Novel Blocker of Onco SK3 Channels Derived from Scorpion Toxin Tamapin and Active against Migration of Cancer Cells

Abstract

Peptide-based therapy against cancer is a field of great interest for biomedical developments. Since it was shown that SK3 channels promote cancer cell migration and metastatic development, we started using these channels as targets for the development of antimetastatic drugs. Particularly, tamapin (a peptide found in the venom of the scorpion Mesobuthus tamulus) is the most specific toxin against the SK2 channel currently known. Considering this fact, we designed diverse tamapin mutants based on three different hypotheses to discover a new potent molecule to block SK3 channels. We performed in vitro studies to evaluate this new toxin derivative inhibitor of cancer cell migration. Our results can be used to generate a new tamapin-based therapy against cancer cells that express SK3 channels.

Figure 1

Alignment of αKTx5 scorpion toxin family and apamin. Sequence chain view of r-tam, r-tam-E25K/K27E, scyllatoxin, BmPO5, and apamin. The mutated amino acids are highlighted in gray.

Figure 2

3D NMR structure of r-tam and K27A mutant. Alignment of mutant (cyan, PDB entry 6D9P) and r-tam (purple, PDB entry 2LU9) from amino acids C3-V29; the K27 amino acid is shown in sticks. K27A is the mutant with the greatest differences in HN protons compared to r-tam. Despite the difference in chemical shifts, both structures conserve the topology.

Figure 3

Interactions of r-tam with (A) SK2 (dark blue) and (B) SK3 (cyan) channels explored by molecular dynamics. Toxin is colored in green. Residues which differ from channels SK2 to SK3 are colored in red. Residues mutated in this study are shown in pink. The PDB data to draw this figure is in the Supporting Information.

Figure 4

Effects of r-tam E25K/K27E mutant on SK2 and SK3 currents. (A, C) SK2 and SK3 current recordings in control condition (black trace), after application of 1 nM r-tam (green trace) or 1 nM E25K/K27E (blue trace) and 100 nM apamin (red trace) to inhibit residual SKCa currents. Whole-cell SK2 currents in HEK293 cells expressing recombinant rat SK2 and whole-cell SK3 currents in HEK293T cells expressing recombinant human SK3 were generated by ramp protocol from −100 to 100 mV for 500 ms from a constant holding potential of 0 mV at pCa 6. (B, D). Currents were analyzed at 0 mV, a membrane potential where the current is only carried by SKCa channels. Lines indicate the median; each point represents the percentage of SKCa currents sensitive to peptides (**p < 0.01, Mann–Whitney test). (E) Concentration–response curves of SK2/SK3 currents to r-tam and E25K/K27E mutant (means ± SEM, n = 3–8, GraphPad Prism). Currents were analyzed at 0 mV.

Figure 5

Effect of E25K/K27E mutant, r-tam, and apamin on SK3-dependent cell migration. Histograms showing the effect of 100 nM E25K/K27E mutant, r-tam, and apamin on MDA-MB-435s migration expressing (SK3 ⁺) or not SK3 channel (SK3 ^–). The normalized cell number corresponds to the ratio of the number of migrating cells in the presence of peptides over the number of migrating cells in control conditions (means ± SEM, N = 4, n = 12, *** p < 0.01, Kruskal–Wallis and post hoc tests).