Toxicology and Pharmacology, KU Leuven, Leuven 3000, Belgium.
Laboratory of Mass Spectrometry-MolSys, University of Liege, Liege 4000, Belgium.
Mar Drugs. 2017 Sep 13;15(9):287. doi: 10.3390/md15090287.
The human ether-à-go-go channel (hEag1 or K10.1) is a cancer-relevant voltage-gated potassium channel that is overexpressed in a majority of human tumors. Peptides that are able to selectively inhibit this channel can be lead compounds in the search for new anticancer drugs. Here, we report the activity-guided purification and electrophysiological characterization of a novel K10.1 inhibitor from the sea anemone . Purified sea anemone fractions were screened for inhibitory activity on K10.1 by measuring whole-cell currents as expressed in oocytes using the two-microelectrode voltage clamp technique. Fractions that showed activity on Kv10.1 were further purified by RP-HPLC. The amino acid sequence of the peptide was determined by a combination of MALDI- LIFT-TOF/TOF MS/MS and CID-ESI-FT-ICR MS/MS and showed a high similarity with APETx1 and APETx3 and was therefore named APETx4. Subsequently, the peptide was electrophysiologically characterized on K10.1. The selectivity of the toxin was investigated on an array of voltage-gated ion channels, including the cardiac human ether-à-go-go-related gene potassium channel (hERG or Kv11.1). The toxin inhibits K10.1 with an IC value of 1.1 μM. In the presence of a similar toxin concentration, a shift of the activation curve towards more positive potentials was observed. Similar to the effect of the gating modifier toxin APETx1 on hERG, the inhibition of Kv10.1 by the isolated toxin is reduced at more positive voltages and the peptide seems to keep the channel in a closed state. Although the peptide also induces inhibitory effects on other K and Na channels, it exhibits no significant effect on hERG. Moreover, APETx4 induces a concentration-dependent cytotoxic and proapoptotic effect in various cancerous and noncancerous cell lines. This newly identified K10.1 inhibitor can be used as a tool to further characterize the oncogenic channel K10.1 or as a scaffold for the design and synthesis of more potent and safer anticancer drugs.
人 Ether-à-go-go 通道 (hEag1 或 K10.1) 是一种与癌症相关的电压门控钾通道，在大多数人类肿瘤中过度表达。能够选择性抑制该通道的肽可以成为寻找新抗癌药物的先导化合物。在这里，我们报告了一种新型 K10.1 抑制剂的活性导向纯化和电生理特性。通过使用双微电极电压钳技术在卵母细胞中表达来测量全细胞电流，筛选纯化的海葵级分对 K10.1 的抑制活性。显示对 Kv10.1 有活性的级分进一步通过 RP-HPLC 纯化。肽的氨基酸序列通过 MALDI- LIFT-TOF/TOF MS/MS 和 CID-ESI-FT-ICR MS/MS 的组合确定，与 APETx1 和 APETx3 高度相似，因此命名为 APETx4。随后，该肽在 K10.1 上进行了电生理特性分析。该毒素的选择性在一系列电压门控离子通道上进行了研究，包括心脏人 Ether-à-go-go 相关基因钾通道 (hERG 或 Kv11.1)。该毒素对 K10.1 的抑制作用的 IC 值为 1.1 μM。在类似的毒素浓度存在下，观察到激活曲线向更正电位的移动。类似于门控调节剂毒素 APETx1 对 hERG 的作用，分离毒素对 Kv10.1 的抑制作用在更正的电压下降低，并且肽似乎使通道保持关闭状态。尽管该肽还会对其他 K 和 Na 通道产生抑制作用，但对 hERG 没有显著影响。此外，APETx4 在各种癌细胞系和非癌细胞系中诱导浓度依赖性细胞毒性和促凋亡作用。这种新鉴定的 K10.1 抑制剂可用于进一步表征致癌通道 K10.1，或作为设计和合成更有效和更安全的抗癌药物的支架。
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