Mutants of the positively charged mu-conotoxin kiiia and uses thereof

Abstract

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. The mutant is obtained by performing C1A, K7R and C15R site-directed mutation on a natural mu-conotoxin KIIIA sequence; and the amino acid sequence of the mutant is shown in the sequence table SEQ ID NO. 1. The application performs systematic structural optimization on a parent peptide KIIIA containing three pairs of disulfide bonds, adopts a strategy of combining disulfide bond deletion with site-directed amino acid mutation, and obtains a synthetic simple mu-conotoxin KIIIA mutant which has a significant inhibitory effect on Na V 1.4 A series of mutants with significant inhibitory effect on the channel. Further by introducing non-natural amino acid Pen in the most active mutant, the stability in vivo is significantly enhanced, thereby prolonging the action time.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a mutant of positively charged μ-conotoxin KIIIA and its applications. Background Technology

[0002] Voltage-gated sodium ion channels (Na V Sodium ions (Na⁺) are a class of key transmembrane proteins that selectively mediate the crossing of sodium ions (Na⁺) across the cell membrane, thereby generating and propagating action potentials in excitable cells and playing a central role in nerve signal transduction and muscle excitation. These channels can be classified into nine subtypes based on the composition of their α subunits (Na⁺). V 1.1 to Na V 1.9), respectively by SCN1A to SCN11A Gene encoding. Different subtypes exhibit specific distributions in tissues and participate in the regulation of various physiological activities and disease processes. Na V The α subunit of the channel consists of approximately 2000 amino acid residues and is a single-chain transmembrane protein. It folds together to form four similar but not identical repeating domains (I–IV), each containing six transmembrane segments (S1–S6). S1 to S4 constitute the voltage-sensing module, responsible for sensing changes in membrane potential. The S5 and S6 helices and their connecting region together form the central pore of the ion conduction channel. The S5–S6 connecting region contains an extracellular vestibule, an ion-selective filter, and the P1 / P2 helix used to stabilize this structure. Compared to voltage-gated calcium and potassium channels, Na+... V The selective filter of the channel exhibits a significant asymmetric configuration, composed of four amino acids (Asp, Glu, Lys, and Ala) located at different positions in four repeating domains, forming a conserved DEKA motif. This structure creates a unique charge distribution environment in space, constituting the molecular basis for sodium ion selective permeability. This ensures that the channel exhibits high recognition and conduction efficiency for Na⁺ under physiological conditions, facilitating subsequent Na⁺ ion permeability. V This provides important structural evidence for the regulation of channel function and drug development.

[0003] In different Na V In the channel subtype, Na V 1.4 It plays a crucial role in the excitation-contraction coupling process of skeletal muscle. Its high voltage sensitivity and rapid dynamic characteristics ensure the timing precision and force control of voluntary muscle contraction. Neural stimulation triggers Na... V 1.4 After the channel opens, rapid influx of Na⁺ causes membrane depolarization, which in turn prompts the sarcoplasmic reticulum to release calcium ions, thereby initiating the muscle fiber contraction response. The channel encoding... SCN4AGene mutations have been proven to cause various muscle-related diseases, including myotonia, periodic paralysis, myasthenia gravis, and congenital myopathy. Current research has found that μ-conotoxins from marine cone snails are a class of toxins that react with sodium... V 1.4 Antagonists with high selectivity and high affinity not only provide molecular probes for elucidating the structure-function relationship of sodium channels, but also offer new therapeutic strategies for drug development in neuromuscular diseases. Among them, μ-conotoxin KIIIA, containing three disulfide bonds, is currently the smallest and most thoroughly studied representative polypeptide, exhibiting strong affinity for sodium channels. V 1.2, Na V 1.4 and Na V All 1.7 isomers exhibit significant inhibitory activity. Their biological effects are highly dependent on the disulfide bond pairing mode, with different isomers displaying distinctly different pharmacological characteristics. Among them, KIIIA isomer 1 (Cys1–Cys5 / Cys2–Cys4 / Cys3–Cys6) shows significant inhibitory activity against Na+. V Channel 1.4 exhibits the highest efficiency and selectivity (IC). 50 = 65 nM). Based on this, the present invention 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 novel peptide neuromuscular blocking agent with improved efficacy and better bioavailability, which is Na V 1.4 This provides new technological approaches for the precise intervention of related diseases.

[0004] Our team previously identified a structurally simplified mutant, KIIIA-1 ([C1A, C15A]KIIIA), and conducted a series of rational designs based on its binding mode. Through site-specific amino acid mutations, we designed and obtained an enhanced-activity analog, KA ([K7R, A15R]KIIIA-1), which exhibits enhanced activity against Na+. V The inhibitory activity of the 1.4 channel was approximately four times higher than that of the parent peptide. However, KA, as a lead compound for neuromuscular blockade drugs, still has certain limitations. On the one hand, its overall bioactivity has not yet reached the ideal pharmacodynamic level; on the other hand, the introduction of positively charged arginine residues leads to insufficient stability in the serum environment and a significantly shortened half-life, thus limiting the further pharmaceutical application of this molecule in the treatment of neuromuscular blockade and related diseases. Therefore, it is urgent to optimize and modify the structure of this type of peptide molecule to maintain or enhance its Na+ activity. V 1.4 While maintaining selectivity and activity, we can also improve its in vivo stability and pharmacokinetic characteristics, laying the foundation for the development of novel, safe, and long-acting peptide muscle relaxants and medical aesthetic products. Summary of the Invention

[0005] Based on the active peptide KA ([K7R, A15R]KIIIA-1), this invention designs 11 mutants by introducing positive charges at different positions to enhance its affinity for Na+. V 1.4 Electrostatic Interactions of the Channel. The results showed that the inhibitory activity of the mutant [S5R]KA was approximately 10-fold higher than that of the parent peptide. However, the high positive charge content of [S5R]KA led to poor serum stability. Therefore, this invention further mutated a single cysteine ​​residue to the amino acid penicillamine (Pen), which has a sterically hindered side chain, obtaining the candidate peptide C4Pen, which possesses both high activity and excellent serum stability, providing a new approach for developing novel long-acting peptide muscle relaxants.

[0006] The technical solution provided by this invention is as follows: a positively charged cone snail toxin KIIIA mutant, which is obtained by performing site-directed mutations of C1A, K7R, and C15R on the natural cone snail toxin KIIIA sequence; the amino acid sequence of the mutant is shown in SEQ ID NO.1. Preferably, 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.

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

[0008] Preferably, the mutation is 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, C4P, C9, C16; the amino acid sequence of the mutant is shown in any one of SEQ ID NO.13-16.

[0009] This invention also provides the application of the mutant described herein 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.

[0010] Furthermore, the disease or other symptoms caused by sodium channel 1.4 abnormality are preferably diseases or other symptoms caused by sodium channel 1.4 over-excitation.

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

[0012] The present invention also provides a formulation for inhibiting sodium channel 1.4, the formulation comprising the mutant described above, with the mutant as the active ingredient, and a pharmaceutically acceptable carrier.

[0013] Furthermore, the formulation includes tablets, capsules, capsule-in-capsule formulations, micropatch systems in-capsule formulations, 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.

[0014] The present invention has the following beneficial effects:

[0015] This invention achieves a structurally simple and resistant Na+ peptide KIIIA by systematically optimizing the structure of the precursor peptide KIIIA containing three disulfide bonds and employing a strategy combining disulfide bond deletion and site-directed amino acid mutation. V 1.4 Mutants exhibiting significant inhibitory activity in the channel. Further, by introducing the non-natural amino acid Pen into the mutant with optimal activity, its stability in vivo was significantly enhanced, thereby prolonging its duration of action. The candidate molecules obtained through the above optimization strategy possess high activity, high stability, and favorable pharmacokinetic properties, demonstrating broad development potential and providing feasible guidance and technical basis for the research and development of novel muscle relaxants and cosmetic wrinkle removal products. Attached Figure Description

[0016] Figure 1 Na in the embodiments of the present invention V 1.4 Binding mode and surface potential diagram of the KA complex; where A. binding mode; B. surface potential diagram;

[0017] Figure 2 In the embodiments of the present invention, KA and its analogues are used to treat Na. V Inhibition rate of 1.4 (10 nM, n≥4);

[0018] Figure 3 In the embodiments of the present invention, KA, [S5R]KA, and [S6R]KA are used to treat Na. V 1.4 Inhibition concentration effect curve (n≥4);

[0019] Figure 4 This invention provides examples of the effects of [S5R]KA and its analogues on Na at different concentrations in embodiments of the present invention. V Inhibition rate of 1.4 (n≥4);

[0020] Figure 5 This invention relates to the stability of [S5R]KA and its analogues in human AB serum in embodiments of the present invention.

[0021] Figure 6In this embodiment of the invention, [S5R]KA and its analogues are used to treat Na. V 1.4 Inhibition concentration effect curve (n≥4);

[0022] Figure 7 The 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

[0023] 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.

[0024] 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.

[0025] Example 1: Design, synthesis and in vitro activity study of KA and its analogues

[0026] 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).

[0027] Table 1 Targeting Na V 1.4 KA series mutant numbers, names, amino acid sequences, and inhibitory activities

[0028] ;

[0029] Note: "*" indicates C-terminal amidation; Dab is represented by X in computer-readable sequence listings.

[0030] 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 IC50 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.

[0031] Example 2: Synthesis of cone snail toxin KA and its analogues

[0032] 1. Synthesis of KA and its mutant linear peptides

[0033] 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.

[0034] 2. The disulfide bond oxidation process of KIIIA-1 and its mutants

[0035] 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.

[0036] Example 3: In vitro activity and serum stability study of [S5R]KA analog

[0037] The half-maximal inhibitory concentration (IC50) of [S5R]KA 50 The value is 5.1 nM, indicating an effect on Na. V 1.4 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.

[0038] Table 2 Targeting Na V 1.4 [S5R]KA series mutant numbers, names, and amino acid sequences

[0039] ;

[0040] Note: "*" indicates C-terminal amidation; Pen is represented by X in computer-readable sequence listings.

[0041] 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.

[0042] Example 4: Animal in vivo activity test

[0043] 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 7 The 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.

[0044] 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. Mutants of the positively charged μ-conotoxin KIIIA characterized in that: The mutant is obtained by site-directed mutation of the sequence shown in SEQ ID NO. 1 at the following positions: S5R; the amino acid sequence of the mutant is shown in SEQ ID NO.

4.

2. Mutants of the positively charged μ-conotoxin KIIIA characterized in that: The mutation is obtained by introducing a non-natural amino acid Pen at any of the following positions of the mutant shown in SEQ ID NO. 4: C2Pen, C4Pen; the amino acid sequence of the mutant is shown in SEQ ID NO. 13 or 14.

3. Use of the mutant of claim 1 or 2 in the preparation of a peptide muscle relaxant drug or a medical and cosmetic product.

4. A formulation for inhibiting sodium channel 1.4, characterized by: The preparation contains the mutant of claim 1 or 2 as an active ingredient, and a pharmaceutically acceptable carrier.

5. The formulation of claim 4, characterized in that: The preparation includes capsules, micro-patch systems within capsules, tablets, Ovules, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, pharmaceutically acceptable gums, chewable tablets, effervescent tablets.

Images