Alpha-conotoxin polypeptide, fluorescent molecular probe thereof and application thereof

Abstract

This invention belongs to the field of biotechnology, specifically relating to an α-conotoxin polypeptide, its fluorescent molecular probe, and its applications. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, wherein position 11 is a D-lysine residue, C-terminus is amidated, and two specific disulfide bonds are formed intramolecularly. The polypeptide exhibits high affinity and excellent selectivity for α6β4 nAChRs and good stability in serum, making it suitable as a pharmacologically active ingredient antagonizing α6β4 nAChRs for the treatment of diseases related to α6β4 nAChR dysfunction, such as depression, Parkinson's disease, and neuropathic pain. This invention also provides a fluorescent molecular probe capable of in-situ visualization and tissue distribution studies of α6β4 nAChRs, and provides a novel tool for drug screening based on this receptor. This invention lays a solid foundation for the development of diagnostic reagents and candidate drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an α-conotoxin polypeptide, its fluorescent molecular probe, and its applications. Background Technology

[0002] nAChRs are ligand-gated ion channels widely distributed in the central nervous system, peripheral nervous system, and non-neuronal cells. These receptors are assembled from five subunits to form a pentamer, primarily including α (α1-α10), β (β1-β4), δ, γ, and ε subunits. Different combinations of subunits form nAChR isomers with unique pharmacological characteristics, exhibiting differences in selectivity for specific agonists / antagonists and biophysical properties (such as Na+). + K + Ca 2+ Significant changes in permeability and desensitization kinetics. nAChRs play different roles in various physiological processes through their subtype-specific regulatory mechanisms. Differentiating nAChR subtypes is crucial for developing drugs to treat pain, memory disorders, Parkinson's disease, schizophrenia, and nicotine addiction. Therefore, exploring the distribution and pathological functions of nAChRs has always been an important goal in drug development.

[0003] α6*nAChRs (* indicates the possible presence of other subunits; other subtype combinations mentioned below, such as α6β4*nAChRs, all have the same meaning, i.e., the possible presence of other auxiliary subunits) are one of the major subtypes of nAChRs, mainly distributed in the ventral tegmental area, nucleus accumbens, and spinal cord. Their important function is to participate in ethanol-mediated activation of dopaminergic neurons in the ventral tegmental area and the mediation and release of γ-aminobutyric acid (GABA). In addition, α6*nAChRs are also expressed in peripheral tissues such as the mouse thymus, enteric nervous system, bronchi, and urinary tract, but their specific nAChR subtypes and physiological functions still need further investigation. In mice, reducing the expression level of α6*nAChRs decreases dopamine release and weakens the stimulatory effects of certain analgesics. In recent years, an increasing number of studies have reported that α6β4*nAChRs participate in pain regulation as a key target for pain perception and transmission.

[0004] α6β4 nAChRs play crucial roles in various physiological and pathological processes, including pain, learning and memory, motor regulation, body temperature maintenance, and depression, addiction, and cardiovascular diseases. However, due to the lack of highly selective ligands or drug tools, our understanding of their physiological functions and pathological mechanisms remains very limited. Therefore, developing novel, highly selective, potent α6β4 nAChR blockers, especially inhibitors that can effectively distinguish them from structurally and distributionally similar α3β4 and α6β2* nAChR subtypes, has significant scientific value and economic potential for elucidating the biological functions of this receptor, advancing research on related disease mechanisms, and developing innovative drugs. Summary of the Invention

[0005] To address the lack of highly selective α6β4 nAChR antagonists in existing technologies, this invention provides an α-conotoxin polypeptide with high selectivity and high affinity, its fluorescent molecular probe, and its applications.

[0006] This invention provides the following technical solutions:

[0007] An α-conotoxin polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1, is: GCCANPVCNGkHC# (where k represents D-lysine and # represents C-terminal amidation).

[0008] Furthermore, two pairs of disulfide bonds are formed within the polypeptide molecule, wherein the first cysteine ​​in the sequence forms a first pair of disulfide bonds with the third cysteine, and the second cysteine ​​forms a second pair of disulfide bonds with the fourth cysteine.

[0009] This invention provides the application of the above-mentioned α-conotoxin polypeptide in the preparation of drugs antagonizing α6β4 nAChRs.

[0010] Furthermore, the drug is used to treat and / or prevent at least one of the following conditions: depression, Parkinson's disease, anxiety, neuropathic pain, addiction, and learning and memory disorders.

[0011] A fluorescent molecular probe comprising the aforementioned polypeptide and a BODIPY-FL fluorescent group covalently linked to the N-terminus of the polypeptide; the probe is formed by coupling the N-terminal amino group of the polypeptide with the BODIPY-FL NHS ester under weakly alkaline conditions.

[0012] This invention provides the application of the above-mentioned fluorescent molecular probe in the preparation of reagents for detecting, locating or imaging α6β4 nAChRs.

[0013] The present invention also provides the application of the above-mentioned polypeptide or the above-mentioned fluorescent molecular probe in constructing a drug screening model for screening candidate substances that regulate the activity of α6β4 nAChRs, wherein the drug screening model is a cell model or an animal model.

[0014] The following provides an explanation of the terminology involved in this invention.

[0015] The fluorescent molecular probe described in this invention refers to a conjugate formed by coupling the polypeptide with a fluorescent group, which can specifically bind to α6β4 nAChRs. By observing the changes in fluorescence signals before and after binding, the target receptor can be traced, located, or imaged.

[0016] In this invention, the term "effective amount" refers to a dose sufficient to achieve therapeutic, preventive, mitigating or alleviating effects on the disease or condition described in this invention in a subject, or to achieve the desired biological effect in an experimental system.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) The α-conotoxin polypeptide [D-Lys] modified with D-type amino acids provided by the present invention 11 LvIC exhibits excellent antagonistic activity and high selectivity against α6β4nAChRs.

[0019] (2) Its fluorescent molecular probe [D-Lys] 11 LvIC-BDP can retain the high affinity and high selectivity of the parent peptide and can be used as a highly sensitive fluorescent probe for in situ distribution studies and visualization detection of α6β4 nAChRs.

[0020] (3) The peptide and its fluorescent molecular probe provide a new and powerful tool for studying the pathological mechanisms of diseases related to α6β4 nAChRs dysfunction, as well as for developing related diagnostic reagents and therapeutic drugs. Attached Figure Description

[0021] Figure 1 : [D-Lys 11 [Flowchart of the two-step oxidation synthesis of LvIC; D-type amino acids are marked in red, cysteine ​​is marked in bold, and # indicates C-terminal amidation.]

[0022] Figure 2 : [D-Lys 11 The purification and identification spectra of LvIC; the left figure is the high performance liquid chromatogram (detection wavelength 214 nm), and the right figure is the mass spectrum.

[0023] Figure 3 : [D-Lys 11Stability evaluation results of LvIC in 20% rat serum.

[0024] Figure 4 L-shaped [Lys] 11 Stability evaluation results of LvIC in 20% rat serum.

[0025] Figure 5 : [D-Lys 11 Concentration-response curves of LvIC to α6β4 nAChRs.

[0026] Figure 6 100μM [D-Lys] 11 [Efficacy test results of LvIC against other subtypes of nAChRs (α3β4, α6β2, α9α10 and α7)]

[0027] Figure 7 : [D-Lys 11 Schematic diagram of LvIC-BDP synthesis; the direction of electron transfer is indicated by red arrows in the diagram.

[0028] Figure 8 : [D-Lys 11 The purification and identification chromatograms of LvIC-BDP are shown; the left figure is the high performance liquid chromatogram (detection wavelength 503 nm), and the right figure is the mass spectrum.

[0029] Figure 9 : [D-Lys 11 Concentration-response curves of LvIC-BDP on α6β4 nAChRs.

[0030] Figure 10 100μM [D-Lys] 11 [Efficacy test results of LvIC-BDP against other subtypes of nAChRs (α3β4, α6β2, α9α10 and α7)]

[0031] Figure 11 Results of fluorescent staining of frozen sections of rat urinary tract tissue. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. The following embodiments are only used to illustrate the present invention, but the scope of protection of the present invention is not limited thereto.

[0033] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0034] Example 1: α-Conotoxin [D-Lys] 11 Synthesis of LvIC.

[0035] The α-conotoxin polypeptide (α-conotoxin [D-Lys] in this embodiment) 11 The polypeptide, LvIC, has the amino acid sequence shown in SEQ ID NO: 1. Derived from the cone snail *Conus lividus*, this 13-amino acid polypeptide has the sequence: GCCANPVCNGkHC# (where k is a D-type lysine and # indicates C-terminal amidation). Specifically, the first cysteine ​​residue at the N-terminus of the polypeptide forms a disulfide bond with the third cysteine ​​residue, and the second cysteine ​​residue forms another disulfide bond with the fourth cysteine ​​residue; the carboxyl terminus is amidated.

[0036] Specifically, [D-Lys] was prepared using a solid-phase synthesis method. 11 LvIC linear peptide, see synthesis procedure (see above) Figure 1 The specific synthesis process is as follows:

[0037] (1) First, the carboxyl group of the C-terminal amino acid is fixed on a solid resin support. Then, according to the target peptide sequence, Fmoc protecting group removal, amino acid activation and coupling are carried out in a cyclical manner to connect each amino acid residue in sequence, and finally the whole sequence is assembled on the resin.

[0038] (2) In the polypeptide sequence, cysteine ​​residues at positions II and IV were protected with acetamide methyl (ACM) as side chain thiol groups to avoid non-specific oxidation; at the same time, the C-terminus of the peptide chain was modified by amidation. After the linear peptide chain was assembled, it was treated with a cleavage reagent to cleave it from the resin and remove the side chain protecting groups, and the crude peptide product was collected.

[0039] (3) The crude peptide was then subjected to stepwise oxidative folding: First, the first pair of disulfide bonds was oriented under potassium ferricyanide conditions to obtain a monooxidized intermediate; then the oxidation environment was adjusted and iodine solution was added to guide the correct pairing of the second pair of disulfide bonds to obtain the target product with a natural spatial conformation.

[0040] (4) After each reaction step, reversed-phase high-performance liquid chromatography was used to separate and purify the crude peptide, monooxidation intermediate, and final double oxidation product, and the molecular weight of the products at each stage was verified by electrospray ionization mass spectrometry (see Figure 2 Meanwhile, its retention time and chemical purity were determined using analytical high-performance liquid chromatography.

[0041] The results showed that the target peptide [D-Lys] was successfully obtained after two steps of oxidative folding and purification. 11LvIC. HPLC showed a single sharp chromatographic peak at a detection wavelength of 214 nm, with a chemical purity > 98%. Electrospray ionization mass spectrometry showed its [M+3H] content. 3+ The mass-to-charge ratio (m / z) of the ion peak is 434.37 (see [reference]). Figure 2 The result is highly consistent with the theoretical calculation value of 434.04, confirming that its amino acid sequence, D-type modification and C-terminal amidation are all correct.

[0042] Example 2: Fluorescent molecular probe [D-Lys] 11 Synthesis of LvIC-BDP.

[0043] The fluorescent molecular probe [D-Lys] in this embodiment 11 The synthesis of LvIC-BDP involves using the [D-Lys] obtained in Example 1. 11 The N-terminal amino group of LvIC undergoes a nucleophilic substitution reaction with the N-carboxysuccinimide of the fluorescent dye BODIPY-FL NHS ester under weakly alkaline conditions (pH=8.5) to form an amide bond (see [link to relevant documentation]). Figure 7 ).

[0044] After the reaction, the product was separated and purified by reversed-phase high-performance liquid chromatography, and its molecular weight was verified by electrospray ionization mass spectrometry (see [link to product]). Figure 8 Meanwhile, its retention time and chemical purity were determined using analytical high-performance liquid chromatography (detection wavelength 503 nm).

[0045] The results showed that [D-Lys¹¹]LvIC successfully underwent a coupling reaction with BODIPY-FL NHS ester. The HPLC chromatogram of the purified product showed a significantly different retention time for the main peak compared to the starting peptide, and a characteristic absorption at 503 nm, indicating that the fluorophore had been attached. Mass spectrometry analysis showed that its [M+2H]... 2+ Ion peak (m / z=788.17) (see) Figure 8 The molecular weight of the probe is consistent with the theoretical molecular weight of the probe, and the chemical purity of the probe after purification is > 95%.

[0046] Example 3: [D-Lys 11 LvIC and D-Lys 11 Activity study of LvIC-BDP on α6β4 nAChRs and other nAChR subtypes.

[0047] Using dual-electrode voltage clamping technology to [D-Lys] 11 LvIC and D-Lys 11 The LvIC-BDP activity was determined, and the specific experimental procedure is as follows:

[0048] (1) To evaluate [D-Lys 11 LvIC and its fluorescent molecular probe ([D-Lys 11 To investigate the bioactivity of LvIC-BDP, we used the two-electrode voltage-clamp technique (TEVC) to determine its inhibitory effect on rat α6β4 nAChRs expressed in Xenopus laevis oocytes and examined its selectivity for other nAChR subtypes.

[0049] (2) After isolating V-VI stage oocytes, inject a 1:1 molar mixture of subunit-specific cRNAs. After culturing in ND96 buffer at 17°C for 2-4 days, dissolve the peptides and serially dilute them with ND96 buffer containing 0.1 mg / mL BSA to a final concentration range of 10. -4 Up to 10 -8 M. The concentration of the polypeptide was determined by HPLC or spectrophotometry.

[0050] (3) During electrophysiological recording, oocytes were clamped at -70 mV and perfused at a flow rate of 4 mL / min. Each 60-second cycle included: 2 seconds of baseline (ND96+BSA), 2 seconds of agonist pulse (acetylcholine concentration depending on subtype), and 56 seconds of elution. Dose-response curves were constructed (see [link to relevant documentation]). Figure 5 , Figure 9 And calculate IC 50 The value is used to quantify the inhibitory effect.

[0051] (4) The efficacy against other nAChR subtypes (α3β4, α6β2, α9α10 and α7) was tested at a concentration of 100 μM, and the results are shown in […]. Figure 6 and Figure 10 .

[0052] The results show that the dual-electrode voltage clamp experiment indicates that [D-Lys] 11 LvIC effectively antagonizes rat α6β4 nAChRs expressed in Xenopus laevis oocytes, and its concentration-response curve is shown in the figure. Figure 5 Calculations show that its IC 50 The value was 8.2 (6.8-10.1) nM, showing potent antagonistic activity. At a concentration of 100 μM, [D-Lys 11 LvIC showed a current suppression rate of less than 10% for nAChRs subtypes such as α3β4, α6β2, α9α10, and α7 (see [link to relevant documentation]). Figure 6 This indicates that it has excellent selectivity for the α6β4 nAChRs subtype.

[0053] Similarly, its fluorescent molecular probe [D-Lys] 11 The antagonistic activity of LvIC-BDP against α6β4 nAChRs was preserved, IC50 The value is 218.1 (164.0-290.7) nM (see [reference]). Figure 9 At a concentration of 100 μM, its inhibitory activity against current induced by other tested subtypes was also significantly lower than its inhibitory activity against α6β4 nAChRs (see [link to relevant documentation]). Figure 10 This confirms that fluorescent labeling did not significantly affect its affinity and subtype selectivity.

[0054] Example 4: [D-Lys 11 Stability evaluation of LvIC

[0055] The target peptide was dissolved in 20% rat serum and incubated at 37°C. Samples were taken at different time points, and the purity changes were analyzed by ultra-high performance liquid chromatography to evaluate the in vitro stability of the peptide. The specific implementation steps are as follows:

[0056] (1) Take 15 nmol of lyophilized peptide powder, dissolve it completely in 5 μL of water, add 120 μL of 20% rat serum, mix thoroughly, and prepare the test sample solution.

[0057] (2) The above sample solution was incubated in a metal bath at 37°C. 10 μL samples were taken at 0, 4, 8, 12, and 24 hours for subsequent ultra-high performance liquid chromatography (UHPLC) analysis. Purity was monitored using an UHPLC system with a gradient elution program set to increase mobile phase B (acetonitrile containing 0.05% TFA) from 5% to 70% within 5 minutes, and a detection wavelength of 214 nm.

[0058] (3) Identify the chromatographic peak corresponding to the target peptide by mass spectrometry. Using the target peak area of ​​the sample at 0 hours as a baseline (100%), calculate the ratio of the target peak area to the initial peak area at each time point as the peptide residual rate at that time point, which is used to characterize its stability (see [reference]). Figure 3 All stability tests were independently repeated at least 3 times.

[0059] The results show that: [D-Lys] 11 LvIC exhibited good in vitro stability in 20% rat serum. Figure 3 As shown, after incubation at 37°C for 24 hours, the residual rate was still as high as 54.9 ± 4.1%. No obvious degradation peaks or fragmentation peaks were observed during the incubation process. These results indicate that the peptides of the present invention have good resistance to enzymatic degradation in a simulated body fluid environment, providing a favorable pharmacokinetic profile for their potential in vivo application.

[0060] Example 5: Investigating the in situ distribution of α6β4 nAChRs in rat tissues using fluorescence imaging

[0061] To characterize the tissue distribution of α6β4 nAChRs, this study used [D-Lys] 11 LvIC-BDP was used as a fluorescent molecular probe for fluorescence imaging analysis of rat urinary tract tissue. The specific steps are as follows:

[0062] (1) Fresh urinary tract tissue from SD rats was flash-frozen in liquid nitrogen and stored at -80°C. The tissue was frozen into 6 μm thick sections by Servicebio, and the sections were baked at 37°C for 20 minutes. The stained areas were delineated with an immunohistochemical pen. Blocking solution (3% BSA, 0.1% Tween 20) was added, and the sections were blocked at room temperature for 30 minutes. The sections were then rinsed twice with PBS for 5 minutes each time and dried at room temperature.

[0063] (2) Set up two processing groups: [D-Lys 11 LvIC-BDP incubation group; [D-Lys 11 [LvIC-BDP and excess unlabeled [D-Lys11]LvIC co-incubation group (competitive experiment). 10 μL of the corresponding peptide solution was added to each group, and incubated at room temperature in the dark for 15 minutes. The sections were washed three times with PBS, 5 minutes each time. After drying, DAPI was added to stain the nuclei for 10 minutes, followed by PBS quenching and mounting with anti-fluorescence agents, and stored at 4°C.

[0064] (3) Images were acquired using a Nikon Eclipse Ti2 and Sunny confocal microscope (see results). Figure 11 Image processing and fluorescence analysis were performed using ImageJ. In the competition experiment, 20 800 × 800 μm regions were selected for statistical analysis of fluorescent spots. Data are expressed as mean ± SEM, and t-tests were performed using GraphPad Prism (P < 0.001). The experiment was independently repeated three times.

[0065] The results show that using [D-Lys] 11 LvIC-BDP staining of frozen sections of rat urinary tract tissue revealed clear and specific green fluorescence signals under a confocal microscope, indicating the distribution of α6β4 nAChRs in the tissue (see [link to original text]). Figure 11 Competitive experimental group (pre-treated with excessive amounts of unlabeled [D-Lys]). 11 The fluorescence signal intensity of [D-Lys(IVIC)] was significantly reduced by approximately 60% compared to the group incubated with the single probe (P < 0.001), indicating that [D-Lys(IVIC)]... 11 The binding of LvIC-BDP to tissues is specific. This result successfully validates that this fluorescent molecular probe can be used for in-situ visualization and distribution studies of α6β4 nAChRs.

[0066] Comparative Example 1: L-type [Lys] 11Synthesis and activity evaluation of LvIC

[0067] To highlight the technical advantages of the D-type amino acid modification of this invention, an L-type [Lys] amino acid was synthesized in this comparative example. 11 LvIC:

[0068] L-shaped [Lys] 11 LvIC, whose sequence is GCCANPVCNGKHC (K represents L-lysine, i.e., L-lysine at position 11, C-terminal amidation, and disulfide bond connection as in this invention), and whose amino acid sequence is shown in SEQ ID NO: 2.

[0069] The control peptides were prepared using the same solid-phase synthesis and oxidative folding method as in Example 1. The purity was confirmed to be >95% and the structure was correct by HPLC and mass spectrometry.

[0070] Using the exact same experimental methods as in Examples 3 and 4, the L-type [Lys] was determined. 11 The residual rate of LvIC (L-lysine at position 11) after incubation in 20% rat serum for 24 hours, and its IC50 response to α6β4 nAChRs. 50 The values ​​were measured; the inhibition rates of the α3β4, α6β2, α9α10 and α7 subtypes at a concentration of 100 μM were also tested.

[0071] The results show:

[0072] L-shaped [Lys] 11 LvIC (L-lysine at position 11) has similar antagonistic activity to the D-type [Lys11] LvIC of the present invention, and there is no significant difference in their high selectivity for the α6β4 isoform.

[0073] L-shaped [Lys] 11 LvIC (L-lysine at position 11) exhibits extremely poor serum stability, with a polypeptide residual rate of 28.8 ± 5.8% within 24 hours (see [link to relevant documentation]). Figure 4 The residual rate of the D-lysine-modified polypeptide in this invention reached 54.9 ± 4.1%, indicating that, under the premise of comparable activity and selectivity, the significant improvement in serum stability mainly stemmed from the D-configuration modification of the 11th amino acid.

[0074] Conclusion: By introducing D-lysine at position 11, this invention significantly improves serum stability while maintaining potent antagonistic activity and subtype selectivity.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An α-conotoxin polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The α-conotoxin polypeptide according to claim 1, characterized in that, The lysine residue at position 11 of the amino acid sequence is in the D configuration, and the C-terminus of the polypeptide is modified by amidation.

3. The α-conotoxin polypeptide according to claim 1 or 2, characterized in that: The polypeptide molecule forms two pairs of disulfide bonds, wherein the first cysteine ​​in the sequence forms the first pair of disulfide bonds with the third cysteine, and the second cysteine ​​forms the second pair of disulfide bonds with the fourth cysteine.

4. The use of the α-conotoxin polypeptide according to any one of claims 1-3 in the preparation of a drug antagonizing α6β4 nAChRs.

5. The application according to claim 4, characterized in that, The drug is used to treat and / or prevent at least one of the following conditions: depression, Parkinson's disease, anxiety, neuropathic pain, addiction, or learning and memory disorders.

6. A fluorescent molecular probe, characterized in that, The polypeptide comprises any one of claims 1-3, and a BODIPY-FL fluorescent group covalently linked to the N-terminus of the polypeptide.

7. The use of the fluorescent molecular probe according to claim 6 in the preparation of reagents for detecting, locating or imaging α6β4 nAChRs.

8. The use of the polypeptide according to any one of claims 1-3 or the fluorescent molecular probe according to claim 6 in constructing a drug screening model for screening candidate substances that regulate the activity of α6β4 nAChRs, characterized in that, The drug screening model is a cell model or an animal model.

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