Mu-conotoxin KIIIA mutant rich in positive charges and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to a mutants of mu-conotoxin KIIIA rich in positive charges and application of the mutants. The mutant is obtained by carrying out site-directed mutagenesis on C1A, K7R and C15R on a natural mu-conotoxin KIIIA sequence; the amino acid sequence of the mutant is shown as SEQ ID NO. 1 in a sequence table. According to the invention, a series of mutants which are simple and convenient to synthesize and have a remarkable inhibition effect on a NaV1.4 channel are obtained by performing systematic structural optimization on a parent peptide KIIIA containing three pairs of disulfide bonds and adopting a strategy of combining disulfide bond deletion and amino acid site-directed mutagenesis. Furthermore, non-natural amino acid Pen is introduced into the mutant with the optimal activity, so that the stability of the mutant in vivo is remarkably enhanced, and the action time is prolonged.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a mutant of mu-conotoxin KIIIA rich in positive charges and application thereof. BACKGROUND

[0002] Voltage-gated sodium ion channels (Na V ) are a class of key transmembrane proteins that selectively mediate the movement of sodium ions (Na⁺) across the cell membrane, thereby generating and propagating action potentials in excitable cells and playing a core role in the process of nerve signal transmission and muscle excitation. This class of channels can be divided into nine subtypes (Na V 1.1 to Na V 1.9) according to the composition of the α subunit, which is encoded by SCN1A to SCN11A genes. Different subtypes show specific distribution in tissues and are involved in the regulation of various physiological activities and disease processes. The α subunit of Na V channel is composed of about 2000 amino acid residues and is a single-chain transmembrane protein, which folds into four similar but not identical repeat domains (I-IV) as a whole. Each domain contains six transmembrane segments (S1-S6), of which S1 to S4 constitute the voltage-sensing module responsible for sensing changes in membrane potential; the S5 and S6 helices and the connecting region between them together form the central pore diameter of the ion conduction channel. The S5-S6 connecting region contains an extracellular vestibule, an ion-selective filter, and P1 / P2 helices for stabilizing the structure. Compared with voltage-gated calcium channels and potassium channels, the selective filter of Na V channel presents a significant asymmetric configuration, which is composed of four amino acids (Asp, Glu, Lys, Ala) located at different positions of the four repeat domains, forming a conserved DEKA motif. This structure forms a unique charge distribution environment in space, constituting the molecular basis for the selective permeability of Na⁺, ensuring that the channel has high recognition and conduction efficiency for Na⁺ under physiological conditions, and providing an important structural basis for subsequent functional regulation and drug development of Na V channel.

[0003] Among different Na V channel subtypes, Na V 1.4 plays a key role in the excitation-contraction coupling process of skeletal muscle, and its high voltage sensitivity and rapid kinetic characteristics ensure the timing accuracy and force control of voluntary muscle contraction. After the Na V 1.4 channel is opened by nerve stimulation, Na⁺ rapidly flows into the cell, causing membrane depolarization, which in turn promotes the release of calcium ions from the sarcoplasmic reticulum, thereby initiating the muscle fiber contraction response. The SCN4AMutations in genes have been shown to cause a variety of muscle-related diseases, including myotonia, periodic paralysis, muscle weakness, and congenital myopathy. Existing studies have found that μ-conotoxin from marine cone snails is a class of toxins that can selectively block the voltage-gated sodium channel Na V 1.4 Antagonists with high selectivity and high affinity not only provide molecular probes for the structure-function relationship of sodium channels, but also provide new treatment ideas for the development of drugs for neuromuscular diseases. Among them, μ-conotoxin KIIIA containing three pairs of disulfide bonds is the smallest and most studied representative peptide with the smallest known structure, which selectively blocks the voltage-gated sodium channel Na V 1.2, Na V 1.4 and Na V 1.7 subtypes have significant inhibitory activity. Its biological effect is highly dependent on the pairing mode of disulfide bonds, and different isomers show significantly different pharmacological characteristics. Among them, KIIIA isomer 1 (Cys1-Cys5 / Cys2-Cys4 / Cys3-Cys6) shows the highest potency and selectivity (IC V = 65 nM) to Na 50 1.4 channel. Based on this, the present application selects this isomer as a structural template for molecular design, and improves its stability and pharmacokinetic properties through reasonable molecular modification and structural optimization, so as to obtain a new peptide neuromuscular blocker with improved efficacy and better bioavailability, and to provide a new technical approach for precise intervention of Na V 1.4 related diseases.

[0004] The team of the present application found the structure simplified mutant KIIIA-1 ([C1A, C15A]KIIIA) and carried out a series of reasonable designs based on the binding mode. By site-directed amino acid mutation, the analog KA ([K7R, A15R]KIIIA-1) with enhanced activity was designed and obtained, and its inhibitory activity to Na V 1.4 channel is about four times higher than that of the parent peptide. However, KA as a lead compound of muscle relaxant still has certain limitations. On the one hand, its overall biological activity has not reached the ideal efficacy level, and on the other hand, the introduction of positively charged arginine residues leads to insufficient stability in the serum environment, and the half-life is significantly shortened, thereby limiting the further drug application of the molecule in the field of neuromuscular blockade and related disease treatment. Therefore, it is urgent to optimize and modify the peptide molecule to maintain or enhance its Na V 1.4 selectivity and activity, and to improve its in vivo stability and pharmacokinetic characteristics, so as to lay a foundation for the development of new, safe and long-acting peptide muscle relaxants and medical and beauty products. SUMMARY

[0005] The application designs 11 mutants by introducing positive charges at different positions on the basis of the active peptide KA ([K7R, A15R]KIIIA-1) to enhance its binding with Na V 1.4 Channel electrostatic interaction. It is found that the inhibitory activity of the mutant [S5R]KA is about 10 times higher than that of the parent peptide. However, the [S5R]KA rich in positive charges leads to poor serum stability. Therefore, the application further mutates a single cysteine residue into the amino acid penicillamine (Pen) with a large steric hindrance side chain to obtain a candidate peptide C4Pen with high activity and excellent serum stability, thereby providing a new idea for developing new long-acting peptide muscle relaxants.

[0006] The technical scheme provided by the application is as follows: a positive charge-rich conotoxin KIIIA mutant, wherein the mutant is obtained by performing C1A, K7R and C15R site-directed mutation on a natural conotoxin KIIIA sequence; the amino acid sequence of the mutant is shown in the sequence table SEQ ID NO. 1. Preferably, the mutant is obtained by performing site-directed mutation on the mutant shown in the sequence table SEQ ID NO. 1 at any one of the following positions: A1R, N3R, S5R, S6R, W8R, D11R, H12R, S13R, S13K; or introducing a non-natural amino acid Dab at the S5 and S6 positions; the amino acid sequence of the mutant is shown in any one of the sequence tables SEQ ID NO. 2-12.

[0007] Preferably, the non-natural amino acid Dab is (S)-2, 4-diaminobutyric acid.

[0008] Preferably, the mutation is obtained by introducing a non-natural amino acid Pen at any one of the following positions of the mutant shown in the sequence table SEQ ID NO. 4: C2, C4P, C9, C16; the amino acid sequence of the mutant is shown in any one of the sequence tables SEQ ID NO. 13-16.

[0009] The application also provides a use of the mutant in the preparation of a product for treating a disease or other symptoms caused by abnormal sodium channel 1.4, a peptide muscle relaxant or a medical and cosmetic product.

[0010] Further, the disease or other symptoms caused by abnormal sodium channel 1.4 is preferably a disease or other symptoms caused by excessive excitation of sodium channel 1.4.

[0011] Further, the disease caused by abnormal excitation of sodium channel 1.4 is preferably myotonia or myotonia syndrome.

[0012] The present application also provides a preparation for inhibiting sodium channel 1.4, which contains the mutant and takes the mutant as an active ingredient, and a pharmaceutically acceptable carrier.

[0013] Further, the preparation includes tablets, capsules, capsules in capsules, micro-patch systems in capsules, lozenges, tablets, Ovules, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, pharmaceutically acceptable gums, chewable tablets, effervescent tablets or multi-particulate dosage forms.

[0014] The present application has the following beneficial effects: The present application has the following beneficial effects: V 1.4 channel has a significant inhibitory effect. Further, by introducing a non-natural amino acid Pen in the most active mutant, the stability in vivo is significantly enhanced, thereby prolonging the action time. The candidate molecule obtained by the above optimization strategy has high activity, high stability and better pharmacokinetic properties, has broad development potential, and provides a feasible guidance and technical basis for the research and development of new muscle relaxants and medical beauty wrinkle removal products. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Na V 1.4 and KA complex binding mode and surface potential map; wherein, A. binding mode; B. surface potential map; Figure 2 Na V 1.4 inhibition rate (10 nM, n≥4) of KA and its analogues in the embodiments of the present application; Figure 3 Na V 1.4 inhibition concentration effect curve (n≥4) of KA, [S5R]KA, [S6R]KA in the embodiments of the present application; Figure 4 Na V 1.4 inhibition rate (n≥4) of [S5R]KA and its analogues at different concentrations in the embodiments of the present application; Figure 5 Stability of [S5R]KA and its analogues in human serum type AB in the embodiments of the present application; Figure 6 Na V 1.4 inhibition concentration effect curve (n≥4) of [S5R]KA and its analogues in the embodiments of the present application; Figure 7The graph shows the change in forelimb grip strength of rats over time after drug administration and the area under the grip strength curve over 24 hours (*P<0.05, **P<0.01); where A. grip strength change curve; B. area under the grip strength change curve. Detailed Implementation

[0016] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0017] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0018] Example 1: Design, synthesis and in vitro activity study of KA and its analogues Given that KA is in Na V The binding pocket on 1.4 has high electronegativity. Figure 1 This invention introduces positively charged groups at different positions of KA and further designs 11 peptides to enhance their activity (Table 1). Table 1 Targeting Na V 1.4 KA series mutant numbers, names, amino acid sequences, and inhibitory activities ; Note: "*" indicates C-terminal amidation; Dab is represented by X in computer-readable sequence listings.

[0019] This invention conducted electrophysiological activity tests on a series of KA mutants designed in Table 1, and their inhibitory effects at a concentration of 10 nM are shown in Table 1 and... Figure 2 As shown. Test results indicate that the mutants [S5R]KA (SEQ ID NO.4) and [S6R]KA (SEQ ID NO.6) exhibit excellent inhibitory activity. Further Na... V 1.4 Channel half-maximal inhibitory concentration (IC50) 50 ) determination, such as Figure 3 As shown, the results show its IC 50 The values ​​were 5.1 nM and 6.1 nM, respectively, reaching single-digit nanomolar levels, compared to the parent peptide KIIIA (IC). 50 = 65 nM) activity increased by approximately 10 times. Ultimately, this invention selected [S5R]KA for further research and development.

[0020] Example 2: Synthesis of cone snail toxin KA and its analogues 1. Synthesis of KA and its mutant linear peptides This invention provides a method for synthesizing KA and its analogues, which is prepared manually on Rink amide resin via standard Fmoc solid-phase peptide synthesis (SPPS). The specific method includes: activating standard amino acid residues with HCTU in the presence of N,N-diisopropylethylamine (DIPEA); pre-swelling the resin in N,N-dimethylformamide (DMF) for 30 minutes before synthesis; coupling the first amino acid to the resin with 4 equivalents and 8 equivalents of DIPEA; subsequently assembling the peptide chain stepwise through cyclic Fmoc deprotection and amino acid coupling; after the peptide chain is assembled and the terminal Fmoc is removed, cleaving the peptide chain with trifluoroacetic acid / triisopropylsilane / water (TFA / TIPS / H2O, 90:5:5, v / v / v) for 3 hours, and obtaining the crude peptide by filtration through resin, washing with DCM, concentration, cold ether precipitation, and centrifugation. The molecular weight of the crude peptides was confirmed by liquid chromatography-mass spectrometry (LC-MS) and purified by reversed-phase high-performance liquid chromatography (RP-HPLC, C18 column). The mobile phase consisted of buffer A (water / acetonitrile / TFA, 90:10:0.05) and buffer B (acetonitrile / water / TFA, 90:10:0.05), with a linear gradient of 0–70% B applied for 70 min. The elution of the peptides was monitored by UV absorption at 214 nm and 280 nm.

[0021] 2. The disulfide bond oxidation process of KIIIA-1 and its mutants The protecting groups on the cysteine ​​residues of KA and its mutants are triphenylmethyl (Trt) and acetaminomethyl (Acm), respectively. The specific experimental steps are as follows: After dissolving the crude peptide in water, it was first oxidized dropwise with 2,2′-dipyridine disulfide (DPDS) methanol solution for 30 minutes, then stirred at room temperature for 1 hour. After confirming the completion of the reaction by mass spectrometry, it was purified by semi-preparative HPLC. Subsequently, the purified peptide was treated with iodine solution (5 mg / mL acetonitrile) until the solution turned orange, and further oxidation was completed by stirring for 2–3 hours. Finally, excess iodine was reduced with water-soluble ascorbic acid (5 mg / mL H2O) until the solution decolorized, yielding peptide molecules with the target disulfide bonds.

[0022] Example 3: In vitro activity and serum stability study of [S5R]KA analog The half-maximal inhibitory concentration (IC50) of [S5R]KA 50 The value is 5.1 nM, indicating an effect on Na. V1.4 Excellent inhibitory activity of the channel. However, this invention evaluated the half-life of [S5R]KA in human serum and found that less than 50% remained after 4 hours, indicating relatively poor enzymatic stability. Given that [S5R]KA is a polypeptide containing two pairs of disulfide bonds, this invention attempts to enhance its serum stability by replacing Cys with the amino acid Pen. [S5R]KA contains four cysteine ​​residues. To determine the optimal mutation site, this invention replaced all four cysteine ​​residues with Pen (Table 2) and evaluated the changes in activity and stability of these mutants to screen for the most favorable mutants.

[0023] Table 2 Targeting Na V 1.4 [S5R]KA series mutant numbers, names, and amino acid sequences ; Note: "*" indicates C-terminal amidation; Pen is represented by X in computer-readable sequence listings.

[0024] This invention evaluated the effect of each Pen mutant on Na V Regarding the inhibitory activity of 1.4, among the four mutants, C16Pen showed the most significant decrease in activity. This is likely because both methyl groups of the Pen residue face inwards towards the peptide chain, and the resulting steric confinement may interfere with the formation of the optimal binding conformation. In contrast, the activity decreases of C2Pen and C9Pen were relatively mild because their methyl groups are oriented differently and do not directly hinder peptide conformation. C4Pen's activity decreased only slightly because its two methyl groups face outwards towards the binding pocket, minimizing direct interference with receptor binding and maintaining peptide activity. Similarly, the outward-facing methyl groups in C4Pen significantly hinder protease steric hindrance, thus significantly improving the peptide's serum stability and extending its half-life to over 8 hours. Binding concentration-reaction curve experiments showed that while C4Pen significantly improved stability, it also significantly reduced Na+ steric hindrance. V The inhibitory activity of the 1.4 channel decreased by only about two times compared to [S5R]KA, IC 50 The value is 11.1 nM, indicating potential for further development and research in terms of in vivo activity and pharmacological applications, such as... Figure 4 , Figure 5 and Figure 6 As shown.

[0025] Example 4: Animal in vivo activity test This invention uses μ-conotoxin CnIIIC as a positive control to verify the muscle relaxant activity of the two screened high-efficiency peptides [S5R]KA and C4Pen in rat skeletal muscle. Figure 7The experimental results showed that, at a dose of 100 μg per rat, both [S5R]KA and C4Pen exhibited superior muscle relaxant effects compared to the positive control, and the KIIIA analogues showed rapid onset of action, reaching a stable maximum effect within 5 minutes. AUC analysis indicated that these compounds were significantly more potent than the positive control. Although [S5R]KA showed a stronger short-term effect, its serum stability was poor, with a significant decrease in activity 12 hours after administration; in contrast, C4Pen, through optimized serum stability, maintained its efficacy for nearly 24 hours. The prolonged duration of action of C4Pen compensated for its slightly lower activity, resulting in no significant difference in overall efficacy (AUC) between [S5R]KA and C4Pen.

[0026] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant rich in positively charged μ-conotoxin KIIIA, characterized by: The mutant was obtained by site-directed mutagenesis of the natural μ-conotoxin KIIIA sequence at C1A, K7R, and C15R; the amino acid sequence of the mutant is shown in SEQ ID NO.1 of the sequence listing.

2. A mutant rich in positively charged μ-conotoxin KIIIA, characterized by: The mutant is obtained by performing site-directed mutations at any of the following positions on the mutant shown in SEQ ID NO.1: A1R, N3R, S5R, S6R, W8R, D11R, H12R, S13R, S13K; or by introducing the non-natural amino acid Dab at the S5 and S6 positions; the amino acid sequence of the mutant is shown in any one of SEQ ID NO.2-12.

3. The mutant according to claim 2, characterized in that: The non-natural amino acid Dab mentioned is (S)-2,4-diaminobutyric acid.

4. A mutant rich in positively charged μ-conotoxin KIIIA, characterized by: The mutation was obtained by introducing the non-natural amino acid Pen at any of the following positions in the mutant shown in SEQ ID NO.4: C2, C4, C9, C16; the amino acid sequence of the mutant is shown in any one of SEQ ID NO.13-16.

5. The use of the mutant according to any one of claims 1-4 in the preparation of products for treating diseases or other symptoms caused by sodium channel 1.4 abnormalities, peptide muscle relaxants, or medical aesthetic products.

6. The application according to claim 5, characterized in that: The diseases or other symptoms caused by sodium channel 1.4 abnormalities refer to diseases or other symptoms caused by excessive excitation of sodium channel 1.

4.

7. The application according to claim 6, characterized in that: The disease or other symptoms caused by overexcitation of sodium channel 1.4 are myotonia or myotonia syndrome.

8. A formulation for inhibiting sodium channel 1.4, characterized in that: The formulation contains the mutant as described in any one of claims 1-4, and uses the mutant as the active ingredient, as well as a pharmaceutically acceptable carrier.

9. The formulation according to claim 8, characterized in that: The formulations include tablets, capsules, capsule-in-capsule formulations, micropatch systems in capsules, lozenges, tablets, octopuses, solutions, emulsions, suspensions, syrups, slurries, powders and granules for recombination, dispersible powders and granules, pharmaceutically acceptable gums, chewable tablets, effervescent tablets, or multigranule dosage forms.

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