A spider toxin DkTx mutant and its synthesis method and use

Abstract

This invention belongs to the field of biomedicine and relates to a spider toxin DkTx mutant, its synthesis method, and uses. These mutants are a series of products obtained by structurally modifying the spider toxin DkTx, containing one or two pairs of disulfide bonds. The mutants provided by this invention exhibit potent antagonism against TRPV1. Furthermore, the synthesis method provided by this invention is simple, rapid, low-cost, and amenable to large-scale production.

Description

Technical Field

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

[0002] Ion channels are transmembrane protein complexes that regulate the selective transport of inorganic ions across the cell membrane. They are 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. They play a crucial role in inflammatory responses and maintaining homeostasis. The TRP channel family can be further divided into six subfamilies based on sequence homology and physiological functions: TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML. TRPV1 is a representative channel of the TRPV subfamily, involved in inflammatory responses, thermoregulation, and metabolic control. Its abnormal activation or dysfunction is closely associated with chronic pain, neuropathic pain, inflammatory diseases, and cardiovascular disease. TRPV1 channels are tetrameric transmembrane proteins. Each subunit contains six transmembrane helices (S1-S6). S5-S6 form the ion channel pore, while S1-S4 are responsible for temperature sensing and ligand binding. The N-terminus mediates protein-protein interactions and signal transduction, while the C-terminal regulatory site interacts with protein kinases (such as PKA and PKC), affecting channel activation and inactivation. TRPV1 agonists can alleviate chronic pain by inducing desensitization, while antagonists reduce pain signaling by blocking its activity.

[0003] Spiders are members of the Arthropoda class, and are widely distributed in various ecosystems around the world. As successful predators, spiders rely on highly evolved venom systems to capture prey, defend against predators, and aid in digestion. Spider venom is composed of a series of bioactive molecules, including peptides, proteins, enzymes, and small molecule toxins. Spider toxins mainly act on ion channels, G protein-coupled receptors, and other neural signaling proteins, playing a key role in regulating nerve impulse transmission, muscle activity, and pain perception. Spider toxins are usually disulfide-rich polypeptides that exhibit a highly stable three-dimensional folded structure, which makes them highly tolerable and biologically active.

[0004] DkTx is a spider toxin composed of 75 amino acid residues and is the first known spider toxin with a double-knot peptide structure. DkTx is composed of two independently folded ICK domains connected by a flexible peptide, hence the name "double-knot" toxin. Each ICK domain of DkTx forms a stable spatial conformation through a disulfide-rich folding framework. These domains tightly bind to the transmembrane S3-S4 loop of TRPV1, stabilizing the channel's open conformation and leading to a sustained influx of Na⁺ and Ca²⁺, irreversibly activating the TRPV1 channel and exhibiting highly selective and long-lasting effects. Due to its complex structure, the synthesis of spider toxin DkTx has the following problems: (1) Low efficiency of solid-phase synthesis: The traditional solid-phase peptide synthesis (SPPS) method has low reaction efficiency, and the condensation time of a single amino acid is as long as 40-120 minutes, and expensive condensing agents (such as HCTU / HATU) are required, which is costly; (2) Racemization and side reactions: Amino acid racemization is prone to occur during the synthesis process, especially when traditional condensing agents (such as HOBt) are used, which not only affects the purity of the product but also may introduce explosion risks; (3) Disulfide bond folding is difficult: DkTx contains 12 cysteines, which require the formation of a complex disulfide bond network. Existing in vitro refolding methods (such as redox buffer) have low correct folding rates (only about 2%) and are prone to producing isomers, requiring cumbersome HPLC separation and purification of active products; (4) Recombinant expression limitations: The E. coli expression system often causes DkTx to form inclusion bodies or misfolded isomers. Even with the use of soluble fusion tags (such as thioredoxin TrxA), it is still difficult to obtain active toxins in their native conformation; in addition, the expression yield is low (0.4-0.8 mg / L culture medium), making it difficult to meet large-scale needs; (5) Harsh process conditions: Some methods rely on hazardous reagents or special equipment (such as microwave synthesizers), which increases the difficulty and cost of industrial production. Summary of the Invention

[0005] The present invention aims to address the challenges of prior art methods for synthesizing the spider toxin DkTx by providing a DkTx mutant, its synthesis method, and its uses. These mutants are based on the structure of the spider toxin DkTx, resulting in a series of products containing one or two pairs of disulfide bonds. Testing has shown that these mutants have strong antagonistic effects on TRPV1 channels and can be used to prepare TRPV1 channel antagonists. The preparation process is simple and cost-effective.

[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 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.

[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, and SEQ ID NO.5.

[0008] In another aspect of the present invention, the technical solution provided is a mutant containing a pair of disulfide bonds, specifically: a spider toxin DkTx mutant, 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 any one of 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, amino acid Xaa9 at position 15 is introduced at the end, and Xaa9 is P.

[0010] Further preferably, 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 ID NO.11, and SEQ ID NO.12.

[0011] In a third aspect of the present invention, a technical solution is provided for a method for synthesizing a spider toxin DkTx mutant, comprising the following steps:

[0012] (1) Linear peptides with side chain group protection were synthesized on 2-Chlorotrityl resin by Fmoc solid phase synthesis; the thiol groups on the Cys residues at positions 1 and 9 were protected by acetamidomethyl protecting groups; the thiol groups on the Cys residues at positions 6 and 14 were protected by trityl protecting groups.

[0013] 2) Use TFA cleavage buffer to cleave the linear peptide from the resin, resulting in a free carboxyl group at the C-terminus of the peptide chain and an exposed sulfhydryl group;

[0014] 3) oxidizing the sulfhydryl groups at the 1st and 9th Cys residues to disulfide bonds, or oxidizing the sulfhydryl groups at the 1st and 9th Cys residues and the sulfhydryl groups at the 6th and 14th Cys residues to disulfide bonds; and obtaining the product after purification.

[0015] Preferably, the sulfhydryl groups on the Cys residues at positions 1 and 9 are oxidized by iodine oxidation.

[0016] Preferably, the sulfhydryl groups on the Cys residues at positions 6 and 14 are oxidized by DTDP oxidation.

[0017] In the fourth aspect of the present invention, the technical solution provided is the use of the spider toxin DkTx mutant, which is used to prepare an antagonist of the TRPV1 channel.

[0018] In the fifth aspect of the present invention, the technical solution provided is a TRPV1 channel antagonist, the active ingredient of which contains the spider toxin DkTx mutant.

[0019] This invention modifies the structure of the parent peptide, the spider toxin DkTx, which contains three pairs of disulfide bonds. By deleting one or two pairs of disulfide bonds and non-critical amino acids at either end, DkTx mutants are produced, making them simpler and faster to synthesize. These mutants exhibit potent antagonism against TRPV1, and the synthesis method provided by the invention is simple, rapid, and low-cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of the DkTx analog DkTx-1 provided by the present invention; wherein (a) is the spatial structure; (b) is the amino acid sequence;

[0021] Figure 2 Schematic diagram of the structure of the DkTx analog DkTx-1-1 provided by the present invention; wherein (a) is the spatial structure; (b) is the amino acid sequence;

[0022] Figure 3Figure 1 is a diagram showing the binding pattern of the complex between TRPV1 and the DkTx analog DkTx-1 in an embodiment of the present invention; wherein A is a diagram showing the dynamic simulation of the complex between the DkTx analog DkTx-1 and TRPV1; BG are partial magnified views of the interaction between the amino acids at positions 3, 5, 6, 12, 15, and 17 of DkTx-1 and TRPV1; TRPV1 is represented in blue, DkTx-1 is represented in green, hydrogen bonds are represented by yellow dashed lines, and salt bridges are represented by purple dashed lines;

[0023] Figure 4 The antagonistic rate of DkTx-1 mutants on TRPV1 at a concentration of 1 μM;

[0024] Figure 5 The antagonistic rate of DkTx-1-1 mutants on TRPV1 at a concentration of 1 μM;

[0025] Figure 6 This is the concentration-effect curve of the antagonistic effect of the DkTX-1-1 mutant (SEQ ID NO. 12) on TRPV1. DETAILED DESCRIPTION

[0026] To facilitate understanding of the present invention, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments. Preferred embodiments of the present invention are shown 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. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0027] Example 1 Design, Synthesis and Structure-Activity Relationship Study of the Double Disulfide Spider Toxin DkTx Analog DkTx-1

[0028] The present invention deletes a pair of disulfide bonds and non-critical amino acid residues at both ends of the spider toxin DkTxDkTx junction 2 to obtain a spider toxin analog with a novel disulfide bond skeleton, denoted as DkTx-1, whose amino acid sequence is CWXaa1Xaa2Xaa3CXaa4Xaa5CPMXaa6FC. Figure 1 As shown, DkTx-1 contains two pairs of disulfide bonds, namely, disulfide bonds formed by Cys residues at positions 1 and 9, and disulfide bonds formed by Cys residues at positions 6 and 14. A series of mutants can be obtained by performing the following point mutations at the positions indicated by Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, and Xaa6: 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; and Xaa6 is selected from any one of E or D.

[0029] In addition to the above-mentioned site-directed amino acid mutations, deletion mutations at positions Xaa1 or Xaa5 are also included. These mutants can antagonize the activity of TRPV1.

[0030] In order to clarify the structure-activity relationship between DkTx-1 and TRPV1 protein, the molecular docking technology was first used to construct a complex model of TRPV1 and DkTx-1 binding, and a 400 ns molecular dynamics simulation (MD) was performed. Amber22 software was used for MD, and the force field selections were: ff19SB force field for protein, Lipid17 force field for lipid, and frcmod.ionjc_tip3p force field for ion. VMD software was used to analyze the trajectory of conformational changes and calculate the simulated RMSD value. PyMOL software was used to analyze and visualize the binding mode between TRPV1 and DkTx-1. The complex binding mode of DkTx-1 and TRPV1 is shown in the figure. Figure 3 shown.

[0031] In the study of the structure-activity relationship between polypeptides and receptors, a site-directed mutagenesis strategy is usually used to mutate the key amino acid residues of the ligand or receptor, and by analyzing its effect on biological activity, provide guidance for subsequent mechanism analysis and drug optimization. In the present invention, after determining the binding mode of DkTx-1 and TRPV1, analysis of the possible interaction forces showed that the key amino acid residues are S5, K6, D12, etc., and there are hydrogen bonds, salt bridges and other interactions with TRPV1 protein E536, D601, N652, D654 and K656. Combined with the above analysis, the present invention designed 5 mutants, as shown in Table 1. This series of mutants were subjected to electrophysiological activity tests, and their antagonistic activity against TRPV1 at a concentration of 1 μM was as follows. Figure 4 shown.

[0032] Table 1 Numbers and amino acid sequences of DkTx-1 series mutants targeting TRPV1

[0033] SEQ ID NO. sequence 1 CWQHRCRQCPMDFC 2 CWRKCESCPMEFC 3 CWGHKCECPMEFC 4 CWDQKCEQCPMEFC 5 CWDKRCESCPMEFC .

[0034] Example 2 Synthesis of DkTx-1 Series Mutant Lines

[0035] 1. Synthesis of DkTx-1 series mutant linear peptides

[0036] A linear peptide of DkTx-1 was synthesized using Fmoc solid-phase reaction technology. Synthesis was performed on 2-Chlorotrityl resin at a 0.1 mmol scale, with the synthesis direction from C-terminus to N-terminus. Prior to synthesis, the resin was swollen with DCM / DMF (1:1) for 1 hour. Trt-protected Cys (0.4 mmol, 4 eq) and DIPEA (135 μL, 0.8 mmol, 8 eq) were added and reacted for 3 hours. After the reaction, the resin was washed three times with DMF and then washed three times with DCM. Unreacted sites on the resin were blocked by the addition of 5 ml of blocking reagent (methanol:DCM:DIPEA = 2:2:1) and the reaction was repeated twice for 1 hour. The resin was then treated with a 20% piperidine solution in DMF for 30 minutes to completely remove the Fmoc protecting group. The subsequent amino acid (0.4 mmol, 4 eq), HCTU (165 mg, 0.4 mmol, 4 eq), and DIPEA (135 μL, 0.8 mmol, 8 eq) were then added sequentially to DMF and incubated at 30°C for 1 hour to achieve coupling. After each reaction, the resin was washed three times with DMF and then with DCM. Coupling and deprotection reactions were detected by ninhydrin colorimetry. The coupling and deprotection procedures were repeated to extend the peptide chain. After the Fmoc protecting group of the last amino acid was removed, the resin was washed with DCM. Add 10 mL of lysis buffer (TFA: Tips: H₂O = 90:5:5) and react at room temperature for 3 h. Filter, wash the resin with DCM, collect the filtrate, concentrate it by rotary evaporation under reduced pressure, add glacial ether for precipitation, centrifuge (6000 rpm, room temperature, 5 min), and discard the supernatant to obtain the crude linear peptide. The product was verified by LC-MS and purified by RP-HPLC. The peptide semi-preparative and analytical HPLC methods are shown in Table 2:

[0037] Table 2 Semi-preparative HPLC and analytical HPLC methods

[0038] .

[0039] 2. Disulfide bond oxidation process of DkTx-1 series mutants

[0040] The thiol protecting groups of DkTx-1 are Trt and Acm. The thiol groups at Cys residues 6 and 14 were protected with a trityl (Trt) protecting group, which was then oxidized with DTDP (4,4'-bipyridyl disulfide) to form the first disulfide bond, and then purified by semi-preparative liquid phase chromatography. The thiol groups at Cys residues 1 and 9 were protected with an acetamidomethyl (Acm) protecting group, which was then oxidized with iodine to form the second disulfide bond.

[0041] The specific experimental steps are as follows: (1) Oxidation of the first disulfide bond: 0.1 mmol of the crude peptide was placed in a beaker, 40 mL of pure water was added, and a methanol (5 mL) solution of DTDP (22 mg, 0.1 mmol, 1 eq) was slowly added dropwise. The mixture was stirred and reacted at room temperature for 1 h. The reaction was monitored by LC-MS. After the reaction, the product was purified by RP-HPLC on a C18 column to obtain a product containing a single disulfide bond.

[0042] (2) Oxidation of the second disulfide bond: The solution obtained in (1) was lyophilized to obtain a peptide powder, which was dissolved in 80% acetic acid aqueous solution at a concentration of 2 mg / ml. 10 eq of I2 was added and stirred at room temperature. LC-MS real-time monitoring was performed. After the reaction was completed, 6 volumes of diethyl ether were added for precipitation. The mixture was centrifuged (8000 rpm, RT, 5 min), the supernatant was discarded, the precipitate was dissolved in pure water, and purified by RP-HPLC.

[0043] Example 3 Design, Synthesis and Structure-Activity Relationship Study of the Single Disulfide Spider Toxin DkTx Analog DkTx-1-1

[0044] After systematically studying the double-disulfide DkTx analog DkTx-1, the present invention aimed to further reduce synthesis costs. Therefore, a single-disulfide DkTx analog, designated DkTx-1-1, and its linear peptide mutants were designed. Ultimately, a series of mutants that still maintained high activity were obtained. Compared to DkTx-1, DkTx-1-1 only contains disulfide bonds formed by Cys residues at positions 1 and 9, as shown in Figure 2. Figure 2 The amino acid sequence is CWXaa1Xaa2Xaa3Xaa4Xaa5Xaa6CPMXaa7FXaa8. A series of mutants can be obtained by making the following point mutations at the positions indicated by Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, and Xaa8: 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 any one of Q, R, or deletion; Xaa7 is selected from E or Q; and Xaa8 is selected from any one of E, Q, or D. In addition, when Xaa6 is Q, Xaa7 is E, and Xaa8 is Q, amino acid Xaa9 at position 15 is introduced at the end, and Xaa9 is P.

[0045] Starting from DkTx-1-1, a total of 6 mutants were designed and synthesized. The synthesis method is shown in Example 2. The detailed information of the series of mutants of DkTx-1-1 is shown in Table 3. The antagonistic activity of these mutants on TRPV1 channels at a concentration of 1 μM is shown in Table 3. Figure 5 shown.

[0046] Table 3 Numbers and amino acid sequences of the DkTx-1-1 series of mutants targeting TRPV1

[0047] SEQ ID NO. sequence 6 CWSRRNPQCPMEFE 7 CWGERGRCPMEF 8 CWDQRSKQCPMEFQP 9 CWDEKTRQCPMEFD 10 CWSRKNEQCPMEFE 11 CWDRKNKQCPMEFE 12 CWGFRGGRCPMQF .

[0048] By measuring the antagonistic activity of the peptide against TRPV1 at different concentrations, the half antagonistic concentration (IC) of the mutant with the sequence SEQ ID NO.12 against TRPV1 can be calculated. 50 ), the effect curve is as follows Figure 6 As shown, its IC 50 = 572±74nM.

[0049] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A spider toxin DkTx mutant, characterized by: 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, and SEQ ID NO.5; wherein the first and third Cys residues form a disulfide bond; and the second and fourth Cys residues form a disulfide bond.

2. A spider toxin DkTx mutant, characterized by: 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 ID NO.11, and SEQ ID NO.12; wherein the first and second Cys residues form a disulfide bond.

3. The method for synthesizing the spider toxin DkTx mutant according to claim 1, characterized in that: The following steps are involved: (1) A linear peptide with side chain group protection was synthesized on 2-Chlorotrityl resin by Fmoc solid phase synthesis. The thiol groups on Cys residues 1 and 3 were protected with acetamidomethyl protecting groups, and the thiol groups on Cys residues 2 and 4 were protected with trityl protecting groups. (2) Use TFA cleavage solution to cleave the linear peptide from the resin, and the resulting peptide chain has a free carboxyl group at the C-terminus and a naked sulfhydryl group; (3) The sulfhydryl groups on Cys residues 1 and 3, and the sulfhydryl groups on Cys residues 2 and 4 are oxidized to disulfide bonds; the product is obtained after purification.

4. The synthesis method according to claim 3, characterized in that The sulfhydryl groups on Cys residues 1 and 3 were oxidized by iodine oxidation.

5. The synthesis method according to claim 3, characterized in that The sulfhydryl groups on Cys residues 2 and 4 were oxidized by DTDP oxidation.

6. A TRPV1 channel antagonist, characterized in that The active ingredient comprises the spider toxin DkTx mutant according to claim 1 or 2.

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