Abstract:
Propeptides, released from the autocatalytic activation of its zymogen, are potential inhibitors against proteases involved in cancer cell invasion and migration. Our research team previously obtained novel propeptides (SnuCalCpIs) from transcriptome analysis of the medicinal plant Calotropis procera R. Br. and reported them as promising candidates for cancer therapeutics due to their cathepsin L inhibition activity. In the present study, inhibitory activity among SnuCalCpIs was compared with inhibition efficiency and verified by in silico molecular docking analysis. Only SnuCalCpI03 and SnuCalCpI15, expressed in Escherichia coli, showed inhibitory activity against cathepsin L as competitive inhibitors, and the half-maximal inhibitory concentrations (IC50) values of 2.1 nM and 1.6 nM, respectively. They were stable below 70 °C, maintaining more than 90% inhibitory activity over a wide range of pH (2.0-10.0), except at the isoelectric point (pI). The template-based docking simulation models showed that SnuCalCpI02, SnuCalCpI12, and SnuCalCpI16 could not interact with the substrate-binding cleft of cathepsin L even though they possessed the same conserved domain. In contrast, SnuCalCpI03 and SnuCalCpI15 interacted with cathepsin L along the propeptide binding loop and substrate-binding cleft, resulting in obstruction of substrate access to the active site.
摘要:
前肽,从其酶原的自动催化活化中释放出来,是针对参与癌细胞侵袭和迁移的蛋白酶的潜在抑制剂。我们的研究小组先前从药用植物CalotropisproceraR.Br的转录组分析中获得了新的前肽(SnuCalCpIs)。并报道它们由于其组织蛋白酶L抑制活性而成为癌症治疗的有希望的候选者。在本研究中,将SnuCalCpIs的抑制活性与抑制效率进行了比较,并通过计算机分子对接分析进行了验证。只有SnuCalCpI03和SnuCalCpI15在大肠杆菌中表达,显示对组织蛋白酶L作为竞争性抑制剂的抑制活性,半最大抑制浓度(IC50)为2.1nM和1.6nM,分别。它们在70℃以下稳定,在宽范围的pH(2.0-10.0)内保持90%以上的抑制活性,除了在等电点(pI)。基于模板的对接模拟模型显示,SnuCalCpI02、SnuCalCpI12和SnuCalCpI16不能与组织蛋白酶L的底物结合裂隙相互作用,即使它们具有相同的保守结构域。相比之下,SnuCalCpI03和SnuCalCpI15沿前肽结合环和底物结合裂隙与组织蛋白酶L相互作用,导致底物进入活性位点的障碍。