Abstract
The human ether-à-go-go channel (hEag1 or KV10.1) 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 KV10.1 inhibitor from the sea anemone Anthopleura elegantissima. Purified sea anemone fractions were screened for inhibitory activity on KV10.1 by measuring whole-cell currents as expressed in Xenopus laevis 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 KV10.1. 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 KV10.1 with an IC50 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 KV and NaV 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 KV10.1 inhibitor can be used as a tool to further characterize the oncogenic channel KV10.1 or as a scaffold for the design and synthesis of more potent and safer anticancer drugs.
Highlights
Cancer is still one of the leading causes of death worldwide
We present the identification of a novel sea anemone peptide APETx4
The fraction showing inhibitory effect was further purified by reverse-phase high-performance liquid chromatography (RP-HPLC)
Summary
Cancer is still one of the leading causes of death worldwide. In 2012 about 14.1 million new cancer cases and 8.2 million cancer deaths were reported [1]. The WHO reports that in 2015, the number of cancer deaths has risen to 8.8 million. Stichodactyla helianthus, which is able to block both KV 1.1 and KV 1.3 in the low picomolar range An analogue of this peptide, named Dalazatide (Shk-186)) with improved selectivity for KV 1.3 was developed [34] and has currently completed Phase I clinical trials for the treatment of autoimmune diseases [35,36]. We present the identification of a novel sea anemone peptide APETx4. The effect of this peptide on KV 10.1 and other ion channels was electrophysiologically investigated. The antiproliferative, cytotoxic and pro-apoptotic activity of APETx1 was evaluated on various cancerous and non-cancerous cell lines
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