Universidad de Buenos Aires, Facultad de Medicina, Instituto de Investigaciones Médicas A. Lanari, Buenos Aires, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de Buenos Aires, Instituto de Investigaciones Médicas (IDIM), Laboratorio de Parasitología Molecular, Buenos Aires, Argentina.
PLoS Negl Trop Dis. 2020 Jan 21;14(1):e0007481. doi: 10.1371/journal.pntd.0007481. eCollection 2020 Jan.
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.
在血库中，结晶紫（CV）曾被用于消除寄生虫克氏锥虫（Trypanosoma cruzi），以预防输血传播的恰加斯病，该寄生虫在流行地区流行。CV 的一种作用机制涉及脯氨酸摄取的抑制。在 T. cruzi 中，脯氨酸对于宿主细胞感染和细胞内分化等过程是必不可少的，并且可以通过脯氨酸渗透酶 TcAAAP069 获得。
方法/主要发现：CV 抑制了脯氨酸转运蛋白 TcAAAP069，过表达该渗透酶的寄生虫对该化合物的敏感性比对照寄生虫高 47 倍。使用 CV 作为参考分子，通过基于相似性的虚拟筛选方案，通过药物再利用的计算方法，洛他定、赛庚啶、奥氮平和氯法齐明被鉴定为与 CV 具有结构相关性的化合物（结构类似物）。所有这些已批准用于临床的药物均以不同的功效抑制 TcAAAP069 活性，并且在 Y、CL Brener 和 Dm28c T. cruzi 株的epimastigotes、trypomastigotes 和 amastigotes 中也具有杀锥虫作用。最后，通过 Y 株 epimastigotes 和 trypomastigotes 的组合和剂量降低指数值，证明了苯硝唑与 CV 化学类似物之间存在协同作用。
结论/意义：洛他定、赛庚啶和氯法齐明抑制 TcAAAP069 脯氨酸转运蛋白，并对来自三个不同 DTU 的 Y 株的所有 T. cruzi 生活阶段具有杀锥虫作用。这些 CV 结构类似物可以作为设计治疗恰加斯病的治疗替代方案的起点，为旧药物寻找新的适应症。这种方法称为药物再利用，是世界卫生组织推荐的治疗被忽视疾病（如恰加斯病）的策略，联合治疗可能会提高重新定位药物成功的可能性。
