Peptides derived from the venom of the heteroctenus junceus scorpion, its variants, compositions, methods and uses

Abstract

The present invention comprises synthetic peptides derived from the venom of the H. junceus scorpion with anti-metastatic properties, as well as compositions of synthetic peptides derived from the venom of the H. junceus scorpion and its variants, which are obtained from recombinant or synthesis techniques, with anti-metastatic properties and which are effective against tumour cells or small cell lung cancer cells.

Description

[0001] PEPTIDES DERIVED FROM THE VENOM OF THE SCORPION HETEROCTENUS JUNCEUS, THEIR VARIANTS, COMPOSITIONS, METHODS, AND USES Field of the Invention The present invention is generally related to the fields of biochemistry, molecular biology, pharmacy, pharmacology, and physiology. In particular, this disclosure is a novel formulation from the venom of the scorpion Heteroctenus junceus (hereinafter H. junceus) and derived peptides for the treatment of pathological conditions, particularly as an anticancer agent with antimigratory and antiinvasive effects, including antimetastatic effects. The methods and techniques used for obtaining and developing the formulation, as well as the in vitro and in silico experimental results, are described.Background of the Invention: The background of this invention includes prior knowledge of the existence of ion channels, which form pores in the cell membrane and allow the passive flow of ions in the direction of their electrochemical gradient, generating electrical currents. The relevant physiological role of ion channels lies in their participation in cellular processes such as the generation of membrane potential, signal transduction, neurotransmitter release, muscle contraction, hormone secretion, volume regulation, growth, motility, migration, proliferation, and apoptosis, among others. Due to these characteristics, it is relevant that variations in the expression of some of these channels can lead to the development of pathological processes such as cancer.Cancer represents one of the major health problems facing humanity, currently responsible for more than 9.7 million deaths each year. Genomic instability is a primary cause and a fundamental characteristic of human cancer. One of the genes that increases genomic instability is that of ion channels, contributing to the transition to a more aggressive cancer phenotype. Numerous pieces of evidence have accumulated that the overexpression of these ion channels plays a particularly prominent role in key cancer features such as unrestricted proliferation, evasion of apoptosis, neovascularization, tissue invasion, and metastasis (Prevarskaya N, Skryma R, Shuba Y. Ion Channels in Cancer: Are Cancer Hallmarks Oncochannelopathies? Physiol Rev. 2018; 98(2):559-621). Overexpression of the Kv1.1 channel has been identified in several cancer types and is associated with proliferation, migration, cell signaling, and other processes.This characteristic makes it possible to target and reduce or inhibit its activity, and therefore the characteristics associated with cancer development (Ouadid-Ahidouch H, Chaussade F, Roudbaraki M, Slomianny C, Dewailly E, Delcourt P, Prevarskaya N. KV1.1 K(+) channels identification in human breast carcinoma cells: involvement in cell proliferation. Biochem Biophys Res Commun. 2000; 278(2):272-277). Among the different types of K+ channels encoded by more than 80 genes, voltage-gated potassium channels represent the largest and most complex family, with numerous members and are evolutionarily highly conserved. These channels are classified into 12 subfamilies Kv1-Kv12, encoded by at least 40 genes in humans (Alexander et al., 2017). Most subfamilies have distinct channels (e.g., Kv1.1-Kv1.8, Kv2.1-Kv2.2, Kv3.1-Kv3.4, etc.) based on their different biophysical properties and pharmacological profile (Bachmann et al., 2020).From a structural standpoint, potassium channels are composed of four identical α or pore-forming subunits, which assemble to form a complete channel in the cell membrane (Armstrong, 2003). These channels may also contain auxiliary β subunits that can affect both the channel's function and location (Torres et al., 2007). The alpha subunit of voltage-gated potassium channels contains six transmembrane segments (S1-S6). The first four segments (S1-S4) constitute the voltage-sensing region, and the last two contain an extracellular loop that forms the channel pore (S5-S6), known as the pore domain (Armstrong, 2003). These potassium channels are widely distributed in both excitable and non-excitable cells.In excitable cells such as neurons, cardiomyocytes, and muscles, potassium channels play an important role in Ca signaling. 2+Potassium channels are capable of regulating the shape and activation pattern of action potentials as part of their physiological activity. Furthermore, in a wide range of cell types, voltage-gated potassium channels participate in the regulation of cell volume, secretion, proliferation, and migration (Wulff et al., 2009). Several scientific reports indicate that Kv channels play a crucial role in regulating both cell proliferation and death. K+ channels are overexpressed and their activity is upregulated in cells with a high proliferation rate. Kv channel activity involves maintaining the cell membrane potential. This characteristic provides the K+ influx that facilitates Ca2+ influx, as part of the activation mechanism of a wide variety of mitogenic factors that induce cell proliferation (Leanza et al., 2014).In this context, potassium channels are considered modulators of the C influx strength. 2+ and therefore a potential target to be modulated to decrease the effects of Ca 2+and counteract their physiological and / or pathological function (Wulff et al., 2009). Overexpression of several potential-dependent potassium (Kv) channels has been identified in multiple types of cancer (Serrano-Novillo et al., 2019) where they contribute to cell cycle dysregulation with the appearance of an unlimited proliferation rate and resistance to apoptosis, and both processes are part of the main features of cancer development (Oeggerli et al., 2012, Ouadid-Ahidouch et al., 2016, Pérez-García et al., 2018, Urrego et al., 2014). In addition, Kv potassium channels regulate progression through cell cycle checkpoints by mechanisms that are based both on their ion conduction properties and on their interaction with other proteins belonging to signaling complexes in the plasma membrane (Teisseyre et al., 2019, Urrego et al., 2014).On the other hand, voltage-gated potassium channels participate in the regulation of programmed cell death in different cell types and tissues. Numerous studies demonstrate the crucial role of Kv channels in regulating both cell growth and death, associating the activity of these channels with mitogenic factors and proliferation in tumor cells. A distinctive feature of apoptosis is the decrease in cell volume and intracellular K+ concentration. This reduction in cell volume has been linked to the efflux of K+ ions into the extracellular space, which precedes critical events such as mitochondrial depolarization, cytochrome c release, apoptosome formation, cell fragmentation, proteolytic cleavage of procaspase-3, and increased endonuclease activity (Szabo et al., 2010). Several subtypes of ion channels, such as Kv1.1, Kv1.3, Kv1.5, and Kv11, are involved in apoptosis.Among others, these channels have been implicated in cancer progression and apoptosis (Bachmann et al., 2020). In particular, several studies have demonstrated the overexpression of Kv1.3 channels in some cancer samples compared to normal tissue, making them a potentially attractive molecular target for both cancer diagnosis and treatment (Pérez-Verdaguer et al., 2016; Serrano-Albarrás et al., 2018). Increased expression of these channels has been observed in breast, colon, smooth muscle (leiomyosarcoma), skeletal muscle (alveolar rhabdomyosarcoma), and lymph node cancers, as well as in mature neoplastic B cells in chronic lymphocytic leukemia (CLL-B). Therefore, inhibiting Kv1.3 channels in cancer cells could inhibit proliferation and induce apoptosis. The application of margatoxin (MgTX), an inhibitor of Kv1.3 channels (Bartok et al.(Fraser et al., 2014), significantly inhibits the proliferation of the AT-2 cell line of weakly metastatic prostate cancer (Fraser et al., 2000). Similarly, inhibition of Kv1.3 channels by tetraethylamine (TEA), a non-specific inhibitor, reduced the proliferation of breast cancer tumor cells (Teisseyre et al., 2015, Teisseyre et al., 2019). The Kv1.4 channel shows increased activity associated with increased methylation. This evidence correlates with increased tumor grade progression in glioma and gastric cancer patients (Angi et al., 2023). High expression of the Kv1.5 channel in leiomyosarcoma, gastric carcinoma, and colorectal carcinoma has been correlated with a high proliferation rate and cancer aggressiveness. Inhibition of Kv1.5 channel expression results in significant inhibition of proliferation and cell cycle arrest in the G0 / G1 phase in osteosarcoma (Angi et al., 2023). The Kv1 channel.Kv1.1 has been identified in the MCF-7 breast cancer cell line and in the A549 non-small cell lung cancer cell line. In MCF-7, the use of the compound α-dendrotoxin (α-DTX) revealed the existence of potassium currents, specifically that of hKv1.1 (Ouadid-Ahidouch et al., 2000), and in A549 it caused inhibition of lung adenocarcinoma tumor progression in vivo, probably dependent on the presence of this channel in the cell line (Jang et al., 2011). Furthermore, high expression of Kv1.1 has been shown to correlate with a poor prognosis in patients with cervical cancer, and silencing the channel inhibits the proliferation of HeLa tumor cells (Liu et al., 2019). Scientific reports, therefore, confirm that the dysregulation of ion channels is a common characteristic of cancer cells, which contributes to their proliferation, survival and resistance to conventional therapies (Shi et al., 2025).Therefore, focusing therapeutic alternatives on ion channels offers a novel and promising opportunity for treating cancer. The best-known inhibitors of Kv channels are peptides derived from venomous animals. In particular, peptides from scorpion venom have been frequently used in research to determine the structure and function of potassium ion channels and have emerged as compounds that block channels present in cancer cells. Margatoxin, a neuropeptide from the venom of the scorpion Centruroides margaritatus, binds to and inhibits the activity of the Kv1.3 channel with high affinity (Garcia-Calvo et al., 1993). Carybdotoxin, derived from the venom of the scorpion Leiurus quinquestriatus, is a nonspecific inhibitor of Kv1.2 and also interacts with the Kv1.3 and SK4 channels (Banerjee et al., 2013).Also noteworthy are agitoxin-1 (Gross and MacKinnon, 1996) and kaliotoxin, which inhibit Kv1.x channels in the same process. Additionally, the peptide KAaH2, from the scorpion Androctonus australis, is capable of inhibiting Kv1.1 channel activity in glioblastoma cells via the Epidermal Growth Factor Receptor (EGFR) signaling pathway (Aissaoui et al., 2018). Heteroctenus junceus (formerly Rhopalurus junceus) is a widely distributed species endemic to Cuba, belonging to the family Bhutidae. This species is used in traditional medicine for the treatment of some diseases and some scientific reports argue its potential for the treatment of cancer (Diaz-Garcia et al., 2013, Díaz-García et al., 2019, Lozano-Trujillo et al., 2021).To date, only two peptides, C0HJS9 and C0HJT0 (according to the UNIPROT database code), from this scorpion have been isolated, identified, and sequenced. Structurally, these peptides are classified as potassium channel blockers. However, their functional activity and the type of ion channel they recognize are unknown. Given the evidence of the anticancer effect of this scorpion venom, there is a possibility of identifying similar effects in some of these peptides and / or their variants. Summary of the Invention: The proposed invention comprises synthetic peptides derived from the venom of the scorpion H. junceus with anti-metastatic properties, as well as compositions of synthetic peptides derived from the venom of H. junceus and its variants, obtained from recombinant or synthetic techniques, with anti-metastatic properties, effective against tumor cells or small cell lung cancer.Description of the invention The proposed invention further comprises a diagnostic kit for the detection of lung cancer with aggressive characteristics or in the metastatic stage, wherein said kit comprises: specific primers of the KCNA channels (1 to 4), which by detecting mRNA expression of target sequences corresponding to said channels, would allow indicating the different stages of cancer progression or tumor progression state; and wherein, in the case of KCNA1, the presence (overexpression) of the target genes of said primers would indicate metastasis. Another embodiment of the invention comprises: a set of KCNA channel-specific primers (1 to 4), which may be selected from the list comprising: Seq ID No. 8, Seq ID No. 9, Seq ID No. 10, Seq ID No. 11, Seq ID No. 12, Seq ID No. 13, Seq ID No. 14, Seq ID No. 15, and a set of synthetic and / or recombinant peptides which may be selected from the list comprising: SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7; wherein the primer set allows the identification and amplification of gene sequences associated with the expression (overexpression) specifically of said KCNA channels, and wherein the synthetic and / or recombinant peptides allow the validation of the expression of said channels in the tumor cells that express them, thus allowing confirmation, through said overexpression, of the stage of lung cancer, where the overexpression of said channels in said tumor cells is indicative of metastasis, allowing confirmation of said diagnosis. In another embodiment, the primers of the present invention designed to selectively and exclusively identify the mRNA expression of the KCNA1 channel can be selected from the list comprising: Seq ID No. 8, Seq ID No.9, and furthermore, these primers can be used in the diagnosis and / or monitoring of pathologies associated with the ectopic, atemporal, overexpression of the KCNA1 channel, as part of diagnostic kits or other related methodologies, in human and non-human subjects (domestic and farm animals). In one embodiment of the invention, an antimetastatic pharmaceutical composition is provided, comprising at least: i) a set of synthetic and / or recombinant peptides derived from the venom of the scorpion H. junceus, wherein at least one of them corresponds to the synthetic peptide SEQ ID No. 1, ii) pharmaceutical excipients, and iii) distilled water. In an alternative embodiment of the invention, the antimetastatic pharmaceutical composition comprises at least one synthetic peptide derived from the venom of the scorpion H. junceus, wherein the synthetic peptide is SEQ ID No. 1.In another alternative embodiment of the invention, the anti-metastatic pharmaceutical composition comprises at least one set containing at least two synthetic peptides derived from the venom of the scorpion H. junceus, wherein at least the first corresponds to peptide SEQ ID No. 1, and wherein the second can be selected from the list comprising: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7. In another embodiment of the invention, it comprises a peptide isolated from H. junceus scorpion venom, capable of decreasing the activity of the hKv1.1 channel, as well as inhibiting migration and invasiveness in small cell lung cancer. In another embodiment of the invention, it comprises a peptide obtained by recombinant and / or synthetic methods, capable of decreasing the activity of the hKv1 channel.1, as well as inhibiting migration and invasiveness in small cell lung cancer, and wherein said peptide may be selected from the list comprising: peptide SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7. Likewise, the proposed invention is not limited to the aforementioned sequences, but also comprises those variants of peptide SEQ ID No. 1 and peptides SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, which correspond to structural and / or peptidomimetic analogues, wherein said variants exhibit activity on the hKv1.1 ion channel, as well as inhibiting migration and invasiveness in small cell lung cancer. In an alternative embodiment of the invention, the set of synthetic and / or recombinant peptides derived from the venom of the H. scorpion.junceus, wherein said peptides in combination with traditional therapies, which include but are limited to the following: surgery, chemotherapy, radiotherapy, monoclonal antibody therapy, cell cycle checkpoint inhibitors, kinase inhibitors and any other drug or product whose therapeutic indications are aimed at the curative, palliative and / or adjuvant treatment of human and non-human subjects, including also domestic and farm animals, with cancerous pathologies; and wherein furthermore, said peptides may be selected from the list comprising: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7. In an alternative embodiment of the proposed invention, it comprises the use of the peptides SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7.7, including their structural analogues and / or peptidomimetics, administered in suitable excipients, wherein said peptides are useful for the treatment of cancerous and non-cancerous pathologies, associated with ectopic, timeless expression, overexpression and / or increased functional activity of the hKv1.1 ion channel. In another embodiment of the invention, it comprises the use of the peptides SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, their structural analogues and / or peptidomimetics, including as fusion proteins or conjugates to fluorescent compounds, antibodies, dyes, and any other compound that can be conjugated or associated with said peptides, because it serves for diagnosis, monitoring and / or treatment of cancerous and non-cancerous pathologies, associated with ectopic, timeless expression, overexpression and / or increased functional activity of the hKv1.1 ion channel.In another embodiment of the invention, it comprises compositions or the use of any of the peptides of the invention, because said peptides are diluted in appropriate excipients as part of oral, parenteral, topical, nasal, aerosol or other routes of administration used in clinical practice, which inhibit migration, invasiveness and metastasis in small cell lung cancer, as well as in cancerous and non-cancerous pathologies, both in human subjects, as well as in non-human subjects (domestic and farm animals) and wherein said peptides may be selected from the list comprising: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7. highly specific primers for each of these hkv1.x channels (where x ranges from 1 to 8) particular cell marker in specific, related to specific types of cancers. kv1.1 almost exclusively overexpressed in small cell lung cancer. Additionally, this channel is expressed in metastatic cells (an indicator of metastasis in small cell lung cancer - correlation with aggressiveness of small cell lung cancer). The kv1.1 channel is only found to be expressed in brain cells. No expression is detected in other tissues (given the specificity of the detection technique: for example, with 10 to 5 copies, the channel is overexpressed and functions as a diagnostic test). EXAMPLES OF IMPLEMENTATION Example 1 Comparison of the amino acid sequences of the peptide C0HJT0 and scorpion peptides. The peptide C0HJT0 was compared with the peptide sequences of scorpion venoms present in the UNIPROT database (FIGURE 1). The local basic alignment search program (BLAST) of the National Center for Biotechnology Information (http: / / wncbi.(nml.nih.gov) to generate the sequence alignment of the peptides of interest. RESULT The figure shows the sequence alignment of the peptide C0HJT0 with the peptides that showed similarity percentages greater than 70% (Figure 1). The greatest similarity to the sequence of the peptide C0HJT0 was observed with the sequence P0DL43, corresponding to a peptide isolated from the venom of the scorpion Rhopalurus garridoi. This peptide was evaluated in electrophysiological experiments and demonstrated that it is capable of reversibly reducing the current of the human potassium channel hKv1.1 by up to 50% at concentrations of 1 µM. Based on this evidence and the high similarity between both peptides, one hypothesis would consider this channel as a potential target of the peptide C0HJT0. Example 2 Analysis of the molecular docking of the peptide C0HJT0 and the potassium ion channel hKv1.1. The interaction of the peptide C0HJT0 with the ion channel hKv1.1 was analyzed.The human potassium channel Kv1.1 (hKv1.1) was constructed from the amino acid sequence of the α subunit of the hKv1.1 channel, contained in the UniProt database under code Q09470 (UniProt, 2019). Once the hKv1.1 ion channel was constructed, its structure was verified, and molecular docking experiments were performed. The peptide used as a ligand was obtained from the UniProt database and corresponds to the isolated and sequenced peptide from the scorpion Heteroctenus junceus, code C0HJT0 (UniProt, 2019). Both the peptide and the ion channel were prepared for molecular docking experiments by adding hydrogens to each amino acid at pH 7.4 (physiological pH). Energy minimization was then performed to relax all systems and obtain a local minimum of the structures under study. This procedure was performed using the UCSF Chimera program version 1.16 (Pettersen et al., 2004). To perform the sampling, the size of the grid (space in which the peptide will interact with the protein) was defined as 80x80x80 Å. 3 , centered at coordinates x=135,962, y=140,199 and z=113,200. These coordinates were taken from the center of mass of the Kv1.1 channel. The grid size is 80x80x80 Å 3The design was defined to explore the largest possible conformational space. In all molecular docking procedures, a grid area of ​​0.375 Å was used, the number of modes was 10, and the energy range was set at 1 kcal / mol. The best docking poses were selected by analyzing the most negative peptide-channel binding energy (kcal / mol). The algorithm used for all molecular docking experiments was HDOCK (Yan et al., 2020a, Yan et al., 2020b). This algorithm is specialized in the study of protein-protein interactions. The HDOCK algorithm samples the binding modes between two proteins through a global search method based on the Fast Fourier Transform (FFT) and then evaluates the sampled binding modes with an enhanced scoring function based on iterative knowledge of protein-protein interactions.The Confidence Score (CSV) variable was used to select the best poses with a CSV greater than 0.7, indicating that the two molecules are likely to bind stably. With a CSV between 0.5 and 0.7, the two molecules could bind; and with a CSV less than 0.5, binding is unlikely (Huang and Zou, 2014). The HDOCK algorithm performed 20 docking runs of 100 samples each for 2000 conformations of the ion channel-peptide complex. The ion channel-peptide complex with the highest CSV was analyzed, and residues with crucial interactions were highlighted using a contact map of the amino acid sequence with a maximum threshold of 5 Å. The generated complex structures and interactions were subsequently visualized using the molecular visualization software PyMOL. RESULTS Figure 2 shows the best pose from the 100 molecular docking runs. The peptide adopted both horizontal and vertical positions in the pore region.The best horizontal position showed the lowest energy values ​​(-215.76 kcal / mol), suggesting that the complexes between the peptide C0HJT0 and the hKv1.1 channel in this position are the most stable. In the HDOCK simulation, the peptide oriented itself toward the base of the pore within the human hKv1.1 channel, suggesting that its interaction obstructs the passage of K+ ions. +through physical blockage of the pore, as described for other potassium channel-blocking scorpion peptides (Bergeron and Bingham, 2012; Swartz, 2013). The molecular docking score was 0.86, indicating a high probability of interaction between the peptide and the human hKv1.1 channel. Stability of the peptide-channel complex through hydrogen bonding occurs between the carboxyl group of cysteine ​​28 (Cys28) of the peptide and the aromatic NH group of histidine 355 (His355:D) at a distance of 1.9 Å. The other strong interaction stabilizing this complex occurs between the phenolic OH group of tyrosine 379 (Tyr379:D) and the carboxyl group of isoleucine 37 (Ile37) of the peptide (2.7 Å). Another important interaction is that which occurs between histidine 355 (His355:B) and methionine 21 (Met21) of the peptide at a distance of 2.9 Å.It is relevant to highlight that in the hydrogen bond interactions between the peptide and the channel, amino acids are identified that are present almost exclusively in the structure of the Kv1.1 potassium channel. On the other hand, the spatial structure of the peptide-channel complex allows visualization of the orientation of the amino group (NH2). +The presence of arginine 25 (Arg25), which is positively charged at physiological pH, is located towards the interior of the pore. This can generate electrostatic repulsion, preventing the passage of the ion through the ion channel. The location of Arg25 is uncommon in potassium ion channel blocking peptides, as the conformation of scorpion peptide structures primarily locates lysine 18 (Zhu et al., 2011) or lysine 27 (Banerjee et al., 2013; Chen and Chung, 2013) inside the pore. Example 3: Synthesis of the C0HJT0 peptide and electrophysiological evaluation of its interaction with the hKv1.1 channel. The result of the molecular coupling between the C0HJT0 peptide and the hKv1.1 channel will be experimentally verified through electrophysiological assays. The peptide was obtained using the Solid Phase synthesis technique, due to the low quantity required for the tests, the speed of the synthesis and the ease of handling.The Fmoc method was used, in which the amino acids are protected with the F-moc group to prevent the amino group from anchoring, leaving the carboxyl group free to anchor to the resin (Colombo, 1982). Amino acids were added covalently until the peptide sequence was complete. The protecting group of the alpha amino acid was removed using a 20% piperidine in 80% DMF solution. The peptide was purified by HPLC using a C18 reversed-phase column (10 × 250 mm). Purity was determined by analytical HPLC using a C18 column (4.6 × 250 mm). The mobile phases used were: phase A: HPLC-grade water with 0.1% TFA; phase B: acetonitrile with 0.1% TFA. The flow rate for each chromatographic run was performed in a linear gradient from 0 to 45% acetonitrile for 45 min at 1 mL / min. The column temperature was 26°C and the signal was detected at 230 nm.The peptide in solution was lyophilized and stored at -20°C until use. Confirmation of the correct synthesis of the C0HJT0 peptide was performed by mass spectrometry. The sample was dissolved in a solution containing 8 M urea in 25 mM NaHCO₃. The sample was reduced with 20 mM DTT in 25 mM NH₄HCO₃ for 1 h at room temperature. Additionally, the samples were alkylated in a solution of 20 mM ICH₂CONH₂ in 25 mM NH₄HCO₃ and incubated for 1 h in the dark at room temperature. Subsequently, the sample was diluted in 25 mM NaHCO₃, cleaned using a Sep-Pak C18 column (Waters), and dried in a rotary concentrator at 1000 rpm at 10°C overnight. 200 ng of the peptide were injected into a nanoElute nanoUHPLC (Bruker Daltonics) coupled to a timsTOF Pro mass spectrometer (Bruker Daltonics) using an Aurora UHPLC column (25cm x 75μm ID, 1.6μm C18, IonOpticks, Australia).Liquid chromatography employed a 90-min gradient from 2% to 35% buffer B (0.1% formic acid / acetonitrile). Data collection was performed using TimsControl 2.0 software (Bruker Daltonics) under 10 PASEF cycles, with a mass range of 100–1700 m / z, capillary ionization at 1.5 kV and 180°C, and a time-of-flight frequency of 10 kHz at a resolution of 50,000 FWHM. Data collected by mass spectrometry were analyzed using PEAKS Studio X+ software (Bioinformatics Solutions) with the PEAKS IMS protein identification module. Mass tolerance parameters of 50 ppm were used, employing monoisotopic masses and 0.05 Da ionic fragments. The non-specific digestion mode was selected, and a maximum of 2 loss cleavages per peptide were chosen.Post-translational modifications (PTMs) used were: cysteine ​​carbamidomethylation (fixed PTM), methionine oxidation (M), N-terminal acetylation, asparagine and glutamine deamination (NQ), and lysine and N-terminal carbamilation (K, X, N), as variable PTMs. All peptides were filtered using an ALC (Average Local Confidence) >80%. ALC refers to the average confidence that an amino acid is present in the peptide de novo at a particular position, expressed as a percentage. For electrophysiological assays, adherent HEK-293 cells were used, maintained in DMEM-LG 5% fetal bovine serum (FBS), 1% Fungizone, and 1% penicillin-streptomycin, subcultured, and plated on 35 mm plates with 12 mm coverslips. Prior to completing 24 h post subculture, the cells were transfected with the pcDNA3.1 plasmid vector containing the gene encoding the hKv1 channel.The gene encoding GFP was linked to KCNA1 using lipofectamine 3000. Cells were then incubated for 24–48 h at 37°C with 5% CO2. Cells containing the transfected channel and exhibiting green fluorescence under a fluorescence microscope were used to obtain electrophysiological recordings. Transfected cells cultured on coverslips were placed in a recording chamber (RC-25, Warner Instruments) mounted on a vibration-damping table under an inverted Olympus IX71 microscope. A Patch Clamp L / M-EPC 7 amplifier was used to amplify the current signals, which were digitized using a Digidata 1550B and acquired via pClamp 10.7 software. Pipette movement and position were controlled with a Scientifica Patchstar micromanipulator.Recording electrodes were fabricated from borosilicate capillaries using a horizontal drawr (P-87, Shutter Instruments), exhibiting an electrical resistance between 2 and 3 MΩ, and filled with internal solution. The patch-clamp technique in whole-cell mode was used to obtain current recordings using the following protocol: initial current at -80 mV for 100 ms, followed by a 0 mV pulse for 200 ms, and finally a pulse at -50 mV for 150 ms, under constant perfusion at 1 mL / min. Changes in current were recorded by adding different concentrations of the peptide C0HJT0 (4, 40, 400 nM) to the external solution prior to the protocol. Recordings were taken after 30 s of current stabilization with external solution only, and each concentration was immediately applied for 1 min. Each recorded cell was subjected to only one condition and then discarded (unpaired data).The initial 30 s of recording was taken as the control current and the changes in the maximum current (I) were evaluated. maxThe concentrations of the peptide C0HJT0 were evaluated at different levels after 1 minute of infusion. The composition of the internal solution in mM was as follows: 140 KCl, 4 NaCl, 1 CaCl2, 1 EGTA, 10 HEPES, 5 MgATP, 0.3 GTP, pH adjusted to 7.2 with KOH and an osmolarity of approximately 300 mOsm. The composition of the external solution in mM was as follows: 135 NaCl, 4 KCl, 2.5 CaCl2, 2 MgCl2, 10 HEPES, 20 Glucose, pH adjusted to 7.4 with NaOH and an osmolarity of 300 mM. RESULTS The synthetic peptide C0HJT0 was obtained using the Fmoc method, and its purity was analyzed by HPLC and its identity by MS / MS mass spectrometry. The chromatogram showed a high degree of purity of 95% (Figure 3A) while the MS / MS spectrum showed a monoisotopic mass of 3945.55 Da. Complete sequencing of the peptide resulted in the 37 aa sequence TVIDVKCTSPKQCVPACKAAMGTVRAKCMNGKCKCYI (SEQ ID HJP001).This result is 100% consistent with the peptide sequence from the scorpion H. junceus, with UniProt code C0HJT0, previously deduced by proteomic analysis (Rodriguez-Ravelo et al., 2015), demonstrating the correct synthesis and high purity of the synthetic peptide. Chemical synthesis has the advantage of offering greater structural diversity than biotechnological methods, as it allows the use of non-natural amino acids among the wide variety of chemical modifications that can be introduced (Amblard et al., 2006; Vlieghe et al., 2010). Solid-phase synthesis allows the covalent bonding of the C-terminal amino acid to an insoluble support and the growth of the peptide chain by incorporation of the remaining amino acids through successive steps of coupling and deprotection of the α-amino group. The protection of the amino group is carried out using two methods: Boc and Fmoc.The use of the tert-butyloxycarbonyl (Boc) group for temporary protection of the α-amino group is based on its ability to hydrolyze in acidic media. However, this is not limited to this compound (Merrifield, 1969), as other protecting groups can be used in combination with tert-butyloxycarbonyl, such as acetamidomethyl (Acm), 2,4-dinitrophenyl (Dnp), formyl, and 3-nitro-2-pyridinosulfenes (Npys), which are stable in strong acids and can be removed before or after unanchoring. Permanent protection of amino acid side chains can employ, but is not limited to, substituted benzyl groups (urethanes, esters, and ethers), cyclohexyl (cHex), and p-toluenesulfonyl.The Boc group of the alpha-amino acid can be removed by treatment with trifluoroacetic acid (TFA) at concentrations between 25–50% in dichloromethane (DCM), while the side chains are protected by treatment with stronger acids such as liquid hydrogen fluoride and trifluoromethanesulfonic acid. Another method for temporary protection of the α-amino group uses the 9-fluorenylmethyloxycarbonyl (Fmoc) group, which requires less aggressive conditions than the Boc method and is labile in basic media (Stuber et al., 1989). In this second method, permanent protection of the amino acid side chains involves the use of different tert-butyl groups (urethane, ester, and ether). The trityl group (Trt), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 2,4,6-trimethoxybenzyl (Tmob) are also used, among others, whose purpose is the elimination of the protection of the amino acid side chains.Fmoc is removed from the alpha amino acid by treatment with primary or tertiary amines. These solutions include a 20–50% piperidine solution in dimethylformamide (DMF) or a 2% solution of 1,8-diazabicyclo(5,4,4)undec-7-ene (DBU) in DMF with 2% piperidine (Stuber et al., 1989). Any solutions compatible with Fmoc can be used in the deprotection process and final peptide synthesis. Furthermore, any chemical reagents developed for the same purpose can be considered in the peptide synthesis process using either method. The whole-cell patch-clamp technique was used to determine the effect of the synthetic peptide C0HTJ0 on potassium currents from the hKv1.1 channel expressed in HEK-293 cells. Depolarizing pulses in the cells showed potassium currents that are activated, with absence of inactivation, characteristic of this channel (Figure 4A).Different concentrations of the C0HTJ0 peptide showed an increase in peak current inhibition corresponding to increasing concentration (Figure 4A), indicating that the C0HTJ0 peptide inhibits the currents in this channel, consistent with observations from bioinformatics analyses. It is important to note that the effect of the C0HTJ0 peptide occurs at nanomolar concentrations, starting with 20% peak current inhibition at 10 nM and reaching 80–90% peak current inhibition at concentrations of 400 nM (Figure 4B). Example 4: Isolation, identification, and electrophysiological assays of the peptide with activity on the hKv1.1 channel. The venom of the scorpion Heteroctenus junceus was separated by reversed-phase high-performance liquid chromatography (HPLC) using a C18 column (intersustain c18, pore size 5 µm, length 4.6 mm ID x 250 mm, GL Science Inc. Japan).A 2 mL loop was used, and 6 mg of scorpion venom was injected into each chromatographic run. The mobile phases used were: phase A: H₂O / 0.1% TFA; phase B: acetonitrile / 0.1% TFA. The flow rate for each chromatographic run was a linear gradient of 0–45% acetonitrile for 45 min at 1 mL / min. The column temperature was 26°C, and the signal was detected at 230 nm. The fractions of interest were collected individually and grouped according to retention times. After the runs, all fractions with the same retention times were concentrated overnight at 4°C in a concentrator (Centrivap concentrator system, Labconco, USA). Finally, the fractions were stored at -20°C. Electrophysiological assays were performed on HEK-293 cells transfected with the pcDNA 3.1 plasmid vector containing the gene encoding the hKv1 channel.Cells containing the transfected channel (KCNA1) linked to the gene encoding GFP were incubated for 24–48 h at 37°C with 5% CO2. Cells containing the transfected channel and exhibiting green fluorescence under a fluorescence microscope were used to obtain electrophysiological recordings. Current changes were recorded similarly to Example 3. Extracellular solution perfusion involved the individual application of each of the following samples: H. junceus scorpion venom (VC) at 1 mg / mL, F5.1, and F5.3 (the concentration of each was based on its proportion in the venom at 1 mg / mL). In all cases, these were added to the external solution prior to the protocol and adjusted to pH 7.4.The currents of each sample were recorded after 30 seconds of current stabilization using only external solution, and each sample was immediately applied for 1 minute. Each recorded cell was subjected to only one condition and then discarded (unpaired data). The initial 30 seconds of recording were used as the control current, and changes in the maximum current (I) were evaluated. max) at the different concentrations evaluated after 1 min of infusion. For mass spectrometry analysis of the fraction exhibiting activity on the hKv1.1 channel, it was dissolved in a solution containing 8M Urea in 25 mM NaHCO₃ and the procedure was similar to Example 3. RESULTS Figure 5 shows the chromatogram obtained from the injection of R. junceus scorpion venom. The selected fractions correspond to the isolation zone of the peptides previously published (Rodriguez-Ravelo et al., 2015). The data collected in the mass spectrometer were analyzed using PEAKS X+ software with the PEAKS IMS protein identification module. The MS / MS spectral analysis showed the identification of a major peptide present in fraction F5.3.The identified peptide sequence was compared with peptide sequences of the order Scorpiones listed in the UniProt database and revealed 100% similarity only with the alpha-KTx 2.19 peptide with the UniProt code C0HJT0 from the venom of the scorpion Heteroctenus junceus. This result confirms that the peptide is among the components present in H. junceus scorpion venom, as previously described (Rodriguez-Ravelo et al., 2015). The patch-clamp technique was used to determine the effect of scorpion venom and the F5.1 and F5.3 subfractions on potassium currents from the hKv1.1 channel expressed in HEK-293 cells. Depolarizing pulses in the cells showed potassium currents that were activated, without inactivation, characteristic of this channel (Figure 6). Perfusion of whole scorpion venom resulted in an 87% inhibition of the potassium current. In the case of the F5 subfractions.At concentrations equivalent to 1 mg / mL, inhibitions of the maximum current of 23% and 90%, respectively, were obtained for subfractions 1 and F5.3 (Figures 6A and 6B). The inhibition values ​​for the scorpion venom current and the F5.3 fraction are very similar, confirming that the C0HJT0 peptide is predominantly found in the F5.3 subfraction. Additionally, this finding confirms for the first time that the C0HJT0 peptide inhibits the hKv1.1 channel currents. Example 5 KCNA1 gene expression in the Cancer Cell Line Encyclopedia (CCLE) database. The expression patterns of the KCNA1 gene were analyzed in the Cancer Cell Line Encyclopedia (CCLE) database using the Broad DepMap Portal (https: / / depmap.org / portal). Gene expression levels from RNA sequencing data (RNAseq) were illustrated in log2-fold transcripts per million (log2 (TPM+1) (Ghandi et al., 2019).Results: KCNA1 Gene Expression in Tumor Cell Lines. Gene expression data (RNA-seq) from all cell lines included in the Cancer Cell Line Encyclopedia (CCLE) database were used. Figure 7 shows the KCNA1 gene expression behavior in all cell types. The expression levels of the KCNA1 gene, which encodes the hKv1.1 channel, vary depending on the cell type, with lung cancer exhibiting the highest expression levels (Figure 7). Kv1.1 channel expression commonly occurs in the central and peripheral nervous systems, primarily in the hippocampus, cerebellum, and peripheral nerves (D'Adamo et al., 2020).Therefore, the expression of this channel in lung cancer cells would be considered an ectopic overexpression in a tissue that does not normally express it and may represent a treatment opportunity for compounds that limit or inhibit its activity in this context. Primary lung cancer originates in epithelial cells and can be divided into two major histological groups. The most common is non-small cell lung cancer (NSCLC), which accounts for 85%, followed by small cell lung cancer (SCLC), which accounts for approximately 13% of all primary lung cancers (Smolarz et al., 2025). Based on this stratification, the same database was used to identify the mRNA expression levels of the gene encoding hKv1.1 in these cancer cell subtypes.Figure 8 shows the KCNA1 gene expression levels comparatively between small cell lung cancer, non-small cell lung cancer, and non-cancerous cells. SCLC cell lines exhibited the highest KCNA1 gene expression levels (TPM>6) (Figure 8). Conversely, NSCLC showed very low levels of expression of this gene, with only 3 cell lines showing any expression (0.2). <TPM<3). Adicionalmente, las células epiteliales no cancerosas no mostraron niveles de expresión. El cáncer de pulmón de células pequeñas particularmente se diferencia de otros tipos histológicos en muchas características biológicas y clínicas (alta tasa de proliferación, corto tiempo de duplicación de la masa tumoral, marcada tendencia a la metástasis temprana, quimiosensibilidad y relativa radiosensibilidad) (Thai et al., 2021).The high proliferation rate, its tendency to develop early metastases, and low survival rates make it a cancer with limited treatability. Small cell lung cancer (SCLC) is only considered curable when the disease is confined to one hemithorax, which occurs infrequently and is usually treated with chemoimmunotherapy, with or without consolidation radiotherapy. Extensive disease has proven more intractable, with even combination therapies showing no improvement in clinical trials compared to monotherapies, and is associated with complications from the early development of metastases (Thai et al., 2021). In recent years, there have been no significant changes beyond the benefits of immune checkpoint inhibitors, which extend survival by several months (Ko et al., 2021).Additionally, approximately 70% of patients with SCLC already present with metastatic disease at the time of diagnosis, with frequent macrometastases in lymph nodes, brain, liver, and bones (Ko et al., 2021). Therefore, identifying new ways to limit metastatic spread and target metastases is fundamental for the development of new therapies to treat SCLC (Sabari et al., 2017). Ion channels could represent new therapeutic targets as a therapeutic option that could contribute to reducing the high rate of proliferation and metastatic spread. Example 6: Relative quantification of expression levels of voltage-gated potassium channels in the NCI-H1299 and NCI-H446 tumor cell lines. Primer design and qPCR standardization.Based on the high expression of the KCNA1 gene in SCLC cell lines, the NCI-H446 cell line was selected as a model cell for experimental analysis, as it exhibits the highest levels of KCNA1 expression (TPM>6). The treatment of metastases remains a challenge. Therefore, early cancer detection and the development of tumor biomarkers are crucial for developing new metastasis management strategies. Molecules produced by tumors or by the body's response to tumors during their onset or progression have demonstrated critical and promising value in detection, early diagnosis, and prognosis (Liu et al., 2024). The discovery of new molecules linked to the development of tumor metastases, such as the hKv1 channel, is a promising area of ​​research.1. This introduces the possibility of its detection, particularly in small cell lung cancer, where the expression levels observed in in silico studies make it a potentially attractive target. Determining the mRNA levels of the genes of interest (Kv1.1-4) required prior standardization of the PCR conditions. This process was performed individually and following the same methodology for all primer pairs used. In the case of PCRs to determine the different potassium channel variants, the DNA from the channel vectors was used as a template. These vectors were designed by cloning each of the potassium channel sequences into the pcDNA3.1(+) vector, flanked by the HindIII and BamHI cleavage sites. Primer design was performed using the digital tool PrimerBlast® (Primer designing tool).The mRNA sequences available in the GeneBank database, using the accession numbers KCNA1 (NM_000217.3), KCNA2 (NM_001204269.2), KCNA3 (NM_002232.5), and KCNA4 (NM_002233.4), were used as templates for primer design. Exons were selected as templates for primer design. To increase efficiency, common regions between several channels were selected to allow the use of a single common-sense antisense primer for multiple PCRs. Therefore, the same antisense sequence was used to detect KCNA1 and KCNA3. The OligoAnalyzer™ Tool and Primer Designer Tool were used to evaluate the quality of the designed primers. Table 1 shows the sequences of the designed primers. Table 1. Characteristics and sequences of primers for qPCR used in this research. Primers Size Temperature Gene Annealing amplicon sequence. The amplicon resulting from the PCR was extracted from the 1% agarose gel, quantified, and then serially diluted from 10 ng / µL to 10 pg / µL. These dilutions were amplified again by qPCR and used as a calibration curve for the relative quantification of mRNA levels in the samples under study. The NCI-H446 (small cell lung cancer) and NCI-H1299 (non-small cell lung cancer) cell lines were seeded the day before in 30 mm cell culture plates at a confluence of 70–80%. After 18 hours, RNA was extracted using TRIzol® (Life Technologies, Inc.) according to the manufacturer's instructions. Before complementary DNA (cDNA) synthesis, the samples were treated with DNase H I (Invitrogen, Inc.) to remove genomic DNA.cDNA was reverse transcribed from a total of 200 ng of RNA using RT-PCR with the ImProm-II™ Reverse Transcription System (Promega) synthesis kit, according to the manufacturer's instructions. A semi-quantitative real-time PCR was then performed using the Applied Biosystems StepOne thermocycler (Applied). The 18S rRNA gene was used as a control for proper RNA extraction. PCR reactions were performed using SYBRgreen (Agilent Technologies) according to the manufacturer's instructions. Subsequently, electrophoresis was performed on 1% agarose gels, and melting curves were analyzed to confirm the specificity of the PCR products obtained. Two oligonucleotides were used for each gene: one forward (FOR) and one reverse (REV). Results The expression level of the mRNAs of the ion channels hKv1.1, hKv1.2, hKv1.3 and hKv1.4 was analyzed.To determine expression levels, a calibration curve was generated for the relative quantification of mRNA levels (Figure 9). The cell lines used in this study have different expression levels of voltage-gated potassium channels, which are of interest in this research. In particular, the NCI-H446 cell line showed high expression levels of hKv1.1 channel mRNA compared to the NCI-H1299 cell line (Figure 10). This result is consistent with the in silico analyses performed and makes it a suitable model for evaluating compounds or drugs that act on this ion channel. The remaining ion channels showed significantly lower expression levels compared to hKv1.1 (Figure 10). In this regard, the primers designed to identify hKv1 channel mRNA expression...1. They demonstrated high specificity in detecting the gene, compared to other channels in the hKv1.x family, so they could be considered for the development of diagnostic methods and / or treatment monitoring, or integrated with existing or future traditional detection methods (Liu et al., 2024). Example 7. Protein expression of the potential-dependent potassium channel Kv1.1 in the NCI-H1299 and NCI-H446 tumor cell lines by immunoblotting (Western blot). The expression of the hKv1.1 potassium channel protein was determined in the NCI-H1299 and NCI-H446 tumor cell lines. The cells were subcultured and seeded at 70% confluence. After 18 hours, the proteins were extracted by digestion using a lysis buffer containing NaCl (150 mM), EDTA (1 mM) pH 8.0, TRIS (50 mM) pH 7.4, Triton X-100 (1%), Sodium Orthovanadate (1 mM) and Protease Inhibitors 1X.A mouse brain protein extract was used as a positive control. Samples were run on a 12% and 10% acrylamide-bisacrylamide discontinuous gradient gel electrophoresis gel and subjected to a potential difference of 100 V for 2 hrs. After electrophoresis, the samples were transferred to a nitrocellulose membrane and incubated at a potential difference of 110 V for 1 hr. Following blocking of free sites with 5% BSA (for Kv1.1) and 5% skim milk (for GAPDH) in 1X PBS for 1 hr, the membranes were incubated with anti-Kv1.1 monoclonal antibodies (#APC-161, Alomone Labs, Israel) and an anti-GAPDH monoclonal antibody (Novus Biological) for 16 h. oC. Signal development was performed using the following secondary antibodies: anti-mouse HRP 7076S (CST), anti-rabbit Alexa Fluor 546, A21085 (Life Technologies). The relative abundance of the chemiluminescently labeled proteins (post-incubation with the secondary antibody) was determined using a series of photographs taken with a photodocumentation system (ChemisScope 3200 Mini, Clinx Science Instrument Co.). This series of photographs allowed for the determination of different exposure points of the nitrocellulose membrane over time, enabling the construction of a saturation curve. Subsequently, using ImageJ software, the bands of interest were selected, and their pixel density (the selected area, calculated as the average number of pixels) was measured by subtracting the background pixel density.Subsequently, the photo number and pixel density obtained were plotted, allowing the construction of a curve whose upper limit corresponds to the saturation point. Finally, the slope of the resulting line was calculated, ensuring that it corresponded to the linear region of the photodocumentation. The slope ratio of the protein of interest to the housekeeping protein (GAPDH) was then used to determine the relative abundance of each protein. Results: The results show differences in the protein expression levels of the potential-dependent potassium channel hKv1.1 between the cell lines used in this research (Figure 11). These results are consistent with those observed in the mRNA expression levels in both cell lines and confirm the usefulness of the NCI-H446 cell line for evaluating the effect of compounds and drugs on hKv1 channel activity.1 and its relationship to proliferation, migration, invasiveness, and metastasis in this cell line. Example 8 Effect of the synthetic peptide C0HJT0 on the viability, migration, and invasiveness of the NCI-H446 small cell lung cancer cell line. Cell lines The NCI-H446 cell line (AddexBio Catalog No. C0016054) was used for the transwell chamber viability and migration experiments. Cells were maintained in RPMI-1640 90% (w / v) medium with inactivated fetal bovine serum (FBS), 10% (v / v), penicillin (100 U / mL), streptomycin (100 μg / mL), and fungizone (100 μg / mL). Cell viability The effect on cell viability was assessed using the MTT assay. NCI-H446 cells were seeded in a 96-well cell culture plate at a density of 1×104 cells / well at 37°C with 5% CO2. After 24h of incubation, various concentrations of the synthetic peptide C0HJT0 were added (0, 6.25, 12.5, 25, 50, 100 µM).The 96-well plates were incubated for 72 h at 37°C with 5% CO2. Then, 10 μL of MTT solution (5 mg / mL) (Merck, USA) was added to the 96-well culture plates, and the cells were incubated for 3 h at 37°C with 5% CO2. The culture medium was decanted from the plate, and 150 μL of DMSO (100%) was added to each well. A560 nm values ​​were obtained using the Synergy™ HTX multimode microplate reader (Agilent BioTek, USA). The percentage decrease in viability was calculated as (Absorption of negative control - Absorption of treated wells / Absorption of negative control) x 100 for each cell line at each concentration. The experiments were repeated three times, and three technical replicates were used. Transwell chamber migration and invasiveness assay The Transwell chamber migration assay was used to determine the effect of the C0HJT0 peptide on the migratory capacity of NCI-H446 cells.Cells dissolved in RPMI-1640 culture medium without FBS were seeded at 2x10. 5Cells were seeded into 300 µL of a Transwell chamber in the upper part of the Transwell chamber. The chambers were then placed inside a well of a 24-well plate containing 600 µL of RPMI-1640 / 10% FBS medium with the synthetic peptide added individually. Three peptide concentrations (10, 50, and 100 µM) were used, with two replicates per well, and the experiment was repeated three times. Cells seeded in the Transwell chamber with RPMI-1640 / 10% FBS added to the well of the 24-well plate served as a migration control. Cells incubated at 37°C for 24 h were fixed with 4% paraformaldehyde for 5 min, 100% methanol for 15 min, and finally stained with 1% crystal violet. For the invasiveness assays, 100 µL of Matrigel was added to the transwell membrane and incubated for 2 h at 37°C. After this time, the same procedure as for the migration assay was performed and the synthetic peptide was added at 100 µM.In all cases, images were taken with an Olympus microscope at 4X magnification and analyzed using ImageJ software. Statistical Analysis: Data analysis was performed using the GraphPad Prism10 statistical program. The results were plotted as mean ± standard error. Statistical significance was determined using Student's t-test. Significant differences were always considered for p-values ​​≤ 0.05. RESULTS: Cells incubated for 72 h with different concentrations of the peptide did not show a decrease in cell viability in NCI-H446 (Figure 12). The NCI-H446 cell line is a metastatic cell line obtained from the pleural fluid of a patient with small cell lung cancer (Carney et al., 1985). Since no effects on cell viability were observed, experiments were performed to determine if the C0HJT0 peptide has an effect on the migration of these cells.The effect of the C0HJT0 peptide on the migration capacity of NCI-H446 cells was evaluated using the transwell assay with RPMI-1640 / 10% FBS medium as a chemoattractant. Figure 13 shows images of the migrating cell population in each group, stained with 1% crystal violet. The synthetic peptide incubated for 24 h with NCI-H446 cells significantly reduced their migration at concentrations of 50 µM and 100 µM compared to the control (p<0.05) (Figure 13B). Additionally, the synthetic peptide incubated for 24 h in the transwell chamber with Matrigel reduced the invasiveness of NCI-H446 cells at a peptide concentration of 100 µM (Figure 13C, 13D). Metastasis is the most lethal stage of cancer development and progression, in which cells reach and colonize organs distant from the primary site of growth (Gerstberger et al., 2023).Metastasis is a systemic disease that affects multiple organs, either by directly colonizing them and compromising their function, or indirectly, in addition to acquired resistance to existing therapies. Metastasis comprises several stages, including migration, invasion, survival during transit through the circulatory system, and colonization of distant organs (van Zijl et al., 2011). Specifically, the migration and invasion of cancer cells into adjacent tissues and intravasation into blood and lymphatic vessels are essential stages in the appearance and development of metastases in adenocarcinomas, which are among the most common cancers in humans (Yamaguchi et al., 2005). Cancer cell migration can occur singly or in cell clusters, and in most cases, associated with the extracellular matrix of proteins.Regardless of the various migration patterns, all cell migrations require the presence of chemoattractants that allow them to reach the bloodstream (Liu et al., 2024; Yamaguchi et al., 2005). The cell migration process has been used as a model for the development of migration and invasiveness assays, such as the Boyden chamber or transwell assay (Justus et al., 2023), which enable the discovery of new treatment strategies and / or compounds or drugs that can interfere with the migratory potential of tumor cells, as a preliminary step toward reducing or eliminating the development of metastases (Kramer et al., 2013). The migratory and invasive capacity of the NCI-H446 small cell lung cancer cell line, which ectopically expresses the hKv1.1 ion channel, decreased due to exposure to the synthetic peptide C0HJT0.The NCI-H446 cell line has been used in several studies to measure the migratory, invasive, and metastatic capacity of various compounds. These compounds have demonstrated that in vitro inhibition of migration and invasiveness correlates with a reduction in metastasis in animal models of this cell line (Yang et al., 2012; Zheng et al., 2019). The positive result of the peptide C0HJT0 in reducing migration and invasiveness of NCI-H446 cells suggests an anti-metastatic potential in experimental animal models. This characteristic raises the possibility of treating cancerous pathologies with ectopic expression or overexpression of the hKv1.1 channel using this peptide or variants thereof. Example 9 Electrophysiology of the synthetic peptide C0HJT0 on the ion channels Kv1.1, Kv1.2 and Kv1.3 The ion channel genes that encode for the Kv1.1, Kv1.2 and Kv1.3 channels.Three ion channels were individually inserted into the pcDNA 3.1 plasmid vector containing the gene encoding GFP. Each vector was individually transfected into HEK-293 cells using lipofectamine 2000. The transfected cells, cultured on coverslips, were placed in a recording chamber to measure the effect of the synthetic peptide on the inhibition of the currents in each ion channel. The current recording conditions were similar to those established for the hKv1.1 channel in Example 3. Currents were recorded 24–48 h after transfection with the channel-containing vector. Current inhibition was assessed for 8–10 min. After recording for each channel, the cells were washed for 2 min with extracellular fluid. Increasing concentrations of the synthetic peptide were used for the dose-response curves (10, 50, 75, 100, 175, 250, 500, 750 nM).RESULTS Several experiments were performed to analyze the effect of the synthetic peptide C0HJT0 on the potassium currents of the hKv1.1, hKv1.2, and hKv1.3 channels, expressed in HEK-293 cells. Figure 14 shows the dose-response curve of the C0HJT0 peptide on the three ion channels. The dose-response curves allowed for the calculation of EC values. 50 of the synthetic peptide for each of the ion channels. The hKv1.3 channel (EC 50 =68.33 nM) and hKv1.2 (73.91 nM) showed EC values 50 Similar values. The hKv1.1 channel showed EC values. 50 significantly higher than hKv1.2 and hKv1.3 with an EC 50=149.75 nM. On the other hand, after washing with the extracellular solution by perfusion, it was observed that after 60 s the ionic currents of the hKv1.2 and hKv1.3 channels recovered 100% and returned to the values ​​of the control currents (Figure 15). Conversely, the hKv1.1 channel currents only recovered 50% compared to the control currents. This evidence suggests that the synthetic peptide has a higher affinity for the hKv1.1 channel. The fact that the synthetic peptide C0HJT0 remains interacting with the hKv1.1 channel for a much longer time would lead to a more persistent inhibition of the channel current, preventing or reducing the effects associated with this channel in diseases or pathologies linked to ectopic expression or overexpression of this channel. Example 10 Molecular dynamics of the peptide C0HJT0 in the ion channels Kv1.1 and kv1.2 The analysis of the molecular dynamics of the peptide C0HJT0 and the ion channel hKv1.Step 1 was performed taking into account the best peptide-channel complex from Example 2. The human potassium channel Kv1.2 (hKv1.2) was constructed from the amino acid sequence obtained from the Protein Data Bank (Berman et al., 2000) with code PDBid: 2A79, which is resolved to 2.90 Å (Long et al., 2005). Once the hKv1.2 ion channel was constructed, its structure was verified, and molecular dynamics experiments were performed. For the molecular dynamics study of the peptide with both ion channels, all simulations were performed using the CHARMM36 force field (Feng et al., 2023; Huang et al., 2017). The hKv1.2-C0HJT0 complexes, both horizontal and vertical, and the hKv1.1-C0HJT0 complex were introduced into a lipid bilayer formed by the POPC lipid model, which was constructed using the CHARMM-GUI server (Lee et al., 2019). This lipid bilayer has 6758 lipid molecules.The hKv1.2-C0HJTO complexes together with the membrane were introduced into a 15×15×15 Å water box. 3of the TIP3P type (Boonstra et al., 2016) such that waters are present in both the extracellular and intracellular sections. Waters in contact with the membrane were removed. The systems were neutralized with a 0.15 M KCl solution to ensure physiological conditions and subjected to 100,000 steps for energy minimization using the conjugate gradient methodology to reduce any close contacts, 2.0 ns of equilibration, and 40 ns of molecular dynamics simulation at 300 K using the NAMD 2.13 program (Acun et al., 2018). The RMSD, radius of gyration, number of hydrogen bonds, and occupancy levels were used as stability criteria. RESULTS Molecular dynamics studies with the hKv1.2 channel were performed with the peptide C0HJT0 in the 2 most probable interaction positions (vertical and horizontal), which were observed in the molecular docking study in the hKv1.1-C0HJT0 complex (Figure 16).The RMSD parameter was evaluated as a stability criterion to analyze the dynamic behavior of the peptide-channel complexes. The hKv1.1-C0HJT0 complex began to stabilize after 3 ns of trajectory with minimal fluctuations. The RMSD value was 1.6069 ± 0.2617 Å. The RMSD value of the horizontal and vertical hKv1.2-C0HJT0 complexes remained stable over time with average values ​​below 2 Å (RMSD hKv1.2-C0HJT0HORIZONTAL = 1.6118 ± 0.2962 Å and hKv1.2-C0HJT0HORIZONTAL = 1.6118 ± 0.2962 Å). VERTICAL=1.5856±0.2934 Å) and similar to the hKv1.1-C0HJT0 complex. Several authors suggest that for a system to remain stable, this parameter must remain below 3 Å. This result suggests that the peptide C0HJT0 contributes to the stability of the complexes. The radius of gyration (Rg) was also analyzed for all systems, which determines the stability of the complexes by analyzing their level of compaction. The radius of gyration is defined as the mean square distance of the mass of a set of atoms (channel-peptide complex) from a common center of mass. Higher levels of compaction result in smaller radii of gyration. The hKv1.1-C0HJT0 complex exhibited almost constant behavior during the Rg phase, with minimal fluctuations throughout the path, with a value of Rg (hKv1.1-C0HJT0) = 9.677±0.0657 Å. This parameter showed very similar fluctuations for both complexes (Rg (hKv1.2-C0HJT0HORIZONTAL)=19.6019±0.7638 Å and Rg (hKv1.2-C0HJT0 VERTICAL)=19.4652±0.9266 Å with high values ​​for this variable, much higher than that of the hKv1.1-C0HJT0 complex. This result shows that the hKv1.2-C0HJT0 complexes did not remain compact over time and that, therefore, the peptide-channel interaction tends to be lost, suggesting a reversibility of the peptide's effect on the hKv1.2 channel. Another parameter analyzed was the number of hydrogen bond interactions formed throughout the path. The percentage of hydrogen bond occupancy, representing the time the interaction lasted below 3 Å, was determined throughout the path. The hKv1.1-C0HJT0 complex had an average of 3.68±2.14 hydrogen bonds throughout the path. While the hKv1.2-C0HJT0 systems showed large fluctuations and the lowest average levels of hydrogen bonds generated (0.94±0.81), they would not be able to persist over time (Table 2). Table 2.Analysis of the stability and strength of hydrogen bond interactions during the 40 ns path of Molecular Dynamics. O. cu ncia Distance of Lys388-NH--N-Asp4-JTO 50.78% 4.7333±2.0334 JTO-Val2-NH--O-Gln357 34.36% 5.6208±0.8323 The hKv1.1-C0HJT0 complex had more stable interactions with two occupancies above 50% formed by Lys6-Glu353 (87.65%) and Asp4-Gly376 (54.48%). On the other hand, the hKv1.2-C0HJT0 complex VERTICALOnly the Lys34-Glu355 interaction (79.92%) showed a stable interaction, with an interaction distance of less than 3 Å. It is worth noting that other interacting pairs of the hKv1.2-C0HJT0 complex showed high occupancy rates, such as Lys18-Asp379 (92.64%) and Asp4-Lys388 (50.78%), although with interaction distances greater than 3 Å. Based on this criterion, the vertical position of the peptide was more stable than the horizontal position. The calculated interactions did not remain stable over time, further supporting evidence that the channel blockage is not permanent but reversible, as demonstrated in the patch-clamp assays. However, given the therapeutic potential observed in this peptide, it would be necessary to reduce interactions with other ion channels, such as those observed for the hKv1.2 channel.Additionally, hydrogen bonding interactions were able to demonstrate the differences in interaction between the peptide C0HJT0 and the hKv1.1 and hKv1.2 channels. Given the basic nature of most of these peptides, which are derived from scorpion venom, other intermolecular interactions such as salt bridges, cation-π interactions, and hydrophobic interactions must be considered. These interactions, taken together, modulate the stability, affinity, selectivity, and potency of these peptides in their final effect on ion channels and should be taken into account in channel modification strategies. The two main amino acid residues of the peptide C0HJT0 that participate in the interaction with the hKv1.2 channel (Lys18 and Lys34) do not appear to participate in the formation of hydrogen bonds in the hKv1.1-C0HJT0 complex.Therefore, these amino acids could represent the basis for potential modifications of this peptide that increase its selectivity and contribute to increasing its potency on the hKv1.1 channel. Example 11 Molecular docking of the peptide C0HJT0 and its variants in the ion channels Kv1.1 and Kv1.2 The structures of the hKv1.1 and hKv1.2 channels were obtained following the procedure described in Examples 2 and 9 of this document. The peptide C0HJT0 was used to make the modifications and obtain the new variants. The peptide and the constructed hKv1.1 and hKv1.2 channels were prepared for the molecular docking experiments in a manner similar to Example 2. The mutations to the peptide C0HJT0 were made at positions K18 and K34. Five variants with single mutations (K18A, K18D, K18D, K18F, K34A) and five variants with double mutations (K18AK34A, K18FK34A, K18FK34F, K18DK34R, K18EK34R) were analyzed.All mutant variants were prepared following the same computational procedure as that used in Example 2. All mutants underwent 5000 steps of energy minimization using the CHARMM36 force field (Feng et al., 2023; Huang et al., 2017) and the NAMD 3.0 program (Acun et al., 2018). For each run of the molecular docking experiments, the 100 best poses were selected based on the Docking Score and Confidence Score variables. RESULTS The molecular docking results show that all poses, both for C0HJT0 and for the mutations for both channels, block the pore. However, there were differences in affinity between the two ion channels. The peptide C0HJT0 and its variants have significantly greater affinity for the hKv1.1 channel than for hKv1.2, exhibiting more negative energies when comparing results with the hKv1.1 channel compared to hKv1.2 (Table 3). Table 3.Results of the best molecular docking pose of the hKv1.1 and hKv1.2 channels with the peptide C0HJT0 and its mutated variants. Poses with the greatest differences in both ion channels are highlighted in black. Potassium Channel Peptide Interaction Energies Confidence Score C0HJT0 -215.76 0.7884 C0HJT0. K18A -213.51 0.7808 C0HJT0 K18AK34A -214.33 0.7836 C0HJT0 K18FK34A -219.30 0.8000 K 18FK3 Kv1.1 C0HJT0 4F -231.77 0.8369 C0HJT0 K18F -231.26 0.8355 C0HJT0 K18D -208.85 0.7644 C0HJT0 K18E -210.46 0.7702 C0HJT0 K18DK34R -224.52 0.8161 C0HJT0 K18EK34R -226.65 0.8224 COHJT0 -182.01 0.6548 C0HJT0 K18A -191.00 0.6942 C0HJT0 K34A -184.84 0.6675 C0HJT0 K18AK34A -196.54 0.7172 C0HJT0 K18FK34A -195.80 0.7142 Kv1.2 C0HJT0 K18FK34F -210.90 0.7717 C0HJT0 K18F -212.13 0.7760 C0HJT0 K18D -174.40 0.6196 C0HJT0 K18E -174.55 0.6203 C0HJT0 K18DK34R -202.42 0.7405 C0HJT0 K18EK34R-202.24 0.7398 The analysis of interaction energies in the hKv1.1 channel shows that the most negative interaction energies corresponded to the simple mutant C0HJT0 K18F (-231.26) and the double mutant C0HJT0 K18FK34F (-231.77). The double mutants C0HJT0 K18EK34R (-226.65) and C0HJT0 K18DK34R The C0HJT0 mutants (-224.54) showed the second-highest energies, lower than those observed for the original peptide C0HJT0 (-215.76). The interaction energy values ​​of the aforementioned mutants suggest greater stability in the complexes formed compared to the original peptide. On the other hand, the lowest interaction energies of the complexes with the hKv1.2 channel corresponded to the simple C0HJT0 mutants. K18E (-174.55) and C0HJT0 K18D(-174.40). These activation energies were the only ones shown to be higher than those observed for the original peptide C0HJT0 (-182.01) for the hKv1.2 channel, which translates into lower stability of these mutants in the channel-peptide complex. The interaction patterns of the mutants also showed differences compared to the original peptide. Docking studies of the original peptide C0HJT0 identified that it is located in a horizontal position (Figure 2), forming hydrogen bonds between Cys28-His355 (1.9 Å), Ile37-Tyr379 (2.7 Å), and Met21-His355 (2.9 Å), with Arg25 of the peptide inserted inside the pore. In the case of the double mutant C0HJT0 K18FK34FThis complex was positioned horizontally in the channel pore, but the two phenylalanines introduced at positions 18 and 34 cause the loss of interaction with the Tyr379 residues of the four domains of the hKv1.1 channel (Figure 17A). This complex may be stabilized by the Ile37-Tyr379 interaction only in domains A and D of hKv1.1. In the case of the C0HJT0 mutant K18F It was also positioned horizontally, covering the pore of the hKv1.1 channel, but diagonally with respect to the K ions + The introduction of the K18F mutation resulted in the loss of interaction with the Tyr379 residues of all four domains. Stabilization of this complex would include the Lys34-His355 interaction (3.3 Å) in domain I and the Ile37-Tyr379 interaction only in domains A and B (Figure 17B). In both cases, the peptide's interaction with the pore via Arg25 is completely lost. The C0HJT0 variants K18DK34R and C0HJT0 K18EK34RThey showed similarity in the interaction of Arg25, with respect to the original peptide, in the hKv1.1 channel. In all docking experiments the variant C0HJT0 K18DK34R It was positioned horizontally and perpendicular to the K ions + (Figure 18A). Structural analysis reveals that the strongest interactions were found in the interacting pairs Arg25-Gly376 of subunit-IV (2.1 Å), Arg25-Gly375 of subunit-III (2.6 Å), and Arg34-Hist355 of subunit-II (3.2 Å) (Figure 18A). The double mutant C0HJT0 K18EK34R showed stronger interactions than the double mutant C0HJT0 K18DK34R , with the interacting pairs Arg25-Gly376 (2.0 Å), Arg25-Tyr375 (2.6 Å), both belonging to subunit III, Arg25-Tyr375 (2.6 Å) from subunit II, and Arg34-Asp377 (3 Å) with distances within 3 Å, in the formation of hydrogen bonds (Figure 18B). The single mutant C0HJT0 K18DIt was positioned horizontally on the cytoplasmic face, passing through the pore of the hKv1.1 channel, but longitudinally with respect to the K ions + (Figure 19). Although Arg25 was located in the pore perpendicular to the K ions + The hydrogen bonding interactions were somewhat distant at Arg25-Tyr379 (3.6 Å) (Figure 19A). The remaining probable hydrogen bonding interactions were greater than 3 Å. On the other hand, the C0HJT0 variant K18E showed similar behaviors to C0HJT0 K18D The main difference was the existence of an interacting Arg25-Tyr375 pair (2.1 Å) with distances less than 3 Å (Figure 19B). Based on the analysis of hydrogen-bond-mediated stability, the interactions of the C0HJT0 variants K18FK34F and C0HJT0 K18FWith the hKv1.2 channel, they showed a loss of interactions with respect to the hKv1.1 channel. Only one interacting pair was obtained, which corresponded to Gly22-Asp379 (2.8) in domain II for the C0HJT0 K18FK34F and Ile37-Val381 of subunit II (3 Å) in the C0HJT0 variant K18F Both are in the hKv1.2 channel (Figure 20A). It appears that the introduction of phenylalanine at position 18 favors the loss of hydrogen bonding interactions in both cases. The C0HJT0 variants K18DK34R and C0HJT0 K18EK34R They were located at a certain inclination with respect to the K ions + (Figure 20B). This location, in both cases, causes Arg25 to be significantly further from the pore compared to what was obtained with hKv1.1. The strongest interactions were found between Arg25-Asp363 (1.8 Å), Arg25-Gln375 (2.2 Å), Arg25-Asp363 (2.3 Å), and Arg25-Met380 (2.5 Å), all in the D domain of the channel (Figure 20B) for the C0HJT0 variant K18DK34Rand the interacting pairs Arg25-Asp363 (2.3 Å and 2.7 Å), Arg25-Glu357 (2.4 Å) and Arg25-Met380 (2.8 Å), all in the A domain of the hKv1.2 channel and Arg34-Tyr377 (2.8 Å) also in the A domain for the C0HJT0 variant K18EK34R of the hKv1.2 channel (Figure 20B). The C0HJT0 variants K18D (A) and C0HJT0 K18EThey had the least negative interaction energies of all the mutants studied for the hKv1.2 channel, significantly less negative than these same mutants interacting with the hKv1.1 channel (Table 3). In both variants, Arg25 was significantly displaced from the channel pore (Figure 20C). In no case could hydrogen bond interactions shorter than 3 Å be computed. Since Arg25 appears to represent a distinctive amino acid in the interaction of this peptide with the channel pore, the loss of its interaction could correlate with reduced stability of the channel-peptide complexes for the hKv1.2 channel. Example 12 Study of the interactions between the hKv1.1 channel and the peptide C0HJT variants C0HJT0 K18E , C0HJT0 K1 0 and its v 8F and C0HJT0 K18EK34R through Molecular Dynamics simulations. Molecular dynamics simulations with K18E K18F K18EK34 the variants C0HJT0, C0HJT0 and C0HJT0 R(Table 4) were performed similarly to those previously performed with the hKv1.1 channel and the native peptide C0HJT0. Dynamics for all complexes were analyzed using the CHARMM36 force field (Huang et al., 2017), for both the peptide and its variants, as well as for the hKv1.1 channel. Each complex was embedded in a lipid bilayer constructed from the POPC lipid model using the CHARMM-GUI server (Feng et al., 2023; Lee et al., 2019). This lipid bilayer contains 65,140 lipid molecules. Each complex, along with its membrane, was then placed in a 15 × 15 × 15 Å water box. 3of the TIP3P type (Boonstra et al., 2016, Huang and Zou, 2014) such that water was present in both the extracellular and intracellular sections, and water in contact with the membrane was removed. The four complexes were neutralized with a 0.15 M KCl solution to ensure physiological conditions and subjected to 100,000 steps for energy minimization using the conjugate gradient methodology to reduce any close contact, 2.0 ns equilibration time, and 40 ns simulation time for the hKv1.1-C0HJT0 complex and 100 ns for the three mutants at 300 K using the NAMD 2.13 program (Acun et al., 2018). Table 4. Sequences of the C0HJT0 peptide variants. Peptide Final amino acid sequence C0HJT TVIDVKCTSPKQCVPACKAAMGTVRAKCMNGKCKCYI* K0 C0HJT0 18E TVIDVKCTSPKQCVPACEAAMGTVRAKCMNGKCKCYI* RESULTS Molecular dynamics simulations were performed to analyze the behavior and stability of the native peptide and its variants on the hKv1.1 channel. The RMSD parameter was evaluated as a stability criterion to analyze the dynamic behavior of the peptide-channel complexes. In all four cases, the peptide-channel complexes began to stabilize after 3 ns of travel with minimal fluctuations (Figure 21). The RMSD value was very similar for the hKv1.1-C0HJT0 complex (1.6069 ± 0.2617 Å). K18E (1.6027±0.2569 Å) and C0HJT0 K18 ) and the va EK34R (1.60059±0.2560 Å) (fig While the variant C0HJT0 K18 to 21). FIt showed the lowest RMSD values ​​(0.4090 ± 0.0720 Å). Therefore, all the systems were below 2 Å for this parameter, and among these, the hKv1.1-C0HJT0 complex proved to be the most stable (Figure 21). Several authors suggest that for a system to remain stable over time, the RMSD values ​​must remain below 3 Å (Velazquez-Libera et al., 2020). Hydrogen bond formation was analyzed for each of the complexes as part of the parameters that contribute to stability. Figure 22 shows the hydrogen bond interactions in the four systems. A large fluctuation in the number of hydrogen bond interactions was observed in the c Ko 1 8 m Eplejos form K 1 a 8d E K or 3s 4 Rby the channel and the native peptide C0HJT0 and the variants C0HJT0 and C0HJT0. However, the lowest average values ​​in hydrogen bond formation were obtained for the complexes formed HJT0 K with variant C0 18F (2.055±1.2119) and the one formed with the native peptide (3.6808±2.1433) (Figure 22). This behavior indicates that these interactions were not maintained stably over time or are not the main interactions that stabilize these iodine-channel complexes. Conversely, the hKv1.1-C0HJT0 complexes K18E and hKv1.1-C0HJT0 K1 jos pept 8EK34RThey showed significant differences in the number of hydrogen bonds formed compared to the previous ones, with values ​​of 8.9052 ± 3.8352 and 8.8130 ± 3.5696, respectively, suggesting that these complexes could be stabilized primarily by hydrogen bond interactions (Figure 22). The radius of gyration (Rg) was the third parameter analyzed as part of the stability determinants of the peptide-channel complexes. Figure 23 shows that the Rg of all complexes behaved similarly, with very little fluctuation of this variable with respect to its mean value. The small variation of this parameter indicates a high degree of compactness of these systems, which translates into high stability for these complexes.Molecular dynamics simulation studies reveal that the mutant variants are more stable than the native peptide C0HJT0. This conclusion is based on the fact that significant differences were observed in some cases in the number of bridges formed (C0HJT0). K18E , C0HJT0 K18EK hydrogen 34R ) and in other cases showed the ability of the complex (C0HJT0 K ayor est 18FIn all cases, the variants showed a different mode of interaction than the native peptide, which is probably the main reason for the greater overall stability observed in the variants, due to the mutations made. For the purposes of this document, variants are generally considered to be those peptides containing 37 amino acids aligned in the sequence with the code UNIPROT C0HJT0, which are up to 80% identical with sequence changes of up to 7 amino acids and which include, but are not limited to, non-natural amino acids, derivatizations of one or more amino acids, cyclization, or even the addition of organic groups at the amino and / or carboxyl ends.Similarly, compounds whose functional equivalence from a structural point of view shares the same interaction determinants are included, such that they confer stabilities and / or affinities similar to the examples in this document, and thus behave as peptidomimetics or variants thereof. The interaction determinants represent those functional groups whose three-dimensional arrangement in a compound coincides with the spatial structure of any of the sequences in this document and that exhibit stabilities and / or affinities and / or specificity for hKv1.1, which could therefore be partially similar or overlap in the behavior of the sequences or some of their amino acids.The modifications could also include conservative or neutral substitutions, such as those described herein, as well as substitutions of methylated, acetylated, and other amino acids at certain positions to reduce proteolytic degradation. The amino acids considered non-essential in the sequences described herein could lead to reduced variants that may even provide more resistant analogs with greater specificity and / or greater stability. The variants designed and analyzed in this work demonstrate that varying at least one amino acid improves the selectivity and specificity of the interaction between the peptide and the Kv1.1 channel. Therefore, these variants, shown as examples of the present invention, far from being limiting, allow us to predict the design of any other functional analogs of the C0HJT0 peptide that improve selectivity and specificity for the Kv1.1 channel.Example 13 Design and Production of the Recombinant Peptide C0HJT0 Based on the C0HJT0 peptide sequence, an expression vector was designed to enable recombinant production of the peptide in the bacterial strain E. coli Rosetta gami (DE3). This strain is intrinsically resistant to chloramphenicol and allows for greater expression of large quantities of the fusion protein in the cytoplasm once it is bound to thioredoxin (Trx). The expression vector pET32a+p1 was selected; this vector allows for the soluble expression of Trx-bound fusion proteins in the cytoplasm of E. coli bacteria. Additionally, a rigid spacer sequence, consisting of the tandem repeat of the sequence (EAAAK), is included. 3,followed by a cleavage site for Enterokinase (sequence: DDDDK). This vector includes 6-amino-acid histidine sequences, which can be used for subsequent purification of the recombinant protein. Additionally, the C0HJT0 sequence and the fusion protein were inserted flanked by two cleavage sites for KpnI / NcoI. The C0HJT0 expression vector sequence in Rosetta gami (DE3) is shown below. (Figure 24) Chemocompetent bacteria were generated using 0.1 M CaCl2 + 10% Glycerol, according to the previously described protocol (Chung and Miller, 1988). 50 µL aliquots of the bacteria were prepared in cryopreservation tubes stored at -80°C (Ros-Q). 50 µL of bacteria were taken and transformed with 1 µL of the vector pET32a+C0HJT0 (5ng / µL), incubated on LB+Agar+Chloramphenicol (34 µg / mL) / Ampicillin (100 µg / mL) plates at 30°C for 18 hrs.After this time, several colonies were selected and cultured in LB+Chloramphenicol (34 µg / mL) medium for 16 hrs at a temperature of 30°C with shaking at 15 rpm. The inoculum from one of the colonies was transferred to a 2.7 L bioreactor of LB+ampicillin (100 µg / mL) medium and incubated at 30°C with shaking (200 rpm), pH 7.0, 2 LPM of aeration until reaching an OD. 600nmThe expression of the recombinant protein was induced by the addition of 0.5 mM IPTG, with stirring at 300 rpm, pH 7.0, and 5 LPM of aeration for 6 h. The biomass obtained from the induced cultures was resuspended at a ratio of 1 g of biomass per 40 mL of lysis solution (Triton X-1000, 1% in PBS). This biomass was broken down in a BKBM-V0.4 ball mill (BIOBASE) using 0.5 mm zirconium silicate beads. Three breakdown cycles were performed at 530 rpm for 10 min each. The resulting product was centrifuged at 10,000 rpm for 10 min, and the breakdown pellet was resuspended in a volume of lysis solution equivalent to the volume of the breakdown supernatant obtained. Finally, the samples were stored at -20°C until use. Sample quantification was performed using a 2 mg / mL albumin standard as a reference. Analysis of C0HJT0 expression in E.The expression of the recombinant protein C0HJT0 was analyzed by electrophoresis and immunoblotting on a 15% polyacrylamide gel (SDS-PAGE) under reducing conditions. Samples were denatured at 95°C for 5 min. Electrophoresis was performed at 100 V for 2 hrs at room temperature. After electrophoresis, the proteins separated by SDS-PAGE were stained with Coomassie Blue R-250 for 40 min and washed with a solution containing 20% ​​methanol / 10% glacial acetic acid for 2 hrs. The gel was then immobilized on a 0.45 µm nitrocellulose membrane using a semi-wet transfer machine (BioRad) with transfer buffer (Tris-Glycine 20% methanol) at 250 mA for 30 min. The membrane was blocked with 5% milk in 1X Tris saline buffer (TBS) solution and incubated for 2 hrs.The primary anti-histidine antibody (1:5000) was dissolved in 5% milk / 0.1% TBS with tween-20 and incubated for 1 hour at room temperature with shaking. The membrane was then washed three times with TBS-tween buffer (0.1% SDS, 0.1% Tween-20, 1X TBS) for 5 minutes. The membrane was incubated with the secondary anti-mouse IgG (H+L) Alexa Fluor® 680 (Jackson) antibody (1:5000) in TBS-tween (5% milk, 0.1% Tween-20, 1X TBS) for 1 hour at room temperature with shaking. It was washed three times with TBS-tween buffer (0.1% SDS, 0.1% Tween-20, 1X TBS) for 5 minutes. The polyacrylamide gel and nitrocellulose membrane were developed using an Odyssey CLx Infrared Imaging System-LICOR Biosciences. Enterokinase (EK) Digestion Assay: To check peptide expression, digestion was performed with the Enterokinase enzyme in a final volume of 50 µL, with an Enterokinase concentration of 1 IU / µL.The samples were incubated at 37°C and 22°C for 20 hours. The breakthrough supernatant was purified by metal ion chromatography (IMAC). The sample was diluted in PBS pH 7.4 with 300 mM NaCl and passed through a matrix (Sepharose Fast Flow) containing nickel, previously equilibrated with PBS pH 7.4 and 300 mM NaCl. It was eluted with 500 mM imidazole and subsequently dialyzed in PBS pH 7.4 ON buffer. The samples were resuspended in 100 µl per vial and applied to an HPLC column C18 (250 x 4.6 mm), 5 µm, Restek, cat# 9103565. Migration assay of recombinant C0HJT0 peptide. Transwell migration assays were performed similarly to example 8. The recombinant peptide C0HJT0 was used at 140 µM. Results. Figure 25 shows the 15% acrylamide gel electrophoresis where the expression of Rosetta gami bacterial proteins is observed under IPTG induction conditions (Figure 25).Differences in fusion protein expression were observed between the control Rosetta gami bacterium (which does not contain the expression vector) and the transformed Rosetta gami. The fusion protein (Trx-linker-C0HJT0) appears at 25 kDa (Figure 25A). Figure 25B shows the band corresponding to the fusion protein, detected using an anti-histidine antibody, which allows identification of the fusion protein only in the lanes corresponding to the transformed Rosetta gami. The expression and induction conditions established in this protocol allow for obtaining high quantities of the fusion protein (Trx-linker-C0HJT0). The established strategy for the expression of a highly cross-linked peptide in the culture supernatant of this E. coli strain is efficient. The fusion protein was subjected to enzymatic cleavage with enterokinase at 1 IU / µL, and two incubation temperatures were evaluated.Figure 26 shows the 15% polyacrylamide gel electrophoresis of samples from transformed Rosetta gami, after IPTG induction and enterokinase cleavage, at 22°C and 37°C. Lanes 1 and 2 show the intense band at 25 kDa, corresponding to the fusion protein. Lane 3 shows the enzymatic cleavage, yielding a protein band near 15 kDa (Trx-linker) and a band larger than 5 kDa, corresponding to the recombinant peptide (C0HJT0). In lane 3, the appearance of these bands coincides with a decrease in the major band at 25 kDa. Lanes 4-6 show the enterokinase cleavage controls (Figure 26). Lane 7 shows the result of cutting the fusion protein with enterokinase incubated at 22ºC, which indicates that under these cutting conditions a similar result is obtained (Figure 26).Enterokinase cleavage efficiently separates the peptide of interest from the fusion protein. Final separation of the recombinant peptide and verification of its purity were performed by HPLC using a reversed-phase C18 column and an acetonitrile gradient for 45 min. The chromatographic profile of the fraction eluting at 27.35 min corresponds to the recombinant peptide C0HJT0 (Figure 27A). Figure 27B shows images of the migrating cell population for the control group and the group treated with the recombinant peptide, stained with 1% crystal violet (Figure 27B). The recombinant peptide incubated for 24 h with NCI-H446 cells significantly reduced cell migration to 140 µM compared to the control (p<0.05) (Figure 27C). Recombinant production methods for obtaining scorpion peptides have been widely used.The most commonly used systems are bacteria and yeast, with bacteria accounting for over 80% of production. The use of bacteria is due to their rapid growth, simplicity, and low cost. Given that these are conventional methods for obtaining peptides, and that bacterial expression was the method used in this document, any technical modifications to this method, or the use or improvement of other methods that offer enhanced yield, reduced expression time, or other advantages, could be considered for obtaining the scorpion peptides and their variants discussed herein. Brief description of the figures: Figure 1. Alignment of the peptide C0HJT0 with scorpion venom peptides grouped in the UNIPROT database. Residues other than C0HJT0 are indicated in red. S: percentage of similarity. #: number of amino acids.Figure 2: Graphical representation of the interaction between the peptide C0HJT0 and the human Kv1.1 channel (hKv1.1). The magnified image shows the structural analysis of the peptide-channel complex and its probable hydrogen bonding interactions between the amino acids of the peptide C0HJT0 (represented in red and yellow) and the human Kv1.1 channel (blue). Figure 3. Analysis of the peptide C0HJT0 by HPLC and mass spectrometry. (A) Purity analysis of the synthetic peptide C0HJT0 on a reversed-phase C18 column coupled to HPLC. (B) Mass spectrum of the peptide C0HJT0 obtained using an ESI-TOF-TOF mass spectrometer. Figure 4. Electrophysiological recordings of potassium currents in the Kv1.1 channel expressed in HEK-293 cells. A) Recording normalized by the I. maxFigure 5: Chromatogram of H. junceus scorpion venom, identifying fractions F5.1 and F5.3. 6 mg of venom was injected into a reversed-phase C18 column coupled to an HPLC system. The run was performed for 45 min at 1 mL / min in a 0-45% ACN gradient. The dashed line corresponds to the percentage of acetonitrile during the run time. Figure 6. Electrophysiological recordings of potassium currents in the hKv1.1 channel expressed in HEK-293 cells. A) Recording normalized by the I maxA) Potassium current control subjected to VC (Scorpion Venom) 1 mg / ml and fractions F5.1, F5.3 (at concentrations proportional to the VR concentration). B) Bar graph of A considering the percentage of inhibition at the maximum current for each condition. Figure 7: KCNA1 gene expression in all cell types included in the Cancer Cell Line Encyclopedia (CCLE) database. Each point represents a cell line. Gene expression levels are presented as TPM in logarithmic expression. Lines represent the magnitude of KCNA1 gene mRNA expression. TPM: transcripts per million. Figure 8: KCNA1 gene expression in all lung cancer cell subtypes. Each circle represents a cell line. Gene expression levels are presented as TPM in logarithmic expression. Lines represent the magnitude of KCNA1 gene mRNA expression. TPM: transcripts per million. Figure 9.Amplification of specific primers for KCNA1-4, using DNA from expression vectors at different concentrations as a template (standard curve). A: Amplification curves of samples positive for KCNA1 voltage-gated potassium channels, top left panel; melting curve. B: Amplification curves of samples positive for KCNA2 voltage-gated potassium channels, top left panel; melting curve. C: Amplification curves of samples positive for KCNA3 voltage-gated potassium channels, top left panel; melting curve. D: Amplification curves of samples positive for KCNA4 voltage-gated potassium channels, top left panel; melting curve. Figure 10. The NCI-H1299 and NCI-H446 cell lines have differences in the expression of voltage-gated potassium channels.A: Amount of potassium channel mRNA; results are shown for all four channels in cell lines NCI-H1299 (1) and NCI-H446 (2). Statistical analysis: Student's t-test. Comparison between both cell lines * p<0.05. B: 1% agarose gel electrophoresis, KCNA1 and KCNA3 amplicons from cell lines NCI-H1299 (1) and NCI-H446 (2). C: 1% agarose gel electrophoresis, KCNA2 and KCNA4 amplicons from cell lines NCI-H1299 (1) and NCI-H446 (2). Figure 11. There are differences in the protein expression levels of hKv1.1 in cell lines NCI-H1299 and NCI-H446. (A): Ratio of slopes of the change in chemiluminescence over time of hKv1.1 / GAPDH. * p<0.0001. (B): Representative image in Immunoblot assay of NCI-H1299 and NCI-H446 cells. Figure 12. Demonstration of the effect of the C0HJT0 peptide on the viability of NCI-H446 cells.Bar chart of the percentage of cell viability at each peptide concentration, calculated from the absorbance values ​​of the MTT assay. (n=3). Figure 13. Analysis of the effect of the C0HJT0 peptide on the migration capacity and invasiveness of the NCI-H446 small cell lung cancer cell line in the transwell migration assay. A) Representative photomicrograph of the migrating cell populations in each of the experimental groups. B) Bar charts of the percentage of migration in each of the experimental groups (n=3). C) Representative photomicrograph of the cell populations that invade through the matrigel in each of the experimental groups. D) Bar charts of the percentage of invasion in each of the experimental groups (n=3). *p<0.05 compared to the control. Figure 14.Dose-response curves of the effect of the synthetic peptide C0HJT0 on the potassium ion channel currents hKv1.1, hKv1.2 and hKv1.3, expressed in HEK-293 cells. I-normalized recording. max Figure 15 shows the current recordings of the hKv1.1, hKv1.2, and hKv1.3 channels after application of 750 nM of the synthetic peptide. At the end of the treatments, the cells were washed with extracellular solution for up to 2 min. Figure 16 shows the structures of the hKv1.2-C0HJT0HORIZONTAL and hKv1.2-C0HJT0 complexes. n=3. VERTICAL for molecular dynamics studies. The C0HJT0 peptide is represented in a dotted circle. Figure 17. Interaction of the C0HJT0 mutants K18FK34F (A) and C0HJT0 K18F(B) with the hKv1.1 channel. The amino acids of subunit-I are shown in light green, those of subunit-II in pink, those of subunit-III in light blue, and those of subunit-IV in yellow. The amino acids corresponding to the C0HJT0 peptide mutants are shown in dark green. K18FK34F (Ile37, Phe18, Phe34) and C0HJT0 K18F (Ile37, Phe18, Lys34). Figure 18: Interaction of the C0HJT0 mutants K18DK34R (A) and C0HJT0 K18EK34R (B) with the hKv1.1 channel. The amino acids of subunit-I are shown in light green, those of subunit-II in pink, those of subunit-III in light blue, and those of subunit-IV in yellow. The amino acids corresponding to the variants of the peptide C0HJT0 are shown in dark green. K18DK34R (Arg25, Arg34) and C0HJT0 K18EK34R(Arg25, Arg34). Figure 19: Interaction of the C0HJT0 mutants K18D (A) and C0HJT0 K18E (B) with the hKv1.1 channel. Amino acids of subunit-I are shown in light green, amino acids of subunit-II in red, amino acids of subunit-III in light blue, and amino acids of subunit-IV in yellow. Amino acids corresponding to the C0HJT0 peptide mutants are shown in light pink. K18D (Asp18, Arg25, Lys34) and C0HJT0 K18E (Glu18, Arg25, Lys34). Figure 20: Interaction of C0HJT0 variants on the hKv1.2 channel. Light green represents the amino acids of subunit I, pink represents the amino acids of domain B, light blue represents the amino acids of domain C, and yellow represents the amino acids of domain D. The amino acids corresponding to the peptide variants are shown in dark green. A: C0HJT0 K18FK34F; B:C0HJT0 K18F ; C:C0HJT0 K18DK34R ; D:C0HJT0 K18EK34R ; E:C0HJT0 K18D ; F:C0HJT0 K18E Figure 21: Behavior of the RMSD parameter during the simulation time. In the center of the graph, towards the top, the mean values ​​and standard deviation of this variable over time are represented for the native peptide and the variants. Figure 22: Graphical representation of the number of hydromate bridge interactions between the native peptide C0HJT0 and its variants C0HJT0 K18E , C0HJT0 K geno for 18EK34R and C0HJT0 K18F Inserted in the graph are the mean values ​​and standard deviation of this variable over time for the native peptide and variants. Figure 23: Radius of Gyration (Rg) of the α-carbon atoms of complex v1.1 and the native peptide C0HJT0 and its variants K e 1j 8o E formed p K 1 or 8r the hK channel F JT0 K18 tantes C0HJT0, C0HJT0 and C0H EK34RFigure 24. C0HJT0 expression vector map in plasmid pET32a+p1. Figure 25. Analysis of recombinant protein induction in E. coli Rosetta gami by 15% SDS-PAGE. A: SDS-PAGE showing C0HJT0 peptide expression in the breakthrough pellet (RP) and breakthrough supernatant (BSN) after 6 hours of induction with 0.5 mM IPTG at 30°C. B: Detection of recombinant protein by Western blot using an anti-histidine antibody. Lane PM: Molecular weight standard. Lane 1: Breakdown pellet from E. coli Rosetta gami induction, 6 hours (control). Lane 2: Breakdown supernatant from E. coli Rosetta gami induction, 6 hours (control). Lane 3: E. coli Rosetta gami breakthrough pellet expressing pET32a+p1-C0HJT0 vector, induction 6 hrs. Lane 4: E. coli Rosetta gami breakthrough supernatant expressing pET32a+p1-C0HJT0 vector, induction 6 hrs. Figure 26. Analysis of C0HJT0 digestion in E.E. coli Rosetta gami by Tris-Tricine gel electrophoresis. Cleavage of the C0HJT0 C.3 peptide in the cleavage supernatant (SBR) after digestion with 1 IU / µL EK at 37°C and 22°C. Lane PM: Molecular weight standard. Lane 1: E. coli Rosetta gami C0HJT0 C.3 induction 6 hrs. Lane 2: E. coli Rosetta gami C0HJT0 C.3 induction 6 hrs (undigested). Lane 3: E. coli Rosetta gami C0HJT0 C.3 induction 6 hrs, digestion with EK at 37°C. Lane 4: Positive control, undigested EK kit. Lane 5: Positive control, EK kit, digestion 37°C. Lane 6: Positive control, EK kit, digestion 22°C. Lane 7: E. coli Rosetta gami C0HJT0 C.3 induction 6 hrs digestion with EK at 22ºC. Figure 27. Analysis of the purity and functional activity of the recombinant peptide C0HJT0. A) Chromatogram of the pure recombinant peptide applied on a C18 reversed-phase column coupled to an HPLC. B) Representative photomicrograph of the migrating cell populations in each of the experimental groups.C) Bar charts of the percentage of migration in each of the experimental groups (n=3). *p<0.05.

[0002] List of Sequences <110> Blue Scorpion Group Inc <120> PEPTIDES DERIVED FROM THE VENOM OF THE SCORPION HETEROCTENUS JUNCEUS, THEIR VARIANTS, COMPOSITIONS, METHODS AND USES <160> 4 Seq ID No.1 <210> 1 <211> 37 <212> PRT <213> HETEROCTENUS JUNCEUS <400> Thr Val Ile Asp Val Lys Cys Thr Ser Pro Lys Gln Cys Val Pro Ala Cys Lys Ala Ala Met Gly 1 5 10 15 20 Thr Val Arg Ala Lys Cys Met Asn Gly Lys Cys Lys Cys Tyr Ile 25 30 35 Seq ID No.2 <210> 2 <211> 37 <212> PRT <213> Artificial <400> Thr Val Ile Asp Val Lys Cys Thr Ser Pro Lys Gln Cys Val Pro Ala Cys Glu Ala Ala Met Gly 1 5 10 15 20 Ile 25 30 35 Seq ID No.3 <210> 3 <211> 37 <212> PRT <213> Artificial <400> Thr Val Ile Asp Val Lys Cys Thr Ser Pro Lys Gln Cys Val Pro Ala Cys Glu Ala Ala Met Gly 1 5 10 15 20 Thr Val Arg Ala Lys Cys Met Asn Gly Lys Cys Arg Cys Tyr Ile 25 30 35 Seq ID No.5 <210> 3 <211> 37 <212> PRT <213> Artificial <400> Thr Val Ile Asp Val Lys Cys Thr Ser Pro Lys Gln Cys Val Pro Ala Cys Phe Ala Ala Met Gly 1 5 10 15 20 Thr Val Arg Ala Lys Cys Met Asn Gly Lys Cys Lys Cys Tyr Ile 25 30 35 Seq ID No.6 <210> 6 <211> 37 <212> PRT <213> ARTIFICIAL <220> <223> IT CAN BE ISOLATED FROM HETEROCTENUS JUNCEUS OR ARTIFICIALLY SYNTHESIZED <220> <221> MISC_FEATURE <222> (18)..(18) <223> Xaa can be any amino acid as long as it does not disrupt the three-dimensional folding of the protein, or even better, Ala, Phe, Gln, or Asp. <220> <221> MISC_FEATURE <222> (34).(34) <223> Xaa can be any amino acid as long as it does not disrupt the three-dimensional folding of the protein, or even better, Ala, Phe, or Arg. <400> Thr Val Ile Asp Val Lys Cys Thr Ser Pro Lys Gln Cys Val Pro Ala Cys Xaa Ala Ala Met Gly 1 5 10 15 20 Thr Val Arg Ala Xaa Cys Met Asn Gly Lys Cys <210> 7 <211> 37 <212> PRT <213> ARTIFICIAL <220> <223> IT CAN BE ISOLATED FROM HETEROCTENUS JUNCEUS OR ARTIFICIALLY SYNTHESIZED <220> <221> MISC_FEATURE <222> (27)..(27) <223> Xaa can be any amino acid as long as it does not disrupt the three-dimensional folding of the protein, or even better, Gly or Ala <400> Thr Val Ile Asp Val Lys Cys Thr Ser Pro Lys Gln Cys Val Pro Ala Cys Lys Ala Ala Met Gly 1 5 10 15 20 Thr Val Arg Ala Xaa Cys Met Asn Gly Lys Cys Lys Cys Tyr Ile 25 30 35 Seq ID No.8 <210> 8 <211> 20 <212> DNA <213> Artificial <220> Starting point in the direction <221> KCNA1 <400> GATGT CTGGG GAGAA CGTGG 20 Seq ID No.9 <210> 9 <211> 19 <212> DNA <213> Artificial <220> Counterintuitive <221> KCNA1 <400> GCAGC CCGGA GATGT TGAT 19 Seq ID No.10 <210> 10 <211> 20 <212> DNA <213> Artificial <220> Partidor in the sense <221> KCNA2 <400> GCATA GCGTG AGGTG CTTCT 20 Seq ID No.11 <210> 11 <211> 19 <212> DNA <213> Artificial <220> Counterintuitive <221> KCNA2 <400> TCTGG AAACT GGGCT AAGG TC 22 Seq ID No.12 <210> 12 <211> 20 <212> DNA <213> Artificial <220> Partidor in the sense <221> KCNA3 <400> GAGGT GGCCG ATGGT GGAG 19 Seq ID No.13 <210> 13 <211> 19 <212> DNA <213> Artificial <220> Counterintuitive <221> KCNA3 <400> GCAGC CCGGA GATGT TGAT 19 Seq ID No.14 <210> 12 <211> 20 <212> DNA <213> Artificial <220> Partidor in the sense <221> KCNA4 <400> TTCCT ACACG GATCT GCTGC 20 Seq ID No.15 <210> 13 <211> 19 <212> DNA <213> Artificial <220> Counterintuitive <221> KCNA4 <400> GGGCC AGAGT TTTCA TTTGG G 21.

Claims

CLAIMS 1. An antimetastatic pharmaceutical composition, CHARACTERIZED in that it comprises at least: a. A set of synthetic peptides derived from the venom of the scorpion H. junceus, wherein at least one of them corresponds to peptide C0HJT0 (SEQ ID No. 1). b. Pharmaceutical excipients, and c. Distilled water.

2. An antimetastatic pharmaceutical composition, CHARACTERIZED in that it comprises at least: a. A synthetic peptide derived from the venom of the scorpion H. junceus, wherein the peptide is SEQ ID No.

1. b. Pharmaceutical excipients, and c. Distilled water.

3. An antimetastatic pharmaceutical composition, CHARACTERIZED in that it comprises at least: a. A set of at least two synthetic peptides derived from the venom of the scorpion H. junceus, wherein at least the first corresponds to peptide SEQ ID No. 1, and wherein the second may be selected from the list comprising: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.

7. b. pharmaceutical excipients, and c.Distilled water 4. The composition of claim 1, CHARACTERIZED in that it further comprises at least one additional peptide, which may be selected from the list comprising: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.

7.

5. An antimetastatic pharmaceutical composition, CHARACTERIZED in that it comprises: a. the peptide SEQ ID No. 1, b. a fraction enriched in the peptide SEQ ID No. 1, and c. a formulation with the isolated peptide, purified and enriched at a suitable and effective concentration; where said composition would have a therapeutic effect on any cell line or cancer that has ectopic expression, or overexpression of the hKv potassium channel v1.

1.

6. A peptide obtained by recombinant and / or synthetic methods, with the sequence of claim 2, CHARACTERIZED in that it decreases the activity of the hKv1.1 channel and inhibits migration and invasiveness in small cell lung cancer, and wherein said peptide can be selected from the list comprising: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.

7.

7. A set of variants of the peptide of claim 2, CHARACTERIZED in that said peptides correspond to the following peptides: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, with activity on the hKv1 ion channel.1, wherein each of said peptides, or combinations thereof, allows for the physical marking, identification, and / or localization of a tumor, independently, as part of surgical procedures, and / or in combination with different types of therapies, including chemotherapy, radiotherapy, and even surgery to directly remove the marked tissue, as a therapeutic or healing treatment.

8. Variants of the peptides of claim 7, CHARACTERIZED in that, given their structural similarity to peptide SEQ ID No. 1, they inhibit migration and invasiveness in small cell lung cancer.

9. Variants of the peptides of claim 7, CHARACTERIZED in that their structural analogues and / or peptidomimetics have activity on the hKv1.1 ion channel.

10. Variants of the peptides of claim 7, CHARACTERIZED in that their structural analogues and / or peptidomimetics, given their similarity to peptide SEQ ID No.

1. Inhibit migration and invasiveness in small cell lung cancer.

11. Use of the peptides of claim 2, 3, their structural analogues and / or peptidomimetics, administered in suitable excipients, CHARACTERIZED in that they are useful for the treatment of cancerous and non-cancerous pathologies associated with ectopic, atemporal expression, overexpression and / or increased functional activity of the hKv1.1 ion channel.

12. Use of the peptides of claim 2, 3, their structural analogues and / or peptidomimetics, as fusion proteins or conjugated to fluorescent compounds, antibodies, dyes, and any other compound that can be conjugated to or associated with said peptides, CHARACTERIZED in that they serve for the diagnosis, monitoring and / or treatment of cancerous and non-cancerous pathologies associated with ectopic, atemporal expression, overexpression and / or increased functional activity of the hKv1.1 ion channel. 13.A diagnostic kit for cancerous and non-cancerous pathologies associated with ectopic, timeless, overexpression and / or increased activity. functional of the hKv1.1 ion channel, CHARACTERIZED in that it comprises at least one peptide derived from the venom of the scorpion H. junceus, wherein said peptide can be selected from the list comprising: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and wherein it further comprises a set of primers that can be selected from the list comprising: Seq ID No. 8, Seq ID No. 9, Seq ID No. 10, Seq ID No. 11, Seq ID No. 12, Seq ID No. 13, Seq ID No. 14, Seq ID No.

15.

14. A diagnostic kit for determining the progression status of lung cancer, CHARACTERIZED in that it comprises: a. a. a set of KCNA channel-specific primers (1 to 4), which may be selected from the list comprising: Seq ID No. 8, Seq ID No. 9, Seq ID No. 10, Seq ID No. 11, Seq ID No. 12, Seq ID No. 13, Seq ID No. 14, Seq ID No. 15, and b. a set of synthetic and / or recombinant peptides which may be selected from the list comprising: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No.3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7; where the primer set allows the identification and amplification of gene sequences associated with the expression / overexpression specifically of said KCNA channels, and where the synthetic and / or recombinant peptides allow the validation of the expression of said channels in the tumor cells that express them, thus allowing confirmation, through said overexpression, of the stage of lung cancer, where the overexpression of said channels in said tumor cells is indicative of metastasis, allowing confirmation of said diagnosis. 15.Peptides of claim 10, CHARACTERIZED in that said peptides are diluted in suitable excipients as part of oral, parenteral, topical, nasal, aerosol or other routes of administration used in clinical practice, which inhibit migration, invasiveness and metastasis in small cell lung cancer in human subjects, and wherein said peptides may be selected from the list comprising: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.

7. 16.Peptides of claims 10 and 11, CHARACTERIZED in that said peptides are diluted in appropriate excipients as part of oral, parenteral, topical, nasal, aerosol or other formulations used in clinical practice, which inhibit migration, invasiveness and metastasis in cancerous and non-cancerous pathologies in non-human subjects (domestic and farm animals), and wherein said peptides may be selected from the list comprising: peptide SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.

7.

17. Peptides of claims 12 and 13, CHARACTERIZED in that said peptides in combination with traditional therapies, which include but are limited to the following: surgery, chemotherapy, radiotherapy, monoclonal antibody therapy, cell cycle checkpoint inhibitors, kinase inhibitors and all those drugs or products whose therapeutic indications are aimed at the curative, palliative and / or adjuvant treatment of human and non-human subjects (domestic and farm animals) with cancerous pathologies, and wherein said peptides may be selected from the list comprising: peptide SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.

7.

18. The primers of the present invention designed to selectively and exclusively identify the expression of the KCNA1 channel mRNA, CHARACTERIZED in that said primers can be selected from the list comprising: Seq ID No. 8, Seq ID No.9, and wherein, furthermore, said primers can be used in the diagnosis and / or monitoring of pathologies associated with ectopic, atemporal, or overexpression of the KCNA1 channel, as part of diagnostic kits or other related methodologies, in human and non-human subjects (domestic and farm animals).

19. A composition for the treatment of tumor cells and / or tumors in human and animal patients, CHARACTERIZED in that it comprises the peptide SEQ ID No. 1, its structural analogues and / or peptidomimetics, and any of its variants, which may be selected from the list comprising: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, as an active component or in specific formulations containing the peptide SEQ ID No. 1 bound to or associated with another cytotoxic molecule.

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