Spider toxin DkTx mutant as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to a spider toxin DkTx mutant as well as a synthesis method and application thereof. The mutant is a series of products containing one or two pairs of disulfide bonds obtained by modifying the structure of spider toxin DkTx on the basis of spider toxin DkTx. The mutant provided by the invention has a relatively good antagonistic effect on the TRPV1; the synthesis method provided by the invention is simple, rapid in synthesis, relatively low in cost and convenient for large-scale production.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a spider toxin DkTx mutant, its synthesis method and use. Background Art

[0002] Ion channels are transmembrane protein complexes on the cell membrane, responsible for regulating the selective transport of inorganic ions across the cell membrane, which is crucial for maintaining membrane potential homeostasis, signal transduction and intracellular homeostasis. Transient receptor potential (TRP) channels are a class of non-selective cation channels that respond to a variety of external physical and chemical stimuli, including temperature changes, mechanical forces, osmotic pressure and chemical molecules, and play important roles in processes such as inflammatory responses and maintenance of internal environmental homeostasis. The TRP channel family can be further divided into six subfamilies according to sequence homology and physiological functions, including TRPC, TRPV, TRPM, TRPA, TRPP and TRPML. TRPV1 is a representative channel of the TRPV subfamily, which is involved in inflammatory responses, body temperature regulation and metabolic regulation. Its abnormal activation or dysfunction is closely related to chronic pain, neuropathic pain, inflammatory diseases and cardiovascular diseases. The TRPV1 channel is a tetrameric transmembrane protein, and each subunit contains six transmembrane helices (S1-S6), where S5-S6 forms the ion channel pore region, and S1-S4 is responsible for sensing temperature and ligand binding. The N-terminus can mediate protein-protein interactions and signal transduction, while the C-terminus regulatory site interacts with protein kinases (such as PKA, PKC), affecting the activation and inactivation of the channel. TRPV1 agonists can relieve chronic pain by inducing a desensitization mechanism, while antagonists reduce pain signal transmission by blocking its activity.

[0003] Spiders are members of the class Arachnida in the phylum Arthropoda and are widely distributed in various ecosystems around the world. As successful predators, spiders rely on a highly evolved venom system to capture prey, defend against natural enemies and assist digestion. Spider venom consists of a series of bioactive molecules, including polypeptides, proteins, enzymes and small molecule toxins. Spider toxins mainly act on ion channels, G protein-coupled receptors and other nerve signal transduction proteins, playing a key role in regulating nerve impulse transmission, muscle activity and pain perception. Spider toxins are usually polypeptides rich in disulfide bonds, showing a highly stable three-dimensional folded structure, making them highly tolerant and biologically active.

[0004] DkTx is a spider toxin composed of 75 amino acid residues, derived from the Taiwanese bird spider Ornithoctonus formosanus, and is the first known spider toxin with a double-knot peptide structure. DkTx consists of two independently folded ICK domains, which are connected by a flexible polypeptide, thus being called a "double-knot" toxin. Each ICK domain of DkTx forms a stable spatial conformation through a disulfide-rich folding framework and can tightly bind to the transmembrane S3-S4 loop region of TRPV1, stabilizing the open conformation of the channel, resulting in continuous influx of Na⁺ and Ca²⁺, irreversibly activating the TRPV1 channel, and showing extremely high selectivity and long-lasting effect. Due to its complex structure, the synthesis of spider toxin DkTx has the following problems: (1) Low solid-phase synthesis efficiency: The traditional solid-phase peptide synthesis (SPPS) method has low reaction efficiency, with the condensation time of a single amino acid up to 40 - 120 minutes, and expensive condensing agents (such as HCTU / HATU) need to be used, resulting in high costs; (2) Racemization and side reactions: Amino acid racemization is likely to occur during the synthesis process, especially when using traditional condensing agents (such as HOBt), which not only affects the product purity but may also introduce explosion risks; (3) Difficult disulfide bond folding: DkTx contains 12 cysteines and needs to form a complex disulfide bond network. The existing in vitro refolding methods (such as redox buffers) have a low correct folding rate (only about 2%) and are prone to generating isomers, and the active product needs to be separated and purified by cumbersome HPLC; (4) Limitations in recombinant expression: The Escherichia coli expression system often leads to the formation of inclusion bodies or misfolded isomers of DkTx. Even when using soluble fusion tags (such as thioredoxin TrxA), it is still difficult to obtain the active toxin with a native conformation; in addition, the expression yield is low (0.4 - 0.8 mg / L culture medium), making it difficult to meet large-scale demands; (5) Harsh process conditions: Some methods rely on dangerous reagents or special equipment (such as microwave synthesizers), increasing the difficulty and cost of industrial production. Summary of the Invention

[0005] The object of the present invention is to solve the problems existing in the synthesis of spider toxin DkTx in the prior art, and provide a mutant of spider toxin DkTx, its synthesis method and uses. This mutant is obtained by modifying the structure of spider toxin DkTx, and a series of products containing one or two pairs of disulfide bonds are respectively obtained. After detection, these mutants have a strong antagonistic effect on the TRPV1 channel and can be used to prepare antagonists of the TRPV1 channel; moreover, the preparation process is simple and the cost is relatively low.

[0006] In one aspect of the present invention, the technical solution provided is a mutant containing two pairs of disulfide bonds, specifically: a spider toxin DkTx mutant, the amino acid sequence of the mutant being CWXaa1Xaa2Xaa3CXaa4Xaa5CPMXaa6FC; the Cys residues at the 1st and 9th positions form a disulfide bond; the Cys residues at the 6th and 14th positions form a disulfide bond; wherein, Xaa1 is selected from any one of G, D, R, Q or deletion; Xaa2 is selected from any one of H, Q or K; Xaa3 is selected from K or R; Xaa4 is selected from E or R; Xaa5 is selected from any one of S, Q or deletion; Xaa6 is selected from E or D.

[0007] Preferably, the amino acid sequence of the mutant is selected from any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5.

[0008] In another aspect of the present invention, the technical solution provided is a mutant containing one pair of disulfide bonds, specifically: a spider toxin DkTx mutant, the amino acid sequence of the mutant being CWXaa1Xaa2Xaa3Xaa4Xaa5Xaa6CPMXaa7FXaa8; the Cys residues at the 1st and 9th positions form a disulfide bond; wherein, Xaa1 is selected from any one of D, G or S; Xaa2 is selected from any one of E, F, R or Q; Xaa3 is selected from K or R; Xaa4 is selected from any one of G, N, T or S; Xaa5 is selected from any one of G, E, P, K, or R; Xaa6 is selected from Q, R or deletion; Xaa7 is selected from E or Q; Xaa8 is selected from any one of E, Q or D.

[0009] Preferably, when Xaa6 is Q, Xaa7 is E, and Xaa8 is Q, the 15th amino acid Xaa9 is introduced at the end, and Xaa9 is P.

[0010] More preferably, the amino acid sequence of the mutant is selected from any one of SEQ ID NO.6, SEQ ID NO.7, SEQ IDNO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12.

[0011] In the third aspect of the present invention, the technical solution provided is a method for synthesizing a spider toxin DkTx mutant, comprising the following steps: (1)Synthesize a linear peptide with side-chain group protection on 2-Chlorotrityl resin by Fmoc solid-phase synthesis method; wherein, the sulfhydryl groups on the Cys residues at the 1st and 9th positions are protected by acetamidomethyl protecting groups; the sulfhydryl groups on the Cys residues at the 6th and 14th positions are protected by triphenylmethyl protecting groups; (2)Use TFA cleavage solution to cleave the linear peptide from the resin, and the C-terminus of the obtained peptide chain is a free carboxyl group with sulfhydryl groups exposed; (3)Oxidize the sulfhydryl groups on the Cys residues at the 1st and 9th positions into disulfide bonds, or oxidize the sulfhydryl groups on the Cys residues at the 1st and 9th positions and the sulfhydryl groups on the Cys residues at the 6th and 14th positions into disulfide bonds; after purification, the product is obtained.

[0012] Preferably, the sulfhydryl groups on the Cys residues at the 1st and 9th positions are oxidized by iodine oxidation method.

[0013] Preferably, the sulfhydryl groups on the Cys residues at the 6th and 14th positions are oxidized by DTDP oxidation method.

[0014] In the fourth aspect of the present invention, the technical solution provided is the use of the spider toxin DkTx mutant, and this mutant is used to prepare an antagonist of TRPV1 channel.

[0015] In the fifth aspect of the present invention, the technical solution provided is an antagonist of TRPV1 channel, and its active ingredient contains the spider toxin DkTx mutant.

[0016] The present invention conducts structural modification on the parent peptide double-knot spider toxin DkTx containing three pairs of disulfide bonds, and deletes one or two pairs of disulfide bonds and non-critical amino acids at both ends respectively to obtain a DkTx mutant with simpler and faster synthesis. The mutant provided by the present invention has a good antagonistic effect on TRPV1; and the synthesis method provided by the present invention is simple, fast in synthesis, and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic structural diagram of the DkTx analog DkTx-1 provided by the present invention; wherein, (a) is the spatial structure; (b) is the amino acid sequence; Figure 2 It is a schematic structural diagram of the DkTx analog DkTx-1-1 provided by the present invention; wherein, (a) is the spatial structure; (b) is the amino acid sequence; Figure 3This is a diagram showing the binding mode of the complex of TRPV1 and the DkTx analogue DkTx-1 in the embodiments of the present invention. Among them, A is a diagram of the kinetic simulation complex of the DkTx analogue DkTx-1 and TRPV1; B-G are respectively enlarged local views of the interactions between the amino acids at positions 3, 5, 6, 12, 15, and 17 of DkTx-1 and TRPV1. TRPV1 is shown in blue, DkTx-1 is shown in green, hydrogen bonds are shown as yellow dashed lines, and salt bridges are shown as purple dashed lines. Figure 4 It is the antagonistic rate of the mutants of DkTx-1 at a concentration of 1 μM against TRPV1. Figure 5 It is the antagonistic rate of the mutants of DkTx-1-1 at a concentration of 1 μM against TRPV1. Figure 6 It is the antagonistic concentration-effect curve of the mutants of DkTX-1-1 (SEQ ID NO.12) against TRPV1. Detailed implementation manners

[0018] To facilitate the understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments. The preferred embodiments of the present invention are given in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0019] Example 1 Design, synthesis and structure-activity relationship study of the double disulfide bond spider toxin DkTx analogue DkTx-1 In the present invention, a pair of disulfide bonds and the non-critical amino acid residues at both ends of the DkTx knot 2 of the spider toxin DkTx are deleted to obtain a spider toxin analogue with a novel disulfide bond skeleton, denoted as DkTx-1, and its amino acid sequence is CWXaa1Xaa2Xaa3CXaa4Xaa5CPMXaa6FC. As Figure 1 shown, DkTx-1 contains two pairs of disulfide bonds, namely the disulfide bond formed by the Cys residues at positions 1 and 9; the disulfide bond formed by the Cys residues at positions 6 and 14. Among them, at the positions shown by Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, and Xaa6, the following point mutations can be carried out to obtain a series of mutants: Xaa1 is selected from any one of G, D, R or Q; Xaa2 is selected from any one of H, Q or K; Xaa3 is selected from any one of K or R; Xaa4 is selected from any one of E or R; Xaa5 is selected from any one of S or Q; Xaa6 is selected from any one of E or D.

[0020] In addition to the above amino acid site-directed mutations, it also includes deletion mutations at the Xaa1 or Xaa5 positions. These mutants can antagonize the activity of TRPV1.

[0021] To clarify the structure-activity relationship between DkTx-1 and TRPV1 protein, first, a complex model of TRPV1 bound to DkTx-1 was constructed using molecular docking technology, and 400 ns of molecular dynamics simulation (MD) was performed. MD was carried out using Amber22 software, where the force fields were selected as follows: ff19SB force field for proteins, Lipid17 force field for lipids, and frcmod.ionjc_tip3p force field for ions. The VMD software was used to analyze the trajectory of conformational changes and calculate the simulated RMSD value. The PyMOL software was used to analyze and visualize the binding mode between TRPV1 and DkTx-1. The binding mode of the complex of DkTx-1 and TRPV1 is as Figure 3 shown.

[0022] In the study of the structure-activity relationship between polypeptides and receptors, the site-directed mutation strategy is usually adopted to mutate the key amino acid residues of ligands or receptors, and by analyzing their effects on biological activities, it provides guidance for subsequent mechanism analysis and drug optimization. In the present invention, after determining the binding mode between DkTx-1 and TRPV1, analyzing the possible interaction forces shows that the key amino acid residues are S5, K6, D12, etc., which have hydrogen bond, salt bridge and other interactions with E536, D601, N652, D654 and K656 of TRPV1 protein. Combining the above analysis, the present invention designed 5 mutants as shown in Table 1. Electrophysiological activity tests were carried out on this series of mutants, and their antagonistic activities against TRPV1 at a concentration of 1 μM are as Figure 4 shown.

[0023] Table 1 Number and amino acid sequence of DkTx-1 series mutants targeting TRPV1 SEQ ID NO. Sequence 1 CWQHRCRQCPMDFC 2 CWRKCESCPMEFC 3 CWGHKCECPMEFC 4 CWDQKCEQCPMEFC 5 CWDKRCESCPMEFC 。

[0024] Example 2 Synthesis method of DkTx-1 series mutants 1. Synthesis of linear peptides of DkTx-1 series mutants Synthesize the linear peptide of DkTx-1 through the Fmoc solid-phase reaction technique. Use 2-Chlorotrityl resin for synthesis at the 0.1 mmol level, and the synthesis direction is from the C-terminus to the N-terminus. Before synthesis, swell the resin with DCM / DMF (1:1) for 1 h; add Trt-protected Cys (0.4 mmol, 4 eq) and DIPEA (135 μL, 0.8 mmol, 8 eq), and react for 3 h. After the reaction, wash the resin three times with DMF and DCM respectively, add 5 ml of blocking reagent (methanol:DCM:DIPEA = 2:2:1), react for 1 h, repeat 2 times to block the unreacted sites on the resin. Subsequently, treat the resin with a DMF solution containing 20% piperidine for 30 minutes to completely remove the Fmoc protecting group; then add the subsequent amino acids (0.4 mmol, 4 eq), HCTU (165 mg, 0.4 mmol, 4 eq), and DIPEA (135 μL, 0.8 mmol, 8 eq) to DMF in turn, and incubate at 30 °C for 1 h to achieve coupling. After each reaction, wash the resin three times with DMF and DCM respectively, and the coupling and deprotection reactions can be detected by the ninhydrin color reaction. Repeat the coupling and deprotection operations to extend the peptide chain. After the Fmoc protecting group of the last amino acid is removed, wash the resin with DCM. Add 10 mL of cleavage solution (TFA:Tips:H2O = 90:5:5), react at room temperature for 3 h, filter by suction, wash the resin with DCM, collect the filtrate, concentrate the filtrate by rotary evaporation under reduced pressure, add ice ether to precipitate, centrifuge (6000 rpm, RT, 5 min), discard the supernatant, and obtain the crude linear peptide. The product is verified by LC-MS and purified by RP-HPLC. The semi-preparative and analytical methods of polypeptide HPLC are shown in Table 2: Table 2 Semi-preparative liquid phase and analytical liquid phase methods 。

[0025] 2. Disulfide bond oxidation process of DkTx-1 series mutants The thiol protecting groups of DkTx-1 are Trt and Acm respectively. Among them, the thiols on the 6th and 14th Cys residues are protected with a triphenylmethyl (Trt) protecting group, and then oxidized by the DTDP (4,4'-dipyridyl disulfide) oxidation method to form the first pair of disulfide bonds, and purified by semi-preparative liquid phase. The thiols on the 1st and 9th Cys residues are protected with an acetamidomethyl (Acm) protecting group; the second pair of disulfide bonds is formed by iodine oxidation method.

[0026] The specific experimental steps are as follows: (1) Oxidation of the first pair of disulfide bonds: Take 0.1 mmol of crude peptide and place it in a beaker. Add 40 mL of pure water, and slowly drip a methanol (5 mL) solution dissolving DTDP (22 mg, 0.1 mmol, 1 eq). Stir and react at room temperature for 1 h. Monitor the reaction by LC-MS. After the reaction is completed, purify it using RP-HPLC on a C18 column to obtain a product containing a single pair of disulfide bonds.

[0027] (2) Oxidation of the second pair of disulfide bonds: Lyophilize the solution obtained in (1) to obtain a polypeptide powder. Dissolve it in an 80% acetic acid aqueous solution at a concentration of 2 mg / ml, add 10 eq of I2, and stir at room temperature. Monitor it in real time by LC-MS. After the reaction is completed, add 6 volumes of ether for precipitation, centrifuge (8000 rpm, RT, 5 min), discard the supernatant, dissolve the precipitate in pure water, and purify it by RP-HPLC.

[0028] Design, Synthesis and Structure-Activity Relationship Study of Monodisulfide Spider Toxin DkTx Analogue DkTx-1-1 After a systematic study on the bisdisulfide DkTx analogue DkTx-1, the present invention aims to further reduce the synthesis cost. Therefore, a monodisulfide DkTx analogue, denoted as DkTx-1-1, and its linear peptide mutants were further designed. Finally, a series of mutants with still high activity were obtained. Compared with DkTx-1, DkTx-1-1 only contains the disulfide bond formed by the Cys residues at positions 1 and 9, as Figure 2 shown. Its amino acid sequence is CWXaa1Xaa2Xaa3Xaa4Xaa5Xaa6CPMXaa7FXaa8. At the positions shown by Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, the following point mutations can be made to obtain a series of mutants: Xaa1 is selected from any one of D, G or S; Xaa2 is selected from any one of E, F, R or Q; Xaa3 is selected from K or R; Xaa4 is selected from any one of G, N, T or S; Xaa5 is selected from any one of G, E, P, K, or R; Xaa6 is selected from Q, R or deletion; Xaa7 is selected from E or Q; Xaa8 is selected from any one of E, Q or D. In addition, when Xaa6 is Q, Xaa7 is E, and Xaa8 is Q, an amino acid Xaa9 at position 15 is introduced at the end, and Xaa9 is P.

[0029] Starting from DkTx-1-1, a total of 6 mutants were designed and synthesized, and the synthesis method refers to Example 2. The specific information of the series of mutants of DkTx-1-1 is shown in Table 3, and the antagonistic activities of these mutants against the TRPV1 channel at a concentration of 1 μM are as Figure 5 shown.

[0030] Table 3 Number and Amino Acid Sequences of DkTx-1-1 Series Mutants Targeting TRPV1 SEQ ID NO. Sequence 6 CWSRRNPQCPMEFE 7 CWGERGRCPMEF 8 CWDQRSKQCPMEFQP 9 CWDEKTRQCPMEFD 10 CWSRKNEQCPMEFE 11 CWDRKNKQCPMEFE 12 CWGFRGGRCPMQF 。

[0031] By measuring the antagonistic activity of the polypeptide against TRPV1 under different concentration conditions, the half-maximal inhibitory concentration (IC 50 ) of the mutant with the sequence of SEQ ID NO. 12 against TRPV1 can be calculated. The effect curve is as shown in Figure 6 , and its IC 50 = 572 ± 74 nM.

[0032] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A spider toxin DkTx mutant, characterized in that: The amino acid sequence of the mutant is CWXaa1Xaa2Xaa3CXaa4Xaa5CPMXaa6FC; the Cys residues at positions 1 and 9 form a disulfide bond; the Cys residues at positions 6 and 14 form a disulfide bond; wherein, Xaa1 is selected from any one of G, D, R, Q or deletion; Xaa2 is selected from any one of H, Q or K; Xaa3 is selected from K or R; Xaa4 is selected from E or R; Xaa5 is selected from any one of S, Q or deletion; Xaa6 is selected from E or D.

2. The spider toxin DkTx mutant according to claim 1, wherein: The amino acid sequence of the mutant is selected from any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.

5.

3. A spider toxin DkTx mutant, characterized in that: The amino acid sequence of the mutant is CWXaa1Xaa2Xaa3Xaa4Xaa5Xaa6CPMXaa7FXaa8; the Cys residues at positions 1 and 9 form a disulfide bond; wherein, Xaa1 is selected from any one of D, G or S; Xaa2 is selected from any one of E, F, R or Q; Xaa3 is selected from K or R; Xaa4 is selected from any one of G, N, T or S; Xaa5 is selected from any one of G, E, P, K, or R; Xaa6 is selected from Q, R or deletion; Xaa7 is selected from E or Q; Xaa8 is selected from any one of E, Q or D.

4. The spider toxin DkTx mutant according to claim 3, characterized in that: When Xaa6 is Q, Xaa7 is E, and Xaa8 is Q, the 15th amino acid Xaa9 is introduced at the end, and Xaa9 is P.

5. The spider toxin DkTx mutant according to claim 4, wherein: The amino acid sequence of the mutant is selected from any one of SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ IDNO.11, SEQ ID NO.

12.

6. The method for synthesizing the spider toxin DkTx mutant according to any one of claims 1-5, characterized in that, Comprising the following steps: (1) Synthesize a linear peptide with side chain groups protected on 2-Chlorotrityl resin by Fmoc solid-phase synthesis method; wherein, the sulfhydryl groups on the Cys residues at positions 1 and 9 are protected with acetamidomethyl protecting groups; the sulfhydryl groups on the Cys residues at positions 6 and 14 are protected with triphenylmethyl protecting groups; (2) Cleave the linear peptide from the resin using TFA cleavage solution, and the C-terminus of the obtained peptide chain is a free carboxyl group with sulfhydryl groups exposed; (3) Oxidize the sulfhydryl groups on the Cys residues at positions 1 and 9 to form a disulfide bond, or oxidize the sulfhydryl groups on the Cys residues at positions 1 and 9 and the sulfhydryl groups on the Cys residues at positions 6 and 14 to form a disulfide bond; after purification, the product is obtained.

7. The synthesis method according to claim 6, characterized in that, The sulfhydryl groups on the Cys residues at positions 1 and 9 are oxidized by iodine oxidation method.

8. The synthesis method according to claim 6, characterized in that, The sulfhydryl groups on the Cys residues at positions 6 and 14 are oxidized by DTDP oxidation method.

9. Use of the spider toxin DkTx mutant according to any one of claims 1-5, characterized in that, This mutant is used for preparing an antagonist of TRPV1 channel.

10. An antagonist of the TRPV1 channel, characterized in that, Its active ingredient contains the spider toxin DkTx mutant described in any one of claims 1-5.

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