Hainan needle scorpion active polypeptide and application thereof

Abstract

The invention provides a Hainan needle scorpion active polypeptide and application thereof. The Hainan double-needle scorpion active polypeptide is derived from Hainan double-needle scorpion toxin protein, and the amino acid sequence of the Hainan double-needle scorpion active polypeptide is shown as SEQ ID NO.1. The full length of the polypeptide toxin is 38 amino acid residues, the polypeptide toxin comprises 6 cysteines, the whole polypeptide is slightly alkaline, the isoelectric point is 11.87, the hydrophilic amino acid residues account for more than 50%, and the theoretical molecular weight is 4200.78 Da. The difference between the actual molecular weight and the theoretical molecular weight is 7Da, which shows that six cysteines in the polypeptide form three pairs of disulfide bonds. The Hainan needle scorpion active polypeptide disclosed by the invention has the activity of selectively inhibiting a voltage-gated potassium ion channel Kv1.3, so that the Hainan needle scorpion active polypeptide can be applied to preparation of polypeptide medicines for treating autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide technology, and specifically relates to an active polypeptide of Hainan double-needle scorpion and its application. Background Technology

[0002] Kv1.3 is a voltage-gated potassium channel that plays a crucial role in cell membrane excitability and electrical signal transduction. Kv1.3 channels are widely expressed in the immune system, nervous system, and vascular smooth muscle cells, but their expression is particularly pronounced in immune cells, especially effector memory T lymphocytes. In recent years, with in-depth research into the structure and functional mechanisms of Kv1.3 channels, scientists have discovered that this channel is closely related to the occurrence and development of various diseases. For example, it is associated with autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, and systemic lupus erythematosus. Furthermore, Kv1.3 inhibitors have been shown to be effective in treating type 1 diabetes, autoimmune hepatitis, and psoriasis. Surprisingly, these drugs have also demonstrated good safety in rodents and primates.

[0003] Therefore, the Kv1.3 channel has become an important target for studying T cell function and developing drugs to treat autoimmune and inflammatory diseases, and developing drugs that specifically inhibit the Kv1.3 channel has become a new strategy for treating these diseases.

[0004] The most representative Kv1.3 inhibitor currently is ShK, which is derived from sea anemones ( Stichodactyla helianthus The polypeptide isolated from the venom. In addition to Kv1.3, ShK also isolated Kv1.1 (IC). 50 =6.7-87 pM), Kv1.4 (IC 50 =0.31 nM), Kv1.6 (IC 50 =0.16 nM), Kv1.2 (IC 50 =9 nM), Kv1.7 (IC) 50 Both Kv1.3 (Kd = 11.5 nM) and KCa3.1 (Kd = 0.03-30 nM) exhibit inhibitory activity. The modified ShK toxin derivative ShK-186 shows a 100-fold increase in selectivity for Kv1.3 compared to Kv1.1, and a 1000-fold increase in selectivity for Kv1.4 and Kv1.6. Among the many developed Kv1.3 inhibitors, ShK-186 is currently the only peptide drug in clinical trials for the treatment of autoimmune diseases.

[0005] It should be noted that although from the perspective of basic research, there are quite a few toxin peptides that have high affinity for Kv1.3, these toxins also show similar activities to other members of the Kv1 family besides Kv1.3. The lack of selectivity is a major challenge in the development of Kv1.3 inhibitors. At present, there is still a lack of selective Kv1.3 inhibitors of peptides. Summary of the Invention

[0006] This invention screened a highly selective and potent toxin peptide inhibitor against the Kv1.3 channel from the abundant venom of the Hainan double-needle scorpion, named LmKTx13, with the amino acid sequence: QKFTDIKCSSSSSCYEPCRGVTGRAHGKCMNGKCTCYY (SEQ IN NO.1). This toxin peptide is 38 amino acid residues in length, including 6 cysteine ​​residues. The peptide is predominantly basic, with an isoelectric point of 11.87, and hydrophilic amino acid residues account for more than 50%. Its theoretical molecular weight is 4200.78 Da. The actual molecular weight differs from the theoretical molecular weight by 7 Da, indicating that the 6 cysteine ​​residues in the peptide form 3 disulfide bonds.

[0007] BLAST analysis showed that the cysteine ​​arrangement pattern of LmKTx13 is similar to that of many scorpion venoms with inhibitory cystine motifs, suggesting that the secondary structure of LmKTx13 also contains an inhibitory cystine motif, with cysteine ​​linkages of C1-C4 / C2-C5 / C3-C6 (which, according to the position of cysteine ​​in the amino acid sequence, are 8-29 / 14-34 and 18-36).

[0008] Patch-clamp assays revealed that LmKTx13 inhibited the half-maximal inhibitory concentration (IC50) of Kv1.3. 50 The concentration was 7.92 ± 3.0 nmol / L. In selectivity analysis, 2 µM LmKTx13 was found to also exhibit inhibitory activity against Kv1.2 and Kv1.7, with an IC50 concentration of 7.92 ± 3.0 nmol / L. 50 The values ​​were 21.8±1.2 nM and 97.5±28.2 nM, respectively, but they did not show any inhibitory effect on potassium channel subtypes Kv1.1, Kv1.4, Kv1.5, Kv2.1, Kv3.1, Kv3.2, Kv3.4, Kv4.2 and Kv4.3.

[0009] In summary, this invention screened a novel polypeptide toxin, LmKTx13, from the venom of the Hainan double-needle scorpion, exhibiting strong inhibitory activity against the Kv1.3 channel. It shows selectivity within the Kv1 subtype but has no effect on other potassium channel subtypes or sodium channels. Further mechanistic studies revealed that LmKTx13 inhibits channel currents by binding to the pore region of Kv1.3, elucidating its mechanism of action. This provides a lead molecule for the development of drugs targeting Kv1.3 and lays the foundation for a deeper understanding of the Kv1.3 channel and the mechanism of action of its inhibitors. Attached Figure Description

[0010] Figure 1 The following are chromatograms showing the separation, purification, and identification of LmKTx13. (A) C18 reversed-phase high-performance liquid chromatography (HPLC) chromatogram of ~3 mg of crude venom collected from *Scorpion hainanensis* by electrical stimulation. The red arrows indicate the target elution peaks containing LmKTx13. (B) The molecular weight of LmKTx13 was determined using high-resolution mass spectrometry (ESI-Obitrap). The molecular weight of the peptide was obtained as 4193.7 Da after deconvolution of the original primary mass spectrum. (C) Sequence alignment of LmKTx13 with several toxin peptides.

[0011] Figure 2 The following graphs show the effects of LmKTx13 on various potassium channel subtypes. (A) Representative current trajectory of the inhibitory effect of 20 nM LmKTx13 on Kv1.3 channels. (B) Concentration-effect curve of LmKTx13 on Kv1.3, IC50. 50 The value was 7.92 ± 3.0 nM. Data are mean ± SEM, n = 4–6. (C) Representative current trajectories of potassium channel subtypes before (black) and after (red) treatment with 2 µM LmKTx13. (D) Concentration-effect curves of LmKTx13 peptide on Kv1.2 (red) and Kv1.7 (blue) channels detected by whole-cell patch-clamp assay. Data are mean ± SEM, with n = 4–6 cells per point. Figure 3 The effect of LmKTx13 on various sodium channel subtypes is shown. Representative current trajectories for each sodium channel subtype before (black) and after (red) treatment with 2 µM LmKTx13 are displayed. LmKTx13 has no effect on most sodium channel subtypes. Data n=3-4.

[0012] Figure 4The effect of LmKTx13 on the Kv1.3 channel dynamics. (A) Representative current trajectories before treatment (black), after treatment (red), and after elution (blue). (B) Combined elution curves of LmKTx13 before treatment (black), after treatment (red), and after elution (blue), with all currents normalized to the maximum stable current before poisoning. Data are mean ± SEM, n = 6-10. (C) Kv1.3 current trajectories before (black) and after (red) treatment with 20 nM LmKTx13 under activation test voltage. (D) IV curves obtained by normalizing the Kv1.3 channel currents before (black) and after (red) treatment with 20 nM LmKTx13 to their own maximum current values. (E) Steady-state inactivation curves of Kv1.3 channels before (black) and after (red) treatment with 20 nM LmKTx13 at the inactivation test voltage. Data are mean ± SEM, n = 6-10. (F) Steady-state activation curves of Kv1.3 channels before (black) and after (red) treatment with 20 nM LmKTx13 at the activation test voltage. Data are mean ± SEM, with n = 6-10 cells per point.

[0013] Figure 5 The pore structure domain of Kv1.3 participates in the interaction with LmKTx13. (A) Sequence alignment results between channel fragments Kv1.2, Kv1.7, Kv1.3, and Kv1.5. The black arrows indicate the amino acid positions 423, 424, 425, 426, 453, 453, and 455 of Kv1.3 from left to right. (B) Representative current trajectory of the interaction between Kv1.5 / 1.3 8 Muts and LmKTx13. Compared with Kv1.5, this mutant acquired new activities. (C) Concentration-effect curves of Kv1.5 / 1.3 8 Muts and LmKTx13. Data are mean ± SEM, n = 4-6. (D) Representative current plot of the interaction between Kv1.3 / 1.5 8 Muts and LmKTx13. (E) Representative current trajectories of D423N, P424Q, T425G, S426T, G427H, H451R, V453I, and I455V before (black) and after (red) LmKTx13 treatment. (F) Concentration-effect curves of LmKTx13 on mutants in each channel of Kv1.3 (red), D423N (blue), P424Q (orange), T425G (green), S426T (purple), V453I (light orange), and I455V (brown). Data are mean ± SEM, and the number of cells corresponding to each point is n = 4-6. Detailed Implementation

[0014] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0015] 1. Materials and Methods 1.1 Experimental Materials HEK293T and CHO-K1 cells were purchased from the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Ion channel plasmids and mutants used in the experiments were all from previous research and development, with some mutants constructed and validated by sequencing later. The molecular weight of the peptide toxins was determined by electrospray ionization (ESI-MS). Analytical high-performance liquid chromatography (Waters 1525, USA) was purchased from Waters Technologies (Shanghai) Co., Ltd. EPC-10USB patch clamps were purchased from HEKA GmbH, Germany. C18 reversed-phase columns and strong cation exchange columns were purchased from Yuexu (Shanghai) Technology Co., Ltd. Ampicillin, kanamycin, and BCA kits were purchased from Shanghai Sangon Biotech Co., Ltd. DMEM high-glucose medium (Opti-MEM), penicillin-streptomycin, and lipofectamine 2000 were purchased from Thermo Fisher Scientific. Primer synthesis, plasmid sequencing services were provided by Hangzhou Youkang Biotechnology Co., Ltd. (Changsha).

[0016] 1.2 Scorpion breeding and scorpion venom collection Hainan double-needle scorpions were collected from Hainan, China. They were fed mealworms 1-2 times per week, with suitable humidity, temperature, and sufficient nutrition. Toxins were extracted using an electrostimulation method. The scorpion's tail segment (second to last) was held with tweezers, and its stinger was secured to a clean "V"-shaped plastic sheet. A pulsed current was then applied to this area to induce venom secretion onto the plastic sheet. The venom was collected in a single container, centrifuged, and then frozen at -80°C for later use.

[0017] 1.3 Separation and purification of crude toxins The crude toxin was diluted with ultrapure water to a concentration of 10 mg / mL, centrifuged at 14000 pm for 30 min, and the supernatant was collected and filtered through a 0.22 μm sterile filter. The supernatant was then separated and purified using a C18 reversed-phase column and preparative high-performance liquid chromatography (HPLC). After loading the sample, the C18 reversed-phase column was first washed with 100% acetonitrile / 0.1% TFA for 10 min, and then eluted by gradually increasing the acetonitrile concentration from 15% to 45% at a flow rate of 1 mL / min over 30 min. Each elution peak was collected and frozen at -80℃, and then dried using a vacuum dryer for later use.

[0018] 1.4 Isolation and purification of LmKTx13 The target elution peaks were further purified using a C18 cation exchange column and reversed-phase high-performance liquid chromatography (Hanbon) for subsequent experiments. Before use, the ion exchange column was pre-filled with 10% methanol solution, then replaced with ultrapure water, and finally equilibrated with 100% acetonitrile. In the ion exchange chromatography separation, phase C consisted of 1 mol / L NaCl / 0.05 mol / L NaH₂PO₄ (pH 6.2, filtered and vented), and phase D consisted of 0.05 mol / L NaH₂PO₄ (pH 6.2, filtered and vented), with gradient elution performed at a flow rate of 1 mL / min. The elution peaks separated by ion exchange chromatography contained a large amount of inorganic salts; therefore, further desalting was performed using a reversed-phase column. The acetonitrile concentration was gradually increased from 5% to 28% at a flow rate of 1 mL / min over 23 min. Finally, each elution peak was collected, frozen at -80℃, and then lyophilized in a vacuum dryer for later use.

[0019] 1.5 Mass spectrometry identification and purity analysis of the target peak The molecular weight of the target peak eluted after separation and purification was identified using ESI mass spectrometry. The purity of the target sample was then analyzed based on the mass spectrometry results.

[0020] 1.6 Cell Culture and Transfection The cell culture incubator was set to 37°C with a carbon dioxide concentration of 5%, and sterile water was added periodically as needed to maintain humidity. The complete culture medium for HEK293T and CHO-K1 cells was prepared by adding 10% fetal bovine serum, 100 pg / mL streptomycin, 100 U / mL penicillin, and 2 mmol / L L-glutamine to DMEM / DMEM-F12 basal medium, and mixing thoroughly. When the cell density reached 70-80%, ion channel plasmids and green fluorescent protein (eGFP) plasmids were co-transfected using Lipofectamine 2000, following the instructions in the product manual.

[0021] 1.7 Electrophysiological Activity Detection Cells exhibiting strong green fluorescence were selected for electrophysiological recording 24 to 36 hours post-transfection. Whole-cell patch-clamp experiments were performed using an EPC-10USB patch-clamp apparatus at room temperature (25 ± 2 °C). Electrophysiological data were acquired using Patch Master software. Among the ion channels recorded by patch-clamp in this study, Kv1.1-1.3, Kv1.5, Kv1.7, hREG, Navl.5, and av1.7 were human; Kv1.4, Kv2.1, Kv3.1-3.2, Kv3.4, Kv4.2, Kv4.3, Navl.2-1.4, and Nav1.8 were rat; and Nav1.6 was mouse.

[0022] 1.8 Statistical Methods Electrophysiological data were analyzed and processed using Igor Pro 6.10A and Patch Master v2x73, and plotted and statistically analyzed using GraphPad Piism 9.5. Data for each point in the electrophysiological experiments were derived from 4–6 experimental cells, and are expressed as mean ± standard error (mean ± SEM).

[0023] 2 Results 2.1 Isolation and purification of LmKTx13 peptide Hainan double-needle scorpion ( Lychas mucronatus Scorpion venom is mainly distributed in Hainan and Guangxi, China, and belongs to the order Scorpionidae, family Scorpionidae, class Arachnidae, phylum Arthropoda. The collected scorpion venom was freeze-dried and then purified multiple times using reversed-phase high-performance liquid chromatography (RP-HPLC), ion-exchange chromatography, and RPI desalting to obtain a high-purity sample. Figure 1 A). Its molecular weight was identified as 4193.7 Da by ESI high-resolution mass spectrometry. Figure 1 B). The peptide was subjected to DTT reduction, alkylation modification, trypsin digestion, and chromatographic separation again. MS / MS TOF secondary mass spectrometry with Mascot library search and scoring, and Denovo sequencing, combined with transcriptome data from the Hainan double-needle scorpion venom, determined its amino acid sequence as: QKFTDIKCSSSSSCYEPCRGVTGRAHGKCMNGKCTCYY (SEQ ID NO. 1). Figure 1 C), named LmKTx13. It has a total length of 38 amino acid residues, including 6 cysteine ​​residues. The polypeptide is generally basic, with an isoelectric point of 11.87. Hydrophilic amino acid residues account for more than 50%, and its theoretical molecular weight is 4200.78 Da. The actual molecular weight differs from the theoretical molecular weight by 7 Da, indicating that the 6 cysteine ​​residues in the polypeptide form 3 pairs of disulfide bonds. BLAST analysis shows that the cysteine ​​arrangement of LmKTx13 is similar to that of many scorpion venoms that inhibit cystine binding motifs. Figure 1 C) It is speculated that the secondary structure of LmKTx13 also contains an inhibitory cysteine ​​knot motif, with the cysteine ​​linkage being C1-C4 / C2-C5 / C3-C6 (according to the position of cysteine ​​in the amino acid sequence, the linkage is 8-29 / 14-34 / 18-36).

[0024] 2.2 Effects of LmKTx13 on potassium channels Patch-clamp analyses were performed in a quiet indoor environment at approximately 25°C. The clamping voltage was set to -90mV, and a single stimulation with a voltage pulse of +30mV lasting 300ms was applied. Further analysis of the activity of LmKTx13 against Kv1.3 yielded the results shown... Figure 2As shown in Figure A, 20 nmol / L LmKTx13 inhibits approximately 60% of the Kv1.3 current. The inhibition of the Kv1.3 current by LmKTx13 is concentration-dependent. The half-maximal inhibitory concentration (IC50) of LmKTx13 for inhibiting Kv1.3 was obtained by fitting the Hill equation. 50 It was 7.9 ± 3.0 nmol / L. Figure 2 B).

[0025] Considering the conservation of structure and sequence among different potassium ion channels, the activity of LmKTx13 in other potassium channel subtypes was further analyzed. Figure 2 C). At a dosage concentration of 2 μmol / L, LmKTx13 did not show any inhibitory effect on potassium channel subtypes Kv1.1, Kv1.4, Kv1.5, Kv2.1, Kv3.1, Kv3.2, Kv3.4, Kv4.2, and Kv4.3. However, at the same concentration, LmKTx13 inhibited the peak currents of the Kv1.2 and Kv1.7 channel subtypes, with an IC50 value of [missing value]. 50 The values ​​were 21.8 ± 1.2 nmol / L and 97.5 ± 28.2 nmol / L, respectively. Figure 2 D). However, compared to Kv1.3, LmKTx13 showed a more significant inhibitory effect, being 2.7 times and 12.3 times that of Kv1.2 and Kv1.7, respectively. hERG channels expressed on the myocardium are an important indicator for evaluating the safety of new drugs; therefore, this invention also investigated the effect of LmKTx13 on hERG channels. Cells were clamped at -80 mV, depolarized to +30 mV for 2 seconds, and then repolarized to -40 mV for 2 seconds. The results showed that 2 μmol / L LmKTx13 also had no significant inhibitory effect on hERG channels (…). Figure 2 C) suggests that LmKTx13 may have certain cardiovascular safety. The above results indicate that LmKTx13 exhibits some selectivity in inhibiting Kv1.3.

[0026] 2.3 Effects of LmKTx13 on different sodium channel subtypes Voltage-gated sodium channels are also a major target of scorpion venom. Previously reported classical scorpion venoms that regulate sodium channels can be divided into α-scorpion venom and β-scorpion venom. Therefore, this invention will further investigate whether LmKTx13 affects sodium channel currents. The cell clamping voltage was set to -100 mV, and a 0 mV stimulation voltage was applied for 50 ms, during which the channel current was recorded. The results are as follows: Figure 3As shown, 2 μmol / L LmKTx13 did not exhibit inhibitory activity on the current of voltage-gated sodium channel subtypes including Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7 and Nav1.8, indicating that it does not have sodium channel activity.

[0027] 2.4 Effect of LmKTx13 on the dynamics of Kv1.3 Figure 4 AB binding and elution curves show that LmKTx13 binds rapidly to the channel, with the current being completely suppressed within one sweep (5 s). Furthermore, the binding is eluted by the external solution, and the current magnitude essentially returns to pre-toxicity levels, with an elution time constant of approximately 30 s. These results indicate that LmKTx13 has a strong and rapid inhibitory effect on the Kv1.3 channel, but the binding is reversible.

[0028] Next, the effect of LmKTx13 on channel activation kinetics was investigated. Cells were clamped at -90 mV, with a step size of +10 mV, and the test voltage was gradually increased from -80 mV to +80 mV. The results showed that at the stimulation voltage, 20 nmol / L LmKTx13 significantly inhibited the Kv1.3 current. Channels in both the control and drug-treated groups began to open at -40 mV stimulation, while the current in the drug-treated group was inhibited by approximately 60% after toxication. Figure 4 CD). Using the conductivity formula G=I / (VV) rev Calculate the conductance G under various conditional voltages, where I represents the peak current of the whole cell under different pulse voltages, and V represents the depolarization pulse voltage. rev This represents the channel's reversal potential. Based on the ion concentrations in the cell bath and electrode solution, the potassium channel's reversal potential is calculated to be -100.5 mV. The calculated conductance at each test voltage is then normalized, i.e., G / G max Then, the Boltzmann equation was used for fitting to obtain the steady-state activation curves of the Kv1.3 channel before and after poisoning. Figure 4 E). Half-maximum activation voltage V of the control group 1 / 2 The activation V of the 20 nmol / L LmKTx13 treatment group was -22.1 ± 2.2 mV. 1 / 2 The V values ​​for both groups are -22.3 ± 2.0 mV. 1 / 2No statistically significant difference was observed. The results indicate that LmKTx13 does not affect the steady-state activation curve of the Kv1.3 channel. To obtain steady-state inactivation curves before and after toxin action on the Kv1.3 channel, cells were clamped at -90 mV and subjected to a 1000 ms conditional pulse with a step size of +10 mV, ranging from -80 mV to 40 mV, to induce channel inactivation. This was followed by a 300 ms test pulse at +60 mV, with an interval of 5 s. The channel tail current under each test pulse was normalized (I / Ig). max The steady-state inactivation curves of the Kv1.3 channel before and after toxication were obtained by fitting the stimulation voltage using the Boltzmann equation. The results show that the half-maximum inactivation voltage V in the control group... 1 / 2 The V value for the 20 nmol / L LmKTx13 treatment group was -33.7 ± 3.5 mV. 1 / 2 It changed to -37.1±4.9mV, but the V values ​​of the two groups... 1 / 2 No statistically significant difference was observed. Figure 4 F). It can be determined that the toxin has no effect on the inactivation mechanics of the Kv1.3 channel. In summary, the steady-state activation and inactivation curves of the channel before and after LmKTx13 treatment showed no significant changes. It is speculated that LmKTx13 may not inhibit the Kv1.3 current by regulating the voltage-sensitive region of the channel, but rather by hindering ion outflow in a manner similar to "blocking the pores," thereby exerting an inhibitory effect.

[0029] The 2.5 Kv1.3 channel region is the key area for the action of LmKTx13. The above studies indicate that LmKTx13 exhibits high selectivity for Kv1.3. Furthermore, analysis of the toxin's effects on channel activation and inactivation mechanics revealed that the toxin does not affect the channel activation / inactivation curves; therefore, it may be a type of toxin that binds to the pore region. To identify key sites of interaction, the study performed sequence alignment between the S5-S6 regions of the pore regions of Kv1.2 and Kv1.7 (which interact with LmKTx13) and Kv1.5 (which is insensitive to LmKTx13), and the corresponding regions of Kv1.3. Figure 5A). Literature review also revealed that most reported Kv1.3 channels are blocking toxins, with key sites mostly located in the S5-S6 junction region of Kv1.3. Therefore, based on a comprehensive analysis of literature and sequence alignment data, it is inferred that key sites for toxin binding and selectivity exist at D423, P424, T425, S426, G427, H451, V453, and I455 in the Kv1.3 channel. Firstly, previous studies have confirmed that 2 μmol / L LmKTx13 did not inhibit the Kv1.5 channel. To preliminarily verify whether these eight sites contain key amino acids with toxic effects, the genes encoding amino acids D423, P424, T425, S426, G427, H451, V453, and I455 in the Kv1.3 channel were all mutated to their corresponding amino acid encoding genes on Ky1.5. Conversely, the corresponding sites on Kv1.5 were mutated to sites on Kv1.3, resulting in two local region substitutions of 8-mutant chimeras: Kv1.3 / Kv1.58 Muts and Kv1.5 / Kv1.38 Muts. Figure 5 A), followed by patch-clamp verification. Based on the current trajectory diagrams before and after toxication, 200 nmol / L LmKTxl3 can suppress 80-90% of the current in the Kv1.5 / Kv1.3 8Muts channel (…). Figure 5 B), and the concentration-effect curve was calculated to obtain the IC50 of LmKTx13 inhibiting Kv1.5 / Kv1.3 8Muts. 50 The value is 60.8 + 18.3 nmol / L ( Figure 5 C) IC with LmKTx13 to suppress Kv1.3 50 The values ​​were close, differing by approximately 7 times, indicating that the substitution in this region endowed Kv1.5 with the inhibitory activity of the toxin on the channel. However, in the Kv1.3 / Kv1.5 8Muts mutant, this study found that even administration of 20 μmol / L LmKTx13 peptide did not alter the channel current. Figure 5 D) indicates that the mutation of this amino acid at this site on Kv1.3 leads to the loss of LmKTx13 activity. In summary, these results suggest that the S5-S6 region of the Kv1.3 pore structure is a key area for LmKTx13 function.

[0030] Site-directed mutagenesis was constructed targeting the eight mutation regions mentioned above, and patch-clamp analysis was performed. Based on the inhibition-concentration effect curves, the IC50 values ​​of LmKTx13 against the D423N, P4240Q, T425G, S426T, V453I, and I455V mutants were found to be [missing information]. 50The values ​​were 26.1±1.8 nmol / L, 40.3±10.9 nmol / L, 83.7±14.8 nmol / L, 21.2±5.6 nmol / L, 21.1±2.3 nmol / L, and 16.2±2.2 nmol / L, respectively. Simultaneously, analysis based on current trajectory and inhibition rate calculations showed that the current in the G427H and H451R mutant channels was not inhibited at a concentration of 2 μmol / L LmKTx13. Based on these results, this study found that the three channel mutants (T45G, G427H, and H451R) significantly reduced the inhibitory activity of 1 mKTx13 on current, with inhibitory activities weakened by 10.6-fold, 100-fold, and 1000-fold, respectively, compared to the wild-type Kv1.3. This indicates that threonine at position 425, glycine at position 427, and histidine at position 451 on the Kv1.3 channel are key sites for the inhibitory activity of LmKTx13 and are closely related to the selectivity of the toxin. In particular, the mutation at position 451 histidine has the most significant impact on the activity of LmKTx13. The corresponding amino acid for this site on the Kv1.5 channel is arginine.

[0031] 3 Discussion This invention isolates and purifies a novel polypeptide toxin, LmKTX13, from the venom of the Hainan double-needle scorpion. LmKTX13 exhibits significant inhibitory effects on the Kv1.3 channel current. 50 The concentration was 7.9 ± 3.0 nmol / L, and it exhibited some selectivity. Molecular mechanism studies revealed that LmKTx13 did not significantly alter the steady-state activation and inactivation curves of the channel. Figure 4 E and 4F) indicate that it does not affect the activation and inactivation mechanics of the Kv1.3 channel; mutant analysis and patch-clamp detection revealed T425G, G427H and H451R mutations on the S5-S6 Linker in the Kv1.3 pore region, which led to a 10.6-fold, at least 100-fold and more than 1000-fold reduction in the activity of the toxin to the channel, respectively. This demonstrates that T425, G427 and H451 are key sites for LmKTx13 to inhibit channel current. These results indicate that the pore region of Kv1.3 is a key binding region for the toxin.

[0032] Currently, many structurally different peptide toxins, such as ChTk, ShK, and HSTx1, have been reported as inhibitors of the Kv1.3 channel. These inhibitors can inhibit potassium currents at nanomolar or picomolar concentrations, but they all suffer from severe selectivity problems. Therefore, modification based on natural peptides is necessary and significant. The modification of peptide toxin inhibitors is a complex and delicate process aimed at improving the inhibitor's activity, specificity, and stability, making it more suitable as a drug candidate for treating related diseases. Amino acid substitution, structural modification, chemical modification, and molecular docking are all methods that can be used to optimize the performance of Kv1.3 peptide inhibitors to make them closer to the requirements of practical applications. Some peptide toxins have already been modified to obtain better properties. For example, ShK-L5, derived from the sea anemone toxin ShK, although showing a slight decrease in inhibitory activity against Kv1.3, has nearly 260-fold improved selectivity for Kv1.1. ShK-186 is also a modified version of the sea anemone venom ShK. Compared to the original peptide, ShK-186 exhibits significantly enhanced activity and selectivity in inhibiting Kv1.3, and it is currently the only toxin-derived peptide in clinical trials targeting Kv1.3 for the treatment of autoimmune diseases. A series of peptide analogs derived from scorpion venom OSK1 have improved their selectivity for Kv1.3 by progressively truncating the N- and C-terminal sequences. Based on studies of the interaction between scorpion venom BmKTX and channels, the designed and modified ADWX-1 was obtained, which can block Kv1.3 at picomolar concentrations (IC50, 1000 mg / L). 50 =1.9 pmol / L), for Kv1.1 (IC 50 =0.65 nmol / L) and Kv1.2 (IC 50The selectivity of channels with concentrations >100 nmol / L was also significantly improved. This indicates that combining peptide modification and computer molecular simulation with the screening of natural inhibitors is currently an effective strategy for developing selective inhibitors. In this study, the peptide toxin LmKTx13, as a newly discovered selective inhibitor of Kv1.3, showed no activity against any of the 17 tested channels. Although LmKTx13 blocked the currents of Kv1.2 and Kv1.7 channels at nanomolar concentrations, its activity was 2.7 times and 12.3 times weaker than that of Kv1.3, respectively. This characteristic of LmKTx13 has a significant advantage for developing selective inhibitors. In the future, further optimization of peptide performance based on this, such as enhancing activity and selectivity through mutation and modification, may lead to the development of new selective inhibitor drugs for Kv1.3. Although there have been many research results on the development of Kv1.3 inhibitors, only one inhibitor, ShK-186, has entered the clinical research stage. This shows that from a pharmacological perspective and from the perspective of actual clinical drug needs, the search for new Kv1.3 inhibitors still has important practical significance. In summary, highly specific and high-affinity peptide toxins provide important tool molecules for studying the structure and function of ion channels. Investigating the molecular mechanism of the interaction between LmKTx13 and Kv1.3 channels not only lays the foundation for a deeper understanding of the mechanisms of action of Kv1.3 channels and their inhibitors, but also suggests that the strong hydrophilicity and high specificity of LmKTx13 may provide lead molecules for drug development in the treatment of autoimmune diseases such as multiple sclerosis and psoriasis.

Claims

1. A polypeptide, characterized in that, A toxin protein derived from Buthus martensii Karsch, and the amino acid sequence is shown as SEQ ID NO.

1.

2. A polypeptide, characterized in that, An amino acid sequence obtained by substitution, deletion or addition of one or more amino acid residues based on the amino acid sequence shown as SEQ ID NO. 1, and having the activity of selectively inhibiting voltage-gated potassium ion channel Kv1.

3.

3. Use of the polypeptide according to claim 1 or 2 in the preparation of an inhibitor of voltage-gated potassium ion channel Kv1.

3.

4. Use of the polypeptide according to claim 1 or 2 in the preparation of a polypeptide drug for treating autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, type I diabetes, autoimmune hepatitis, psoriasis.

5. A medicament for treating an autoimmune disease, characterized by, A polypeptide drug for treating autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, type I diabetes, autoimmune hepatitis, psoriasis, comprising the polypeptide according to claim 1 or 2.

6. A voltage-gated potassium ion channel Kv1.3 inhibitor, characterized in that, A polypeptide drug for treating autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, type I diabetes, autoimmune hepatitis, psoriasis, comprising the polypeptide according to claim 1 or 2.

7. A method of selectively inhibiting voltage-gated potassium ion channel Kv1.3, characterized by, A polypeptide drug for treating autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, type I diabetes, autoimmune hepatitis, psoriasis, comprising the polypeptide according to claim 1 or 2. A method for treating autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, type I diabetes, autoimmune hepatitis, psoriasis, comprising administering the polypeptide according to claim 1 or 2 to a subject.

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