Crystal violet structural analogues identified by in silico drug repositioning present anti-Trypanosoma cruzi activity through inhibition of proline transporter TcAAAP069

BACKGROUND: Crystal violet (CV) was used for several years in blood banks to eliminate the parasite Trypanosoma cruzi in endemic areas in order to prevent transfusion-transmitted Chagas disease. One mechanism of action described for CV involves inhibition of proline uptake. In T. cruzi, proline is essential for host cell infection and intracellular differentiation among other processes, and can be obtained through the proline permease TcAAAP069. METHODOLOGY/PRINCIPAL FINDINGS: CV inhibited proline transporter TcAAAP069 and parasites overexpressing this permease were 47-fold more sensitive to this compound than control parasites. Using CV as reference molecule, loratadine, cyproheptadine, olanzapine and clofazimine were identified as structurally related compounds to CV (structural analogues) by in silico drug repurposing through a similarity-based virtual screening protocol. All these already-approved drugs for clinical use inhibited TcAAAP069 activity with different efficacies and also presented trypanocidal action in epimastigotes, trypomastigotes and amastigotes of the Y, CL Brener and Dm28c T. cruzi strains. Finally, a synergistic effect between benznidazole and the CV chemical analogues was evidenced by combination and dose-reduction indexes values in epimastigotes and trypomastigotes of the Y strain. CONCLUSIONS/SIGNIFICANCE: Loratadine, cyproheptadine and clofazimine inhibit TcAAAP069 proline transporter and also present trypanocidal effect against all T. cruzi life stages in strains from three different DTUs. These CV structural analogues could be a starting point to design therapeutic alternatives to treat Chagas disease by finding new indications for old drugs. This approach, called drug repurposing is a recommended strategy by the World Health Organization to treat neglected diseases, like Chagas disease, and combination therapy may improve the possibility of success of repositioned drugs.