School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n°, 14040-903, Ribeirão Preto, SP, Brazil.
University of Vila Velha, Av. Comissário José Dantas de Melo, 21, Boa Vista II, 29102-920, Vila Velha, ES, Brazil.
Sci Rep. 2020 Mar 11;10(1):4476. doi: 10.1038/s41598-020-61258-x.
Snake venom serine proteases (SVSPs) are complex and multifunctional enzymes, acting primarily on hemostasis. In this work, we report the hitherto unknown inhibitory effect of a SVSP, named collinein-1, isolated from the venom of Crotalus durissus collilineatus, on a cancer-relevant voltage-gated potassium channel (hEAG1). Among 12 voltage-gated ion channels tested, collinein-1 selectively inhibited hEAG1 currents, with a mechanism independent of its enzymatic activity. Corroboratively, we demonstrated that collinein-1 reduced the viability of human breast cancer cell line MCF7 (high expression of hEAG1), but does not affect the liver carcinoma and the non-tumorigenic epithelial breast cell lines (HepG2 and MCF10A, respectively), which present low expression of hEAG1. In order to obtain both functional and structural validation of this unexpected discovery, where an unusually large ligand acts as an inhibitor of an ion channel, a recombinant and catalytically inactive mutant of collinein-1 (His43Arg) was produced and found to preserve its capability to inhibit hEAG1. A molecular docking model was proposed in which Arg79 of the SVSP 99-loop interacts directly with the potassium selectivity filter of the hEAG1 channel.
蛇毒丝氨酸蛋白酶（SVSPs）是复杂且多功能的酶，主要作用于止血。在这项工作中，我们报告了一种来自 Crotalus durissus collilineatus 毒液的 SVSP，名为 collinein-1，其迄今为止未知的对与癌症相关的电压门控钾通道（hEAG1）的抑制作用。在测试的 12 种电压门控离子通道中，collinein-1 选择性地抑制 hEAG1 电流，其机制与酶活性无关。此外，我们证明 collinein-1 降低了人乳腺癌细胞系 MCF7（高表达 hEAG1）的活力，但不影响肝癌和非致瘤上皮乳腺细胞系（HepG2 和 MCF10A，分别），其 hEAG1 表达水平较低。为了获得对这一意外发现的功能和结构验证，其中一个异常大的配体作为离子通道的抑制剂，我们产生了一种重组的和无催化活性的 collinein-1 突变体（His43Arg），并发现它保留了抑制 hEAG1 的能力。提出了一种分子对接模型，其中 SVSP 99-环的 Arg79 与 hEAG1 通道的钾选择性过滤器直接相互作用。
