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

Abstract 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 strain. Additionally, loratadine, cyproheptadine and clofazimine showed trypanocidal effect on epimastigotes of the CL Brener and DM28c strains. Finally, a synergistic effect between benznidazole and the CV chemical analogues was evidenced by combination and dose-reduction indexes values in epimastigotes 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. 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. Author summary Chagas disease, caused by the parasite Trypanosoma cruzi , affects 7 million people worldwide. Despite there are two drugs available since 50 years ago, the therapy present severe side effects and is not effective in the chronic phase of the disease were most of the patients are diagnosed. Crystal violet (CV) was utilized as additive in blood banks to prevent transfusion-transmitted Chagas disease. Proline is involved in many pathways, like infection establishment and life cycle progression. In this work we first demonstrate that CV has the proline permease TcAAAP069 as one of its molecular targets. Then we search in a database of already-approved drugs for compounds that were structurally related to CV under the premise "similar structure, similar activity". We identified three drugs that inhibit proline transport and present at least the same trypanocidal effect than benznidazole, the current treatment for Chagas disease. Finally we observed a synergistic effect with the multidrug combination therapy. Drug discovery is an expensive and time-consuming process and Chagas disease is associated with poverty. The discovery of new indications to old drugs, called drug repurposing, can facilitate a rapid and more profitable therapy application since preclinical trials and pharmacokinetic studies are already available.