Santana Mota Wanessa, Oliveira Simone S C, Dos Santos-Neto Agenor G, Souza Damião P, Castro Mayara, Pereira Matheus M, Jain Sona, Cardoso Juliana C, Souto Eliana B, Severino Patrícia, Santos André L S
Institute of Technology and Research, Farolândia, Aracaju, Sergipe, Brazil.
Institute of Microbiology Paulo de Góes, Department of General Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Int J Antimicrob Agents. 2025 Sep;66(3):107548. doi: 10.1016/j.ijantimicag.2025.107548. Epub 2025 Jun 4.
Several drugs are commonly used in the management of Leishmaniasis, but their toxicity, cost, and efficacy depend on the form of the disease.The development of new therapeutics remains important to expand treatment options and address existing limitations. Previously, we reported that isopentyl caffeate (ICaf) exhibited potent activity against promastigotes and amastigotes of Leishmania amazonensis without notable toxicity in a murine model. This study investigated the effects of ICaf on the metallopeptidase gp63 (leishmanolysin), a key virulence factor in Leishmania that supports parasite survival by modulating host immune responses, degrading host proteins, and facilitating macrophage invasion.
Molecular docking analysis revealed that ICaf interacted with key amino acids within the active site of gp63, achieving a docking score of -5.5 kcal/mol. The gp63-ICaf interaction was stabilised through a combination of hydrogen bonding, hydrophobic interactions, metal-acceptor interactions and van der Waals forces. Experimental data supported these in silico findings, with ICaf showing a typical dose-dependent inhibition of gp63 proteolytic activity, resulting in an IC of 1.51 µM and a K of 9.89 µM. The formation of a stable gp63-ICaf complex was demonstrated by gel electrophoresis assay. Furthermore, pretreatment of promastigotes with ICaf reduced cell-associated gp63 proteolytic activity, as evidenced by decreased hydrolysis of the fluorogenic peptide substrate Z-Phe-Arg-AMC and gelatin incorporated into electrophoresis gel. Flow cytometry and confocal microscopy, both using anti-gp63 antibodies, further confirmed a decrease in gp63 expression on the parasite surface.
These findings suggest that ICaf is a promising strategy against leishmaniasis, offering a potential new approach to disease treatment.
几种药物常用于利什曼病的治疗，但其毒性、成本和疗效取决于疾病的形式。开发新的治疗方法对于扩大治疗选择和解决现有局限性仍然很重要。此前，我们报道异戊基咖啡酸（ICaf）在小鼠模型中对亚马逊利什曼原虫的前鞭毛体和无鞭毛体具有强大活性且无明显毒性。本研究调查了ICaf对金属肽酶gp63（利什曼溶素）的影响，gp63是利什曼原虫中的一种关键毒力因子，通过调节宿主免疫反应、降解宿主蛋白和促进巨噬细胞入侵来支持寄生虫存活。
分子对接分析表明，ICaf与gp63活性位点内的关键氨基酸相互作用，对接分数为-5.5千卡/摩尔。gp63-ICaf相互作用通过氢键、疏水相互作用、金属受体相互作用和范德华力的组合得以稳定。实验数据支持了这些计算机模拟结果，ICaf对gp63蛋白水解活性表现出典型的剂量依赖性抑制，IC50为1.51μM，Kd为9.89μM。凝胶电泳分析证明了稳定的gp63-ICaf复合物的形成。此外，用ICaf预处理前鞭毛体可降低细胞相关的gp63蛋白水解活性，这可通过荧光肽底物Z-Phe-Arg-AMC和掺入电泳凝胶的明胶水解减少来证明。使用抗gp63抗体的流式细胞术和共聚焦显微镜进一步证实了寄生虫表面gp63表达的降低。
这些发现表明，ICaf是一种有前景的抗利什曼病策略，为疾病治疗提供了一种潜在的新方法。
