Structurally optimized analogs of the retrograde trafficking inhibitor Retro-2cycl limit Leishmania infections

In infected mammalian cells, Leishmania parasites reside within specialized compartments called parasitophorous vacuoles (LPVs). We have previously shown that Retro-2, a member of a novel class of small retrograde pathway inhibitors caused reduced LPV sizes and lower parasite numbers during experimental L. mexicana sp. infections. The purpose of this study was to determine if structural analogs of Retro-2cycl reported to have superior potency in the inhibition of retrograde pathway-dependent phenomena (i.e., polyomavirus cellular infection by polyomavrius and Shiga toxin trafficking in cells) are also more effective than the parent compound at controlling Leishmania infections. In addition to their effects on LPV development, we show that two optimized analogs of Retro-2cycl, DHQZ 36 and DHQZ 36.1 limit Leishmania amazonensis infection in macrophages at EC50 of 13.63+/-2.58μM and10.57+/-2.66μM, respectively, which is significantly lower than 40.15μM the EC50 of Retro-2cycl. In addition, these analogs caused a reversal in Leishmania induced suppression of IL-6 release by infected cells after LPS activation. Moreover, we show that in contrast to Retro-2cycl that is Leishmania static, the analogs can kill Leishmania parasites in axenic cultures, which is a desirable attribute for any drug to treat Leishmania infections. Together, these studies validate and extend the published structure-activity relationship analyses of Retro-2cycl.在受感染的哺乳动物细胞中，利什曼原虫（Leishmania）寄生在名为寄生虫寄生泡（parasitophorous vacuoles，LPVs）的特化区室内。我们此前的研究表明，Retro-2作为一类新型小型逆行通路抑制剂的成员，在实验性墨西哥利什曼原虫（L. mexicana sp.）感染过程中，可缩小LPV体积并降低寄生虫载量。本研究旨在探究Retro-2cycl的结构类似物——据报道这类类似物在抑制逆行通路依赖性现象（即多瘤病毒介导的细胞感染以及细胞内志贺毒素的转运）方面具有更优效力——在控制利什曼原虫感染方面是否也优于其母体化合物。除了对LPV发育的影响外，我们的研究还发现，两种经过优化的Retro-2cycl结构类似物DHQZ 36与DHQZ 36.1，可分别以13.63±2.58μM和10.57±2.66μM的半最大效应浓度（EC50），抑制巨噬细胞中的亚马逊利什曼原虫（Leishmania amazonensis）感染，该EC50值显著低于Retro-2cycl的40.15μM。此外，这些类似物能够逆转利什曼原虫在脂多糖（LPS）激活后对受感染细胞释放白细胞介素6（IL-6）的抑制作用。不仅如此，与仅能抑制利什曼原虫增殖的Retro-2cycl不同，这些类似物可在纯培养（axenic cultures）体系中直接杀灭利什曼原虫，这一特性对于治疗利什曼原虫感染的药物而言是理想的优势属性。综上，本研究验证并拓展了已发表的Retro-2cycl构效关系（structure-activity relationship，SAR）分析结果。