However, because trypanosomatids are auxotrophic for polyamines, they must be obtained from the host by specific transporters. In this investigation, an ensemble of QSAR classifiers able to identify polyamine analogs with trypanocidal activity was developed. Then, a multi-template homology model of the dimeric polyamine transporter of T. cruzi, TcPAT12, was created with Rosetta, and then refined by enhanced sampling molecular dynamics simulations. Using representative snapshots extracted from the trajectory, a docking model able to discriminate between active and inactive compounds was developed and validated. Both models were applied in a parallel virtual screening campaign to repurpose known drugs as anti-trypanosomal compounds inhibiting polyamine transport in T. cruzi. Montelukast, Quinestrol, Danazol, and Dutasteride were selected for in vitro testing, and all of them inhibited putrescine uptake in biochemical assays, confirming the predictive ability of the computational models. Furthermore, all the confirmed hits proved to inhibit epimastigote proliferation, and Quinestrol and Danazol were able to inhibit, in the low micromolar range, the viability of trypomastigotes and the intracellular growth of amastigotes.
Asunto(s)
Enfermedad de Chagas , Tripanocidas , Trypanosoma cruzi , Humanos , Putrescina/uso terapéutico , Ligandos , Danazol/uso terapéutico , Quinestrol/uso terapéutico , Poliaminas/química , Poliaminas/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Proteínas de Transporte de Membrana/uso terapéutico , Tripanocidas/farmacología , Tripanocidas/químicaRESUMEN
BACKGROUND: Hypercholesterolemia causes alterations in platelet function. Platelet hyperaggregation is considered a predisposing factor for atherosclerosis. In this paper, the antiaggregating effect of the polyamines putrescine, spermidine, and spermine was studied on platelets of normal and hypercholesterolemic rabbits. METHODS: New Zealand rabbits were fed with a cholesterol-enriched diet for 10 weeks. Lipids and glucose were determined in serum. The assays of platelet aggregation were carried out using platelet-rich plasma (PRP) obtained from both control and cholesterol-fed rabbits. We used 2.5 micromol /mL ADP and 2 microg/mL collagen as inductors of platelet aggregation. In addition, arginase activity and L-arginine content were determined in PRP. RESULTS: Serum total cholesterol and LDL-cholesterol concentrations were increased from 26.3 +/- 8.1 to 1,485.0 +/- 26.8 mg/dL and from 15.9 +/- 5.9 to 1,383.8 +/- 58.9 mg/dL, respectively, whereas triglyceride concentration increased from 88.3 +/- 35.6 to 411.0 +/- 154.5 mg/dL upon cholesterol feeding. Seventy-five percent of platelet aggregation inhibition was observed with 10 microM of polyamines in PRP of normal rabbits. Spermine inhibited platelet aggregation by 54% in PRP of hypercholesterolemic rabbits when ADP was used as agonist. The order of polyamine action was spermine > spermidine > putrescine. In addition, we found that platelet arginase activity and L-arginine content were unaltered upon hypercholesterolemia. CONCLUSIONS: These results show that the polyamines putrescine, spermidine, and spermine have antagonist action in platelet aggregation and suggest a key role of polyamines in platelet aggregation under normal and hypercholesterolemic conditions.