Moreels L et al. (2017), APETx4, a Novel Sea Anemone Toxin and a Modulat...

XB-ART-54021

Mar Drugs 2017 Sep 13;159:. doi: 10.3390/md15090287.

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APETx4, a Novel Sea Anemone Toxin and a Modulator of the Cancer-Relevant Potassium Channel KV10.1.

Moreels L , Peigneur S , Galan DT , De Pauw E , Béress L , Waelkens E , Pardo LA , Quinton L , Tytgat J .

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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.

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Species referenced: Xenopus laevis
Genes referenced: kcnh2 psd

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	Figure 1. MS/MS spectra characterizing the APETx4 sequence. (A) MALDI-PSD-TOF/TOF of the [M+H]+ species @m/z 4651.02 and (B) ESI-FT-ICR MS/MS (CID) of the [M+4H]4+ species @m/z 1163.76. The combination of the two spectra led to the characterization of the whole sequence (34 peptide bonds broken over 38). The isobaric amino acids L and I have been attributed by sequence homology with APETx1 and APETx3.
	Figure 2. In the upper panel, a multiple sequence alignment of APETx4 and its homologous sea anemone peptides is shown. Peptide names recommended by UniProt and alternative names are given. A Clustal Omega sequence alignment was performed using CLC Main Workbench. Amino acid residues are colored according to the RasMol amino color scheme. Percentages of identity (% ID) were obtained using standard protein BLAST. In the lower panel, the amino acid sequence of APETx4 was modeled on an averaged structured obtained from the solution NMR structure of APETx1 (PDB ID: 1WQK) using Modeller and Chimera. The amino acid residues are colored according to the RasMol amino color scheme. The 5 amino acid residues that differ between APETx1 and APETx4 are displayed as sticks. The C- and N-terminal residues and the cysteine residues (4, 6, 20, 30, 37 and 38) are indicated.
	Figure 3. Concentration- and state-dependent effect of APETx4 on evoked KV10.1 currents. (A) Representative KV10.1 current evoked by a 2 s pulse to 0 mV from the holding potential in control and toxin condition are shown in light gray. An exponential one phase association equation was used to fit the evoked currents (black lines); (B) Concentration-dependency plot fitted with a logistic equation shows the current inhibition (%) in function of APETx4 concentration; (C) The normalized current (I/Imax) was plotted versus the APETx4 incubation time. The time points were fitted with a one-phase decay exponential equation; (D) The state-dependency plot shows the normalized current in control conditions and in toxin condition. During the APETx4 addition the cell membrane potential was clamped at −90 mV for 10 min. All measurements were performed in external ND96 solution.
	Figure 4. Voltage-dependent effect of APETx4 on evoked KV10.1 currents. (A) Normalized currents elicited in ND96 solution (2 mM Ko) were plotted versus the applied pulse potentials (mV) in control (●) and toxin condition (○). The data points were fitted with the Boltzmann equation; (B) Normalized currents elicited in HK solution (96 mM Ko) were plotted versus the applied pulse potentials (mV) in control (●) and toxin condition (○). The data points were fitted with the Boltzmann equation; (C) The inhibited current (%) observed after 1.6 µM APETx4 addition in ND96 (○) and HK (●) solution was plotted versus the applied pulse potentials; (D) Effect of APETx4 on the inactivation properties of KV10.1. The non-inactivating channel fraction (I2/I2,max) was plotted against the corresponding prepulse potential (mV) in control (●) and toxin condition (○).
	Figure 5. Selectivity screening on a panel of NaV and KV channels. The current inhibition (%) observed after addition of 1.6 µM APETx4 to various channels is displayed in a bar graph. Values are shown as average ± SEM of at least 3 independent experiments.
	Figure 6. Voltage-dependent effect of APETx4 on hERG inward tail currents. (A) Normalized inward tail currents were plotted versus the applied pulse potentials (mV) in control (●) and toxin condition (○). The data points were fitted with the Boltzmann equation. (B) The inhibited current (%) observed after 10 µM APETx4 addition was plotted versus the applied pulse potentials.
	Figure 7. Investigation of the proapoptotic effect of APETx4 on various cell lines using a Caspase-3/7 Reagent. The amount of cells in early apoptosis was plotted as Green Object Count (1/mm²) versus time (hours). The data points represented as red triangles were obtained from apoptosis experiments with cells incubated without APETx4, the green squares represent experiments with 20 µM APETx4 and the blue circles were obtained from experiments with 50 µM APETx4.
	Figure 8. A multiple sequence alignment of various gating modifier toxins was performed using Clustal Omega (EMBL-EBI). Cysteine residues are highlighted in grey and the hydrophobic residues; Y4, L5, F6 that are functionally important for the activity of SGTx on the KV2.1 channel.

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Agarwal, The potassium channel Ether à go-go is a novel prognostic factor with functional relevance in acute myeloid leukemia. 2010, Pubmed