TAMAPINE MUTANTS POTASSIUM CHANNEL BLOCKERS FOR INHIBITION OF CANCER CELL MIGRATION.

Abstract

The present invention relates to a recombinant tamapine peptide of SEQ ID NO: 5 for use as an inhibitor of the migration of cancer cells expressing SK3 channels; wherein the recombinant peptide is characterized in that it comprises the E25K / K27E mutant.

Description

Tamapin potassium channel blocker mutants for the inhibition of cancer cell migration FIELD OF INVENTION The present invention relates to the field of biomedical developments and medicinal chemistry, more precisely to peptide-based therapy in the search for tamapine derivatives for new biotechnological alternatives for cancer treatment. BACKGROUND Scorpion venoms consist of a large number of peptides that act on different ion channels, such as sodium, potassium, calcium, and chloride.1 These active peptides are potent neurotoxins for structure-activity studies.2 Ion channels constitute a distinct class of membrane proteins with crucial functions in cellular physiology, involved in neuronal signaling, hormone secretion, muscle contractility, and, more recently, have been linked to cancer. Potassium channels are the most numerous and diverse ion channels in living organisms3 and are an important target for scorpion toxins. Among all the families of K+ channels are the low-conductance, Ca2+-activated K+ channels, which belong to the SK family (also called KCa2) and comprise three members: SK1 (KCa2.1), SK2 (KCa2.2), and SK3 (KCa2.3). The bee toxin apamin is a highly specific ion channel-blocking peptide for the SK family.4In 2002, Pedarzani demonstrated that tamapine, a toxin from the venom of the Indian red scorpion Mesobuthus tamulus, is one of the highly specific scorpion toxins that blocks SK2 channels; native tamapine (aKTx5.4) is a highly selective blocker for SK2 channels with an ECs0 of 24 pM and 100 times less active for SK3 channels with an ECs0 of 1.7 nM,5 see Table 1. Native tamapine is amide-linked at the carboxy terminus. Recombinant tamapine, r-tam, is not amide-linked and features a typical α / β structural motif stabilized by three disulfide bonds (CS-α / β) comprising a double-stranded antiparallel β-helix and β-sheet6. Silatoxin, another toxin in the "KTx5" group, is a slightly better blocker than tamapine for SK3 channels, but not for SK2 channels (Figure 1). Previous work, such as that described in patent WO 2017 / 066444, describes the prevention, improvement, or treatment of learning disabilities or other neurological disorders or diseases, such as those associated with fetal alcohol syndrome (FAS), through the administration of an SK2 channel inhibitor or another SK channel inhibitor. The inventors of that patent have found that these learning disabilities caused by stressors such as alcohol exposure in utero can be treated by administering an SK channel blocker, antagonist, inhibitor, or modifier, such as tamapine. Furthermore, the patent describes that the tamapine compounds it employs can selectively or predominantly block or act on one type of SK channel or act on different SK channels, such as channels comprising SK1, SK2, SK3, and SK4.Such analogues include tamapine isotype 2, peptides having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, deletions, or additions to a native tamapine sequence. Preferably, the tamapine derivative or analogue shall retain at least one functional property of native tamapine, such as the ability to block, antagonize, inhibit, or modify an SK channel or SK channel activity; reversibly block SK channels with selectivity for SK2 over SK1 channels; or block SK2 channels with greater affinity than SK3 channels (affinity for SK2 > SK3 > SK1 > SK4). The document also provides pharmaceutical compositions comprising at least one SK channel blocker, antagonist, inhibitor, or modifier that can be mixed with other pharmaceutically acceptable active ingredients or carriers. Such channels include SK1, SK2, SK3, and / or SK4. WO 2016 / 116156, as well as WO2008088422, describe a composition of formula (X1)a-(F1)d-(X2)b-(F2)e-(X3)c and multimers thereof, wherein X1, X2 and X3 are each independently -(L)tP-(L)g- and f and g are each independently 0 or 1; and P is a peptide toxin of no more than approximately 80 amino acid residues in length, comprising at least two disulfide bonds. Furthermore, P comprises a SK antagonist peptide, which may be apamin, an ScyTx peptide, a BmP05 peptide, a P05 peptide, a tamapin peptide, a P01 peptide, or a TsK peptide, or a peptide analogue of any of these. Moreover, the compounds of this invention have pharmacological activity resulting from their ability to bind to proteins of interest as agonists, mimetics, or antagonists of the native ligands of such proteins of interest. For example, heritable diseases with a known link to ion channels (channelopathies) span various fields of medicine, including neurology, nephrology, myology, and cardiology. A list of hereditary disorders attributed to ion channels includes cancer, where many potassium channel genes are amplified and protein subunits are upregulated.Consistent with a pathophysiological role for the positive regulation of the potassium channel, potassium channel blockers have been shown to suppress the proliferation of uterine cancer cells and hepatocellular carcinoma cells, presumably through inhibition of calcium influx and effects on calcium-dependent gene expression. It has previously been reported that tamapine can control the cell proliferation of cancer cells6. Document WO 2016 / 116156, whose patent application was also filed in Mexico under number MX / a / 2007 / 013031 and granted under patent number MX 286970 to Amgen Inc., describes that the sequence of tamapine is SEQ ID NO 53, and although it does not describe particular mutants or derivatives of tamapine and does not describe the SK2 and SK3 channels, it does describe that the composition of the present invention, which can be used for the treatment of diseases that express SK channels, including cancer, and explaining its possible mechanism, comprises tamapine in its formulation. WO 2015 / 010100 and WO 2015 / 017146 relate to humanized antibodies, which include antibodies comprising an ultralong CDR3 sequence, methods for preparing them, and their uses. Humanized antibodies with ultralong CDR3 sequences may also incorporate non-antibody sequences, such as cytokines or growth factors, into the CDR3 region, so that the resulting humanized antibody is effective, for example, in inhibiting tumor metastasis. Non-antibody sequences may include an interleukin sequence, a hormone sequence, a cytokine sequence, a toxin sequence, a lymphokine sequence, a growth factor sequence, a chemokine sequence, or combinations thereof. Non-antibody sequences may be human, non-human, or synthetic. Examples of toxin sequences include a tamapine sequence.These antibodies bind (e.g., bind specifically or selectively) to a variety of targets, including, for example, protein targets such as transmembrane proteins (e.g., GPCR, ion channels, transporter, cell surface receptors). Melanoma is a major health problem, and its rates are increasing both in the United States and worldwide.7 Melanoma metastasizes early in disease progression, which can occur even in thin primary tumors.8 The disease course is often prolonged, with patients experiencing a disease-free period after surgical excision of the primary tumor only to discover visceral metastases months, years, or even decades later.9 Currently, there are no FDA-approved therapies that significantly improve overall survival for patients with late-stage disease.10 Much work has been done to understand the mechanisms that mediate the complex process of melanoma metastasis. Metastasis accounts for the vast majority of morbidity and mortality associated with melanoma. Evidence suggests that melanoma has a predilection for metastasis to particular organs; in this sense, melanoma cancer is characterized by its high aggressiveness due to cell migration. The SK3 channel has been shown to promote the migration of several cancer cells, including breast cancer and melanoma cells.11,12,13 For example, in 2009,12 inhibition of SK3 channels was found to decrease cell motility in the early stages of metastasis. Furthermore, SK3 channels, but not SK2 channels, are overexpressed in the highly metastatic and invasive melanoma cell line MDA-MB-435s. Previous work has also shown that silencing the SK3 channel in MDA-MB-435s cells reduces their migratory capacity by up to 60% and reduces their ability to form bone metastases in vivo.4 Therefore, further developments in the treatment of metastasis, a mechanism by which cancers such as melanoma migrate between tissues, are still pending, and understanding the biochemistry and the important role of SK3 channels in this phenomenon is of particular interest to the present invention. Patent application MX / a / 2018 / 011703 (Mayorga Flores, M., et al.) described how the use of tamapine mulants inhibits cancer cells possessing SK2 channels, while this specification specifically describes the inhibition of cell motility, but now by blocking SK3 channels, which are involved in metastasis, and how this can be targeted for biotechnological treatments. Tamapine is an excellent SK2 channel blocker, and the high similarity between SK2 and SK3 channels suggested we use recombinant tamapine (r-tam) as a model for creating a more potent SK3 channel blocker. Cancer cells could be either SK3 homotetramers or SK2 and SK3 heterotetramers. Since SK3 and SK2 channels (in melanoma) are implicated in cancer, it may be interesting to use tamapine in cancer as an SK2 blocker and also tamapine mutans as SK3 and SK2 heterotetramer channel blockers. To design this more potent SK3 channel-blocking r-tam derivative, several mutants with modifications in aromatic and positively charged amino acids were synthesized. Here, we describe the electrophysiological results of the best mutant with both channels expressed in HEK cells and present the migration inhibition produced by the most potent r-tam blocking mutant obtained. This novel SK3 channel-blocking peptide could be used as a potential therapeutic compound to control the specific migration of cancer cells. All references cited in the present invention are included by reference in their entirety. SUMMARY OF THE INVENTION The present invention describes a cancer therapy based on SK3 channel-blocking peptides aimed at the development of antimetastatic drugs. It is an object of the present invention to produce and use tamapine-derived mutants to block SK3-type channels in tumor cell lines, particularly for use in inhibiting cell migration of cancer cells expressing SK3-type ion channels, wherein the recombinant tamapine sequences are selected from the group of mutants comprising SEQ ID No. 1-5 and possessing one or more mutations in their sequence, e.g., monomutant sequences or double mutant sequences.The recombinant tamapine monomutant sequence comprises mutations defined by the amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y in any of its amino acid residues and indicated as “X” in the corresponding sequence; whereas the recombinant tamapine double mutant sequence comprises two mutations defined by the amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y or any combination thereof in at least 2 amino acid residues of its sequence and indicated as “X” and “Z” in the corresponding sequence. In a preferred embodiment, the invention proposes various tamapine mutants as novel, potent molecules for blocking SK3 channels. The recombinant tamapine sequences are selected from the group of mutants comprising SEQ IDs No. 1, 2, 3, 4, and 5, wherein said sequences are recombinant tamapine mutants Y31H, Y31A, K27A, K27E, E25K, E25A, K20E, E25K / K27E, and wherein Iη32N, LR30, and ΔR30 / Iη32N are additions and deletions thereof. In a second modality, the invention characterizes the different mutants by NMR and other spectroscopic tools. In a third approach, in vitro and in vivo studies were used to evaluate these novel toxin derivatives as inhibitors of cancer cell migration. The increased blockade of SK3 channels and the decreased blockade of SK2 channels, compared to r-tam, allows us to efficiently modulate the SK3 channel inhibition pattern using the recombinant tamapine sequence selected from the mutant group comprising the E25K / K27E mutant (SEC ID No. 5). Since MDA-MB-435s cancer cells have been shown to cease migrating in the presence of this mutant, it is reasonable to assume that the E25K / K27E mutant of r-tam could be used as an inhibitor of metastasis initiation when cells possess SK3 channels, and this serves as a starting point for studying its pharmacological properties for consideration as a potential drug. The invention can be used to generate a tamapine-based therapy against cancer cells that express SK3 channels, making this therapy highly specific. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Alignment of the αKTδ and apamin scorpion toxin family. Chain sequence view of r-tam, r-tam-E25K / K27E, silatoxin, BmPO5, and apamin. Amino acid changes are shown in gray. Figure 2. 3D NMR structure of r-tam and the K27A mutant. Alignment of the mutant (blue, PDB code: 6D9P) and r-tam (purple, PDB code: 2LU9) of the amino acids C3V29, amino acid K27 is shown in bars. Figure 3. Effects of the r-tam mutant E25K / K27E on the activities of the SK2 and SK3 channels (Taken and modified from: Novel Blocker of Onco SK3 Channels Derived from Scorpion Toxin Tamapin and Active against Migration of Cancer Cells Marlen Mayorga-Flores, Aurélie Chantóme, Carolina Monserrath Melchor-Meneses, Isabelle Domingo, Gustavo Alfredo Titaux-Delgado, Rodrigo Galindo-Murillo, Christophe Vandier, and Federico del Río-Portilla; ACS Medicinal Chemistry Letters Article ASAP; 2020; DOI: 10.1021 / acsmedchemlett.0c00300)): A, B, D, E) Current recordings of SK2 and SK3 under control conditions (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 SK channel currents. Whole-cell SK2 current recordings were performed in the HEK293 cell line expressing recombinant rat SK2, and SK3 current recordings were performed in whole-cell HEK293T cells expressing recombinant human SK3. Both protocols were generated by ramping application in a range of -100 to 100 mV for 500 ms with a constant holding potential of 0 mV to pCa 6. C, F) Histograms showing the % blockade of SK currents by r-tam or E25K / K27E mutant when steady-state inhibition was achieved. Currents were analyzed at 0 mV, a membrane potential where current is only transported by SK channels. Lines indicate the median, each point represents the percentage of SK currents sensitive to the peptides (** p <0.01, MannWhitney test); G, H) Concentration-response inhibition curves of SK2 and SK3 currents performed by r-tam and the double mutant E25K / K27E (means ± SEM, n = 3-8, GraphPad Prism). Currents were analyzed at 0 mV. Figure 4. Effect of the E25K / K27E mutant, r-tam, and apamin on SK3-dependent cell migration. Histograms show the effect of the E25K / K27E mutant, r-tam, and apamin at a concentration of 100 mM on MDA-MB-435 cells expressing the channel (SK3+) or not expressing the SK3 channel (SK3 ). The normalized cell number corresponds to the ratio of the number of cells migrating in the presence of peptides to the number of cells migrating under control conditions (means ± SEM, N = 4, n = 12, *** p < 0.01, Kruskal-Wallis and post hoc tests). DETAILED DESCRIPTION OF THE INVENTION Unless otherwise defined, all technical and scientific terms used in this document have the same meaning commonly understood by an expert in the subject to which this description refers. Peptide-based cancer therapy is a field of great interest for biomedical developments. Tamapine (a peptide found in the venom of the scorpion Mesobuthus tamulus) is the most specific toxin against the SK2 channel currently known; and SK3 channels have been shown to promote cancer cell migration. Considering this, the present invention proposes various tamapine mutants as potent novel molecules for blocking SK3 channels and for using these channels as targets for the development of antimetastatic drugs. Cancer cells may be either SK3 homotetramers or heterotetramers. Since SK3 and SK2 channels (in melanoma) are implicated in cancer, it may be advantageous to use tamapine in cancer as an SK2 blocker and also tamapine mutants as blockers of the SK3 and SK2 heterotetramer channels. The peptides reported in the present invention are highly specific. The recombinant tamapine sequences are selected from the group of mutants comprising SEQ IDs 1-5 and possessing one or more mutations in their sequence, for example, monomutant sequences or double mutant sequences. The recombinant tamapine monomutant sequence comprises mutations defined by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y in any of its amino acid residues and indicated as “X” in the corresponding sequence; whereas the recombinant tamapine double mutant sequence comprises two mutations defined by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y or any combination thereof in at least 2 amino acid residues of its sequence and indicated as “X” and “Z” in the corresponding sequence (see also the sequence listing of the present invention). SEQ. ID. No. Sequence 1 AFCALRRCELSCRSLGLLGKCIGEECKCVPX 2 AFCALRRCELSCRSLGLLGKCIGEECXCVPY 3 AFCALRRCELSCRSLGLLGKCIGEXCKCVPY 4 AFCALRRCELSCRSLGLLGXCIGEECKCVPY 5 AFCALRRCELSCRSLGLLGKCIGEXCZCVPY 6 (r-tam) AFCNLRRCELSCRSLGLLGKCIGEECKCVPY where X and Z are different from the original amino acids and are defined by: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y or any combination thereof The present invention, therefore, relates to recombinant tamapin sequences selected from the group of mutants comprising SEQ ID No. 1, 2, 3, 4 and 5, wherein said SEQ ID No. are recombinant tamapin mutants Y31H, Y31A, K27A, K27E, E25K, E25A, K20E, E25K / K27E. In vitro and in vivo studies were used to evaluate this novel toxin derivative that inhibits cancer cell migration. The present invention describes the electrophysiological results of the best recombinant tamapine mutants with both channels expressed in HEK cells and the migration inhibition produced by the most potent r-tam blocking mutant obtained. The increased blockade of SK3 channels and the decreased blockade of SK2 channels, compared to r-tam, allows us to modulate the SK3 channel more efficiently using E25K / K27E. This novel peptide could be used as a potential therapeutic compound to control the specific migration of cancer cells, for example, melanocytes or melanoma. This mutant can also block cancer cells expressing the SK3 / SK2 heterotetramer. The following section describes in more detail various aspects of the invention, which will be understood based on the description and figures comprising the present invention. Design, construction, and expression of r-tam and mutants We adopted the following strategies to construct r-tam analogs. Five residues in the β-sheet were individually mutated to alanine and oppositely charged amino acids. This was based on three possible situations that could affect the current block: 1. Amidation at the C-terminal of tamapine. 2. Comparison with highly similar proteins, silatoxin, see Figure 1. 3. Taking into account the functional dyad in scorpion toxins, the following mutations were performed: Case 1: We propose the insertion of an asparagine at the C-terminus; this modification could emulate the amide group and its interaction with the SK2 channel. The rationale is that, if the addition of one amino acid could affect activity and Y31 might be more relevant than P30, we decided to remove the P30 residue and insert an asparagine, forming the ΔR30IηN32 mutant. In addition, we obtained the ΔR30 and Y31H mutants to test whether these amino acids might have any relevance.15 Case 2. Within the αKTδ family, silatoxin has a higher affinity for SK3 channels than tamapine. We obtained the mutants K20A, K20E, E25A, E25K, K27E, K27A, and the double mutant E25K / K27E. Case 3. The functional dyad theory16 states that there must be a positively charged residue and an aromatic amino acid close in space, 6.7 A. In this case, residues 20, 27 and 31 were selected to be mutated. Table 1. Electrophysiological activity of the αKTχδ toxin family reported against SK2 and SK3 channels, apamin is included as a reference blocker of these channels. Toxin ECso nM SK2 SK3 Identity REF Tamapine 0.024 1.7 100 5 Silatoxin 0.29 1.1 77 17 BmP05 - 3.8 74 18 Apamine 0.027 1.1 NA 19 Eleven r-tam mutants were designed as described in the Mexican patent application MX / a / 2018 / 011703 (Mayorga Flores, M.) which is included as a reference in its entirety. The biosynthesis procedure allows us to obtain recombinant E. coli peptides with functional folding that includes the same cysteine ​​connectivity for all mutants. The experimental masses obtained by MALDI-TOF are shown in Table 2. Table 2. Characterization by MALDI-TOF, PDB codes and BMRD ID of r-tam risers. SEQ ID No. Mutant r-tam Theoretical Mass Cys-Cys Experimental Mass PDB Code ID BMRB ID 1 Y31H 3433.1 3433.4 6D8Y 30465 1 Y31A 3367.0 3367.0 6D93 3046A 2027A 2027. 3402.3 6D9P 30468 2 K27E 3460.1 3460.0 6D8S 30462 3 E25K 3458.2 3457.9 6D8R 30461 3 E25A 3401.1 3401.0 ln 3049O 3573.2 3573.1 6D8H 30459 ΔΡ30 3362.0 3361.8 6D3T 30454 ΔΡ30 / Ιη32Ν 3476.1 3476.1 6D8Q 30460 4 K 340.1 340.1 6D8U 30464 5 E25K / K27E 3459.1 3458.9 6D8T 30463 6 r-tam 3459.1 3459.0 2LU9 18513 According to the foregoing, the recombinant tamapine sequences of the present invention are selected from the group of mutants qoe comprising SEQ ID no. 1-5 and which possess one or more mutations in their sequence, for example, are monomutant sequences or double mutant sequences. The recombinant tamapin monomutant sequence comprises mutations defined by A, C, D, E, F, G, Η, I, K, L, Μ, N, P, Q, R, S, T, V, W, or Y in any of its amino acid residues; provided that the double mutant sequence of recombinant tamapin comprises two mutations defined by A, C, D, E, F, G, Η, I, K, L, Μ, N, P, Q, R, S, T, V, W, Y or any combination thereof in at least 2 amino acid residues of its sequence. Preferably, the recombinant tamapine sequences are selected from the group of mutants comprising SEQ ID No.1-5, where these sequences are recombinant tamapine mounts Y31H, Y31A, K27A, K27E, E25K, E25A, K20E, were significantly reduced more than SK3 currents after application of 1 nM r-tam. Figure 3C shows that r-tam reduced SK2 currents by 86% and SK3 currents by only 25%. Interestingly, the E25K / K27E mutant (SEQ. ID. No. 5) exhibits the same inhibition efficacy for SK2 (80%) as for SK3 currents (73%) at the same concentration, suggesting that the mutations introduced into the r-tam derivative increase its binding affinity for SK3 channels (Figure 3F). Next, a concentration-response curve was generated to compare the mean inhibitory concentrations (ECs) of r-tam and the E25K / K27E mutant for the SK2 and SK3 currents. As shown in Figures 3G and 3H, the ECs of r-tam for SK2 and SK3 was estimated at 16 pM and 2.6 nM respectively, values ​​very close to native tamapine5.The SK2 and SK3 concentration-response curves for the E25K / K27E mutant (SEQ. ID. No. 5) overlap, with an estimated EC50 of 0.28 nM and 0.38 nM, respectively. In conclusion, the E25K and K27E mutations of r-tam have led to a decrease in its specificity for SK2 (~17-fold) and an increase in its specificity for SK3 channels (~7-fold). Anti-migration effect on cancer cells expressing SK3 channels The SK3 channel in cancer cells has been found to promote cell migration and metastasis development. Previous studies have shown that silencing the SK3 channel in MDA-MB-435s cells reduces their migratory capacity by 60% and their ability to form bone metastases in vivo.14 We compared the potency of each peptide, at 100 nM, in reducing the migration of MDA-MB-435s cells that expressed (SK3+) or did not express (SK3) the SK3 channel. Figure 4 shows that while r-tam has no significant effect on the migration of SK3+ cells, the E25K / K27E mutant (SEQ. ID. No. 5) and apamin reduced the migratory capacity of SK3+ cells by 49% and 46%, respectively. As expected, r-tam, E25K / K27E, and apamin have no effect on SK3-negative cell migration; these cells do not express the SK3 channel (SEQ. ID. No. 5). Therefore, the E25K / K27E mutant, as apamin, reduced SK3-dependent cell migration by more than 75%, Table 3.Considering the high chemical stability of tamapin mutations, the recombinant tamapin sequence selected from the group of mutants comprising the mutant E25K / K27E (SEQ. ID. No. 5) could be a potential drug to slow down the metastasis process. Table 3. Activity of r-tam and E25K / K27E in SK3 channel blockade and cell migration. Peptide Percent current inhibition at InM ECso pM on SK3 Percent total inhibition of migration decrease in MDA-MB-435s cells Percent cell migration inhibition in cells with SK3 r-tam channels 25 2600 No effect No effect E25K / K27E 73 380 45.9 76.5 Apamin Control* 360 48.6 81 *100 nM Tamapin toxin is a peptide highly specific for SK2 channels. Mutations in r-tam conserve the CSa / β motif. Specific point mutations in the r-tam toxin at residues 25 and 27, based on silatoxin activity, increase SK3 channel activity by a factor of 7. At the same time, the activity of the E25K / K27E mutant (SEQ. ID. No. 5) on SK2 channels was reduced by a factor of 17. This increased SK3 channel blockade and decreased SK2 channel blockade, compared to r-tam, allows us to efficiently modulate the SK3 channel with E25K / K27E (SEQ. ID. No. 5). Since MDA-MB-435S cancer cells have been shown to cease migrating in the presence of this mutant, it is reasonable to assume that the E25K / K27E mutant (SEQ. ID. No. 5)5) r-tam could be used as an inhibitor of the initiation of metastasis and is a starting point for studying its pharmacological properties to be considered as a potential drug. The description in this document does not in any way limit how the SK3 blockers and / or the E25K / K27E mutant (SEQ. ID. No. 5) act and / or achieve their effects. The description will be illustrated in more detail in the following examples. Examples Example 1. Construction of expression plasmids Expression of r-tam was performed in E. coli as previously reported.6 Mutants were generated by site-directed mutagenesis and sequenced by overlap extension PCR (Phusion, Bio Labs, Inc.) using the r-tamapine plasmid as a template. Oligonucleotides (Sigma-Aldrich) used to introduce the mutations were designed to be non-complementary. All novel vectors were verified by DNA sequencing (Laragen, Culver City, CA, USA) using T7 terminus primers. Example 2. Expression and purification of toxin-like peptides The Escherichia coli Rosetta strain was transformed with each expression plasmid using thioredoxin as a chaperone, previously constructed to express all mutant toxins. The gene encoding the fusion protein is thioredoxin-His6LVPRGM-tamapin. Cells were grown overnight at 37°C in Luria-Bertani medium. This was used as the primary inoculum for expression in large-scale (1 L) cultures. Freshly seeded cultures were grown at 37°C. Cultures were monitored by periodic optical density measurements at 600 nm. Protein expression was induced by the addition of IPTG (to a final concentration of 0.5 mM) when the Aeoonm reached 0.6–0.8. Cell cultures were allowed to grow for 7 hours at 37°C. The cells were then harvested by centrifugation (6000 X g, 5 min at 4° C) and then suspended in 50 mL with a lysis buffer per gram of pellet cells (Tris 20 mM, pH 8.0 and NaCl 150 mM).Cell pellets were lysed by sonication using a sonicator (Misonix 3000, power level 5) and clarified by centrifugation (16,000 x g, 40 min at 4°C). The soluble protein was purified by metal chelate affinity chromatography. The soluble fraction was applied to an N12+ column (H-Trap®, GE). The fusion protein bound to the column was eluted with 0.3 M imidazole buffer. The protein of interest was dialyzed with protease cleavage buffer (50 mM Tris, 10 mM CaCl2, 200 mM NaCl, pH 8.0). Peptides were released from the fusion protein by thrombin hydrolysis (Sigma-Aldrich). The reaction was carried out at room temperature for 12 h. The protein concentration was determined using the theoretical molar extinction coefficients at 280 nm and then adjusted to 1.0 mg / mL. Approximately 20–30 mg / L of the corresponding fusion protein was obtained in LB medium.The cleavage reaction was monitored by SDS-PAGE electrophoresis, and the fragments containing thioredoxin and His-tag were removed by Ni2+ affinity chromatography. The sequence (LVPRGM) that comprises the thrombin cleavage site provides two additional amino acids (Gly and Met) to the N-terminus. Example 3. Solid Extraction The fraction eluted from the affinity chromatography was concentrated, fractionated, and desalted in a Phenomenex Strata-C18T cartridge. The peptide-containing fraction was eluted with 60% acetonitrile. The protein was then dried by lyophilization. Example 4. Reaction of CNBr Gly-Met residues were removed from the N-terminus using CNBr. A sample of GM toxin was dissolved directly in 50% v / v trifluoroacetic acid to a final concentration of 10 mg / mL. Immediately after the addition of the protein, the oxygen in the solution was purged with nitrogen for 1 hour. Subsequently, 120 equivalents of cyanogen bromide were added for each methionine present in the protein sequence. The reaction was protected from light sources for 24 hours with stirring in a nitrogen atmosphere. The reaction was stopped by adding water at a ratio of four times the reaction volume. This solution was diluted with distilled water to a pH of 2.0. Finally, the sample was recovered from the solution using Strata C18-T cartridges and prepared for purification by reversed-phase HPLC. Example 5. Reversed Phase Chromatography Final purification was performed by reversed-phase HPLC using a C12 analytical column at 25°C (Phenomenex, Jupiter, 250 x 4.6 mm, 4.0 pm particle diameter, 90 Å pore size) fitted with a C12 shielding column (Phenomenex, Jupiter, 10 pm). Elution was carried out at 1 mL / min using solutions A (water + 0.05% v / v TFA) and B (0.05% v / v TFA acetonitrile). A linear gradient from 10% to 30% of solution B was developed over 20 minutes. The peptide absorbance was monitored at 230 nm. The polypeptides obtained from reversed-phase chromatography were >98.0% pure and were subsequently analyzed by mass spectrometry. The selected fraction was lyophilized and then quantified. All peptides described in this work were successfully produced and purified using the same methodology. Example 6. Mass Spectrometry. The molecular mass of the purified peptides was obtained by MALDI-TOF experiments using Bruker Daltonics Microflex LT instruments. For each analysis, 100 nmol of toxin were mixed with 3 pL of matrix (a supersaturated solution of cyano-4-hydroxycinnamic acid in 60% acetonitrile in water with added 0.05% v / v trifluoroacetic acid). One pL of this mixture was then placed on the MALDI plate, and 20 samples were subsequently analyzed. Data were acquired in the m / z range of 0–8000 Da. The reflector mode was used, acquiring 200 exposures. Example 7. 3D structure by NMR The lyophilized toxin was dissolved at 3 mM in 5% (v / v) D2O in H2O. NMR data were acquired at 298 K using a Varian spectrometer operating at proton frequencies of 500 MHz. TOCSY spectra were recorded with mixing times of 80 ms, and NOESY spectra were recorded with mixing times of 150 and 300 ms. All 2D NMR spectra were collected as an array of 1024 complex data points in both dimensions using 32 scans. The NMR data were processed using NMRpipe and a zero-filled, sinusoidal window function prior to Fourier transform. Identification of amino acid spin systems and sequential assignment were performed using the standard strategy described by Wüthrich. Spin systems were identified in the TOCSY spectrum. Sequential assignments were obtained from HN-HN, HN-Hα, and HN-Hβ connectivity analysis in NOESY spectra.The five distance constraints were derived from two-dimensional NOESY with a mixing time of 150 ms. The chemical shift index (CSI) of the secondary structure was calculated according to the Wishart and Sykes method for each residue based on proton resonances. Data analysis was performed using CARA structure calculation. The distance and dihedral angle constraints were derived primarily from the cross-peaks in the NOESY experiment. Structures were generated using CYANA, employing simulated alignment algorithms. A total of 200 structures were calculated, from which 20 structures were selected based on the objective function in CYANA with no unmet constraints to construct the NMR structural ensemble. These 20 structures were refined using Amber to represent the structure in solution.All molecular dynamics simulations and energy minimizations described here were performed using the AMBER-99SB force field and topology parameters. We followed one of the refinement protocols proposed by Lindorff et al. with minor modifications.24 Three-dimensional conformations were inspected and aligned using PyMOL.20 Structure evaluation was performed using PROCHECK. The coordinate file of the final set of all determined structures, along with the distance constraint files, were deposited in the Protein Data Bank as shown in Table 2. Example 8. Cell line culture and stable transfections of the SK channel The human breast cancer cell line MDA-MB-435s was acquired from the American Type Culture Collection (ATCC, LGC Promochem, Molsheim, France) and cultured as previously described.13 This cell line was transduced by a lentivector containing either an SK3-specific interfering shRNA (SK3 cells) or a non-targeted shRNA (SK3+ cells), as previously validated.25 HEK 293 and HEK293T cells were from ATCC, maintained in Dulbecco's modified Eagle medium supplemented with 10% (v:v) fetal bovine serum (Lonza, France). Rat SK2 channel expression in HEK293 cells was performed as previously described.25 Human SK3 channel expression in the HEK293T cell line was generated by transfecting cells with the pPRIPu-hSK3 vector (kindly provided by Dr. P. Martin, Institute de Biologie Valrose, Nice, France) and subjecting the cells to puromycin (1 mg / mL) selection from 48 hours after transfection to 20 days. Example 9. Electrophysiology and cell migration assays The experiments were performed with cells seeded in 35 mm Petri dishes at 2000 cells per cm2. All electrophysiology experiments were performed using the conventional whole-cell recording setup of the patch-recording electrophysiology technique as described above12 13 25. Cell migration was determined as described in the prior art12 13. Example 10. Solutions and drugs The physiological saline solution (PSS) had the following composition (in mM): NaCl 140, MgCh 1, KCl 4, CaCh 2, D-glucose 11.1, HEPES 10; and was adjusted to pH 7.4 with NaOH. The pipette solution for whole-cell recordings contained (in mM): KCl 145, MgCl2 1, Mg-ATP 1, HEPES 10, CaCl2 0.87, EGTA 1 (pCa6). The pH of the solution was adjusted to 7.2 with KOH. Example 11. Statistics Data were expressed as median with quartile or mean ± SEM (N = number of experiments; n = number of cells). Statistical analysis was performed using the Student's t-test for unpaired data or the Mann-Whitney U test. For comparisons between more than two means, we used the Kruskal-Wallis one-way ANOVA followed by Dunn's test. Differences were considered significant when p < 0.05. Statistical analysis was performed using GraphPad Prism 6. Advantages and industrial application of the invention Peptide-based cancer therapy is a field of great interest for biomedical developments. On the one hand, tamapine (a peptide found in the venom of the scorpion Mesobuthus tamulus) is the most specific toxin against the SK2 channel currently known. On the other hand, SK3 channels have been shown to promote cancer cell migration and the development of metastases. Taking this into account, the present invention proposes various tamapine mutants as novel, potent molecules for blocking SK3 channels (composed of SK3 homotetramers and / or SK3 / SK2 heterotetramers). Specifically, the present invention leverages these SK3 channels as targets for the development of antimetastatic drugs. This novel peptide could be used as a potential therapeutic compound to control the specific migration of cancer cells, for example, melanocytes or melanoma cells. In vitro and in vivo studies were used to evaluate this novel cancer cell migration-inhibiting toxin derivative; particularly, using recombinant tamapine sequences selected from the group of mutants comprising SEQ IDs 1, 2, 3, 4, and 5, where said SEQ IDs are...are recombinant tamapine mutants Y31H, Y31A, K27A, K27E, E25K, E25A, K20E, E25K / K27E, and wherein ln32N, LR30 and AP30 / ln32N are additions and deletions thereof; particularly the mutant E25K / K27E (SEQ. ID. No. 5). We compared the potency of each peptide, at 100 nM, in reducing the migration of MDA-MB-435s cells that expressed (SK3+) or did not express (SK3j) the SK3 channel. Figure 4 shows that although r-tam did not have a significant effect on the migration of SK3+ cells, the E25K / K27E mutant and apamin reduced the ability of SK3+ cells to migrate by 46% and 49%, respectively. R-tam still did not have a significant effect on the migration of SK3' cells; in these cells, which did not express the SK3 channel, the effect of apamin and E25K / K27E was suppressed (SEQ. ID. No. 5). Therefore, the E25K / K27E mutant (SEQ. ID. No. 5), like apamin, reduced SK3-dependent cell migration by more than 75%, Table 3.Considering the high chemical stability of tamapine mutations, the E25K / K27E mutant (SEQ. ID. No. 5) could be a potential drug for slowing the metastasis process. The increased SK3 channel blockade and decreased SK2 channel blockade, compared to r-tam, allows for efficient modulation of the SK3 channel using E25K / K27E (SEQ. ID. No. 5). Since MDA-MB-435s cancer cells have been shown to cease migrating in the presence of this mutant, it is reasonable to assume that the E25K / K27E (SEQ. ID. No. 5) mutant of r-tam could be used as an inhibitor of metastasis and lead to a novel tamapine-based therapy targeting cancer cells that express SK3 channels, making this therapy highly specific. The foregoing description of the specific embodiments will reveal as fully as possible the general nature of the invention, so that others, applying knowledge within the art, may readily modify and / or adapt such specific embodiments for various applications without undue experimentation and without departing from the general concept of the present invention. It is therefore intended that such adaptations and modifications be within the meaning and range of equivalents of the described embodiments, based on the teaching and guidance presented herein. It should be understood that the phraseology or terminology in this document is for descriptive purposes and not for limitation; therefore, the terminology or phraseology of this specification should be interpreted by a person skilled in the art in light of the teaching and guidance provided. The scope of the present invention should not be limited by any of the descriptions and exemplary embodiments described above. This work was partially funded by the General Directorate of Academic Personnel Affairs with grant DGAPA-PAPIIT number 210319 and by the General Directorate of Computing and Information and Communication Technologies, Supercomputing Project No. LANCAD-UNAM-DGTIC-145. Part of this work was funded by the University of Tours, the “Région Centre-Val de Loire” INSERM, Canceropôle Grand Ouest, the “Ligue Nationale Contre le Cáncer”, the “CANCEN” Association, and the Tours Hospital Oncology Association (ACORT). We thank E. García24 We thank Ramos, LC Márquez-Alonso, and LM Ríos-Ruiz for their technical support. MMF thanks the National Council of Science and Technology (CONACyT) for grant 289035. We also thank Catherine Leroy for her secretarial support. We also thank M.Sc. Guillermo Roura Pérez of the Institute of Chemistry for his valuable support and suggestions during the drafting of this invention. REFERENCES 1. Luz Morales-Lazaro, S.; Hernandez-Garcia, E.; Serrano-Flores, B.; Rosenbaum, T. Organic Toxins as Tools to Understand Ion Channel Mechanisms and Structure. Curr. Top. Med. Chem. 2015, 15 (7), 581-603. 2. Hübner, C. a; Jentsch, TJ Ion Channel Diseases. Hum. Mol. Genet. 2002, 11 (20), 2435-2445. https: / / doi.Org / 10.1093 / hmq / 11.20.2435. 3. Leanza, L.; Doyle, A.; Venturini, E.; Zoratti, M.; Szegezdi, E.; Szabo, I. Correlation between Potassium Channel Expression and Sensitivity to Drug-lnduced Cell Death in Tumor Cell Lines. Curr. Pharm. 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Claims

1. A recombinant tamapine sequence selected from the group of mutants comprising the E25K / K27E mutant (SEQ. ID. No. 5) for use in SK3 type 5 channel blockade.

2. A recombinant tamapine sequence selected from the group of mutants comprising the E25K / K27E mutant (SEQ. ID. No. 5) for use in inhibiting cell migration of cancer cells expressing SK3 ion channels.

3. A recombinant tamapine sequence selected from the group of mutants comprising the E25K / K27E mutant (SEQ. ID. No. 5) for use in the development of antimetastatic drugs.