Conductive polypeptides targeting ng r1 and uses thereof

Abstract

This invention relates to a conductive polypeptide targeting NgR1 and its applications, belonging to the fields of molecular biology and medicinal chemistry. The polypeptide has the general amino acid sequence formula X1YYX2RYE, where X1 and X2 are W, A, or F, respectively. The polypeptide has the characteristic of targeting NgR1 with high affinity, and therefore, in practical applications, it can regulate the nerve regeneration inhibition signaling pathway at the molecular level, helping to relieve axonal growth limitations. Furthermore, the polypeptide has conductive properties, which can improve the local electrical microenvironment of nerve tissue, enhance the efficiency of nerve signal transmission, and facilitate the functional reconstruction of neural networks.

Description

Technical Field

[0001] This invention relates to a conductive polypeptide targeting NgR1 and its applications, belonging to the fields of molecular biology and medicinal chemistry. Background Technology

[0002] Nerve injury and dysfunction are widespread in various diseases and injury states. They can occur in acute injuries such as trauma, ischemia, and compression, as well as in chronic pathological processes such as neurodegenerative diseases, metabolic abnormalities, and inflammation-related diseases. Once neural structures are damaged, it often leads to impaired nerve signal transmission, resulting in abnormalities in sensory, motor, visual, or other neurological functions, severely impacting patients' quality of life. Unlike other tissues, mature neural tissue has limited self-repair capabilities after injury, making it difficult to effectively rebuild functional neural connectivity networks. Although some progress has been made in recent years in areas such as neuroprotection, nerve regeneration, and neuromodulation, overall, functional recovery after nerve injury still faces many challenges. How to promote nerve regeneration and achieve long-term, stable functional reconstruction remains a crucial problem that urgently needs to be solved in the fields of neuroscience and regenerative medicine.

[0003] Numerous studies have shown that restricted axonal regeneration is closely related to the activation of multiple inhibitory signaling pathways in nerve growth. Among them, the Nogo protein and its receptor Nogo-receptor 1 (NgR1) play a crucial role in the inhibition of nerve regeneration. NgR1 is widely expressed on the surface of various neuronal types and can interact with a variety of inhibitory ligands, including Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). When NgR1 binds to these inhibitory ligands, it can activate a series of downstream signaling pathways, leading to growth cone collapse, cytoskeleton rearrangement, and axonal extension inhibition, thereby restricting nerve regeneration at both the molecular and cellular levels. Therefore, NgR1 is widely considered an important molecular target for restricting axonal regeneration and functional recovery after nerve injury, and effective intervention in it has potential therapeutic value.

[0004] Current interventions targeting NgR1 have been extensively studied, including antibodies, gene regulation technologies, and small molecule inhibitors. However, existing technologies still have certain limitations in practical applications. For example, antibody molecules have limited stability in vivo, are easily degraded, and have low targeted delivery efficiency; gene regulation still faces challenges in terms of safety and controllability; and small molecule drugs generally suffer from insufficient target specificity. Furthermore, most of the aforementioned existing strategies focus on blocking or regulating NgR1-related molecular signaling pathways, while having limited effects on improving the overall electrophysiological function of neural tissue, making it difficult to achieve a holistic recovery of neural function.

[0005] On the other hand, conductive materials are widely used in nerve repair and nerve function regulation because they can improve electrical signal transmission in the neural tissue microenvironment. However, most existing conductive materials are conductive polymers or inorganic conductive materials, and their biocompatibility, biodegradability, and safety for long-term in vivo application are still insufficient. Furthermore, they typically lack the ability to target specific neuroinhibitory receptors, making it difficult to achieve precise regulation of nerve regeneration-related signaling pathways. Therefore, achieving effective targeting of NgR1 while maintaining good biocompatibility and conductivity to synergistically promote nerve axon regeneration and electrophysiological function recovery remains a pressing technical problem to be solved in this field. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a conductive polypeptide targeting NgR1 and its applications. This material can effectively regulate the nerve regeneration inhibitory receptor while improving the local electrical microenvironment of nerve tissue, thereby synergistically promoting nerve axon regeneration and nerve function recovery.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows.

[0008] A conductive polypeptide targeting NgR1, wherein the amino acid sequence of the polypeptide has the general formula X1YYX2RYE, where X1 and X2 are W, A, or F, respectively.

[0009] Preferably, the amino acid sequence is WYYWRYE, AYYWRYE, AYYARYE, or FYYFRYE.

[0010] Preferably, the amino acid sequence of the conductive polypeptide is WYYWRYE.

[0011] Preferably, the structural formula of the conductive polypeptide is:

[0012] .

[0013] A nucleic acid comprising a nucleotide sequence encoding the conductive polypeptide.

[0014] A biological material comprising the nucleic acid, the biological material including an expression cassette, a vector, a transposon, a host cell, or a transgenic cell line.

[0015] A divalent or multivalent assembly formed from the conductive polypeptide.

[0016] Preferably, the divalent or multivalent is formed by covalent linkage of connecting molecules, or by non-covalent linkage with a polymer. More preferably, the polymer is polyethylene glycol (PEG).

[0017] A nanofiber formed by the self-assembly of the divalent or multivalent form under physiological conditions.

[0018] A conjugate comprising the conductive polypeptide and a carrier, wherein the carrier is one or more selected from nanomaterials, liposomes, polymers, and oily compounds.

[0019] The application of the conductive polypeptide, the nucleic acid, the biomaterial, the divalent or multivalent, the nanofiber, and the conjugate in the preparation of products for the prevention and / or treatment of nerve damage or neurological dysfunction.

[0020] Beneficial effects (1) The polypeptide provided by the present invention has the characteristic of targeting NgR1 and has a high affinity. Therefore, in practical applications, it can regulate the nerve regeneration inhibition signaling pathway at the molecular level, which helps to relieve the limitation of axon growth.

[0021] (2) The polypeptide has electrical conductivity, which can improve the local electrical microenvironment of nerve tissue, enhance the efficiency of nerve signal transmission, and facilitate the reconstruction of neural network function.

[0022] (3) The polypeptide material described in this invention has good biocompatibility and degradability, and is more suitable for in vivo application than traditional conductive polymers or inorganic conductive materials.

[0023] (4) This invention achieves the synergistic effect of "molecular signal regulation" and "electrophysiological function improvement", providing a new technical approach for the treatment of nerve injury and nerve dysfunction. Attached Figure Description

[0024] Figure 1 Flowchart for screening NgR1-targeting peptides.

[0025] Figure 2 Here is the chemical structural formula of the polypeptide WE.

[0026] Figure 3 This is the IV curve of the polypeptide.

[0027] Figure 4The surface plasmon resonance (SPRi) method was used to detect the binding affinity of the WE peptide to the NgR1 protein.

[0028] Figure 5 Confocal microscopy images of the binding of cellular-level peptides to Schwann cells (NgR1 overexpression) and 293T cells (NgR1 low expression).

[0029] Figure 6 WE polypeptide hydrogel is used for the repair of damaged primary neurons. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments.

[0031] In a first aspect, the present invention employs a de novo design method to design a conductive polypeptide targeting NgR1, wherein the amino acid sequence of the polypeptide has the general formula X1YYX2RYE, where X1 and X2 are W, A, or F, respectively.

[0032] Preferably, the amino acid sequence is WYYWRYE, AYYWRYE, AYYARYE, or FYYFRYE.

[0033] Preferably, the polypeptide contains L amino acid residues. Type D Type or L D A mixture of types.

[0034] Preferably, the amino acid sequence of the polypeptide is WYYWRYE.

[0035] Preferably, the structural formula of the polypeptide is: .

[0036] Secondly, the present invention also provides a DNA fragment comprising a nucleotide sequence encoding the polypeptide described above.

[0037] Thirdly, the present invention also provides an expression vector comprising at least one copy of a DNA fragment encoding an amino acid sequence of the polypeptide described above.

[0038] As a preferred technical solution, the expression vector of the present invention includes at least one copy of the DNA fragment encoding an amino acid sequence as described in the second aspect of the present invention.

[0039] Fourthly, the present invention also provides a prokaryotic or eukaryotic host cell containing the expression vector as described in the third aspect of the present invention.

[0040] Fifthly, the present invention also provides a divalent or multivalent assembly composed of the polypeptide described in the first aspect of the present invention. The divalent or multivalent assembly of the present invention has the characteristic of targeting NgR1-positive neural cells and neurons.

[0041] As a preferred technical solution, the divalent or multivalent of the present invention is formed by covalent linkage of connecting molecules or by non-covalent linkage after mixing with polymers.

[0042] The polymer can be selected according to specific needs, for example, it can be polyethylene glycol (PEG).

[0043] In a sixth aspect, the divalent or multivalent forms of the present invention can self-assemble into nanofibers under physiological conditions and exhibit electrical conductivity.

[0044] The present invention employs the conjugation of the polypeptides described in the first aspect and the divalent or multivalent polypeptides described in the fifth aspect with nanomaterials, liposomes, polymer materials, etc. The peptides, divalents, or multivalents involved in the present invention can enable the conjugated compounds to be more stably transported to target cells in the body.

[0045] The polypeptides, divalents, or multivalents involved in this invention can also be mixed with oily compounds or mixtures of various oily compounds. The peptides involved in this invention can also enable the resulting mixture to be transported more stably to target cells in the body.

[0046] Seventhly, the use of the polypeptide described in this invention in the preparation of products for the prevention and / or treatment of nerve damage or neurological dysfunction.

[0047] Example 1: Design and Construction of Peptide Libraries 1) Experimental materials and instruments Tentagel resin, Wang resin, N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), N,N-dimethylformamide (DMF), ninhydrin, tetramethylurea hexafluorophosphate (HBTU), vitamin C, hexahydropyridine, phenol, anhydrous ethanol, methanol, anhydrous diethyl ether, various Fmoc-protected amino acids, cyanogen bromide, streptavidin-labeled magnetic nanobeads, biotin-labeled NgR1 protein, skim milk, Tween-20, SPRi naked gold core, peptide synthesis tube, shaker, circulating water vacuum pump, rotary evaporator.

[0048] 2) Solvent preparation Deprotecting agent: 20% hexahydropyridine; reaction solution: N-methylmorpholine, N,N-dimethylformamide = 1:24 (v / v).

[0049] The lysis buffer consisted of trifluoroacetic acid (92.5%), triisopropylsilane (2.5%), ethylenedithiol (2.5%), and ultrapure water (2.5%).

[0050] Ninhydrin test solution: Ninhydrin, Vitamin C, Phenol = 1:1:1 (v / v / v).

[0051] 3) Synthesis of "One Bead, One Component" Peptide Library A peptide library was synthesized using the Fmoc solid-phase peptide synthesis method. The process of synthesizing targeted peptides is as follows: Figure 1 As shown. The specific method involves coupling the amino acids to be protected one by one onto a solid-phase resin, and then cleaving the peptide chain from the resin under strong acid while removing the side chain protecting groups. Weigh 300 mg of Tentagel... Using NH2 resin, following the solid-phase peptide synthesis procedure described above, the resin was divided into three equal parts. 60 mg of Tyr, Trp, and Phe were added to each part, along with an equal amount of HBTU, for coupling. After coupling, the three parts of resin were mixed and deprotected. Then, the resin was divided into five equal parts. 36 mg of Val, Ser, Asn, Gln, Tyr, and an equal amount of HBTU were added to each part, for coupling. After coupling, the five parts of resin were mixed and deprotected. This process was repeated three times. Divide the resin into three equal parts. Add 60 mg of His, Arg, and Lys to each part, along with an equal amount of HBTU, for coupling. After coupling, mix the three parts of resin and deprotect. Divide the resin into five equal parts. Add 36 mg of Val, Ser, Asn, Gln, and Tyr to each part, along with an equal amount of HBTU, for coupling. After coupling, mix the five parts of resin and deprotect. Divide the resin into two equal parts. Add 90 mg of Asp and Glu to each part, along with an equal amount of HBTU, for coupling. After coupling, mix the two parts of resin and deprotect. After the above displacement and shrinkage steps, the resin is dried under vacuum to obtain a dry resin loaded with peptide libraries for later use.

[0052] Positive peptide beads are identified by the binding reaction between streptavidin-labeled magnetic beads and biotin on proteins. The surface of the positive peptide beads is coated with a layer of magnetic material, making them magnetic and thus captured by a magnetic field. The peptide beads at the bottom of the tube are carefully transferred to another tube using a pipette. Individual peptide beads are cleaved using hydrogen bromide and processed via MALDI. TOF MS identification yielded the corresponding sequence information. The FITC and MALDI markers were then resynthesized based on the sequence. TOF identification and HPLC purification were used for subsequent experiments.

[0053] The polypeptide sequence of the present invention was obtained by chemical synthesis as follows: WE: WYYWRYE (SEQ ID No. 1).

[0054] AWE:AYYWRYE (SEQ ID No. 2).

[0055] AAE:AYYARYE (SEQ ID No. 3).

[0056] FFE: FYYFRYE (SEQ ID No. 4).

[0057] Experimental Example 2: IV curve of the peptide Peptides WE, AWE, AAE, and FFE were dissolved in aqueous solution at a concentration of 500 μmol / L. After vortexing for 1 minute to ensure complete dissolution, all peptides were placed at 4°C for 48 hours to self-assemble. Before testing, the interdigitated electrodes were cleaned sequentially with ultrapure water and ethanol in an ultrasonic bath for 15 minutes, and then air-dried. 2 μL of the freshly prepared peptide solution was added dropwise to the electrode surface and dried overnight in a 20°C oven to obtain the peptide-coated electrode. Current measurement: After completing the peptide coating, IV characteristics were tested using a dual-electrode configuration connected to a stage and a manual measurement probe, with real-time data monitoring using a Keysight B1500A device. Measurements were performed in 10 mV steps, with a 2-second scan delay and a maximum current limit of 100 mA. Each sample was measured at least three times, and the data showed a consistent trend. The current value was reported as the average of the three measurements.

[0058] Depend on Figure 3 It can be seen that peptides WE, AWE, AAE and FFE are all conductive. Among them, the limiting current of peptide WE can reach the μA level, and its conductivity is calculated to be 222 mS / m.

[0059] Experiment 3: Testing the binding affinity of peptides to NgR1 at different NgR1 protein concentrations.

[0060] Surface plasmon resonance imaging (SPRi) was used to detect the affinity of NgR1 affinity peptides. SPRi analysis was performed on a plexaPlexArray HT system (plexa LLC, Bothell, WA) using a bare gold SPRi chip (nanocaptured gold chip, gold layer thickness 47.5 nm). Purified peptides were imprinted onto the gold chip surface via thiol groups on cysteine ​​residues. The printed chips were then incubated overnight in a humidified chamber at 4°C. Before use, the SPRi chips were rinsed in PBS with 5% (m / v) skim milk and plugged overnight. The SPRi analysis procedure followed the following injection cycle: run buffer (PBST, baseline stabilization); sample (five protein concentrations, binding); run buffer (PBST, washing); 0.5% (v / v) H3PO4 in deionized water (regeneration). NgR1 protein was diluted with PBST to 16 nM, 32 nM, 64 nM, 128 nM, and 256 nM. The binding signal was recorded and analyzed in real time using PlexArray HT software, and the dissociation constants of the target peptide ligand and NgR1 protein were obtained as follows: Figure 4 As shown.

[0061] Example 4: Confocal microscopy images of cells with different NgR1 expression levels using peptides 293T and Schwann cells were cultured in DMEM / Highglucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin in a 37°C incubator (5% CO2). 1×10⁻⁶ cells were cultured... 5 mL -1 293T cells and Schwann cells were seeded into culture dishes and cultured overnight for cell adhesion. FITC-labeled targeting peptides were then added at 5 × 10⁻⁶. -5 The concentration of M was dissolved in cold PBS. The cells were incubated in the dark at 4°C for 30 min with FITC-labeled peptide solution (200 μL, with Hoechst 33342 (1 mM)). Finally, the cells were washed three times with cold PBS. Fluorescence confocal imaging was performed using a confocal laser scanning microscope as shown below. Figure 5 As shown. Throughout the experiment, a 488 nm laser was used as the excitation source for FITC, with an emission wavelength of 520–620 nm. The Hoechst 33342 laser was excited at 50 mW with an excitation wavelength of 405 nm and an emission wavelength of 472 nm. The imaging objective was a Leica STELLARIS 8 63× oil immersion objective.

[0062] Depend on Figure 5It can be seen that the WE peptide can bind to NgR1-expressing cells, which indicates that WE not only has a high affinity for NgR1-positive cells, but also has good specificity. This shows that the peptide of the present invention has a high affinity for NgR1-positive cells when used alone, and can be used as a peptide targeting NgR1.

[0063] Experimental Example 5: Peptide WE hydrogel for the repair of damaged primary neurons To establish a neuronal injury model, the primary neuronal cell culture medium was replaced with low-glucose DMEM medium, and the cells were placed in an anaerobic gas-generating bag and incubated for 1 hour to establish a hypoxic neuronal injury model. When evaluating the effect of peptides in the primary neuronal cell model, WE solution prepared with 1 mol / L PBS was added to the cells, and the cells were cultured for 48 hours, with electrical stimulation applied every 24 hours. The control group was maintained in standard culture medium. The electrical stimulation group was maintained in standard culture medium while receiving the same electrical stimulation as the WE peptide culture group. After incubation, the cells were washed with PBS, and FITC-labeled phalloidin staining was used to visualize the cytoskeleton structure. The cell nuclei were stained with DAPI. The signals of the FITC channel (excitation wavelength 488 nm, emission wavelength 525 nm) and the DAPI channel (excitation wavelength 350 nm, emission wavelength 460 nm) were observed using confocal fluorescence microscopy.

[0064] Depend on Figure 6 It can be seen that primary neurons treated with WE and electrical stimulation simultaneously experienced axonal growth and neuronal fiber structure regeneration, indicating that WE can repair damaged primary neurons.

[0065] From Experiment Example 1 5. It can be concluded that the polypeptide of the present invention has the characteristics of targeting NgR1-positive damaged primary neurons and being able to transmit electrical signals. Therefore, in practical applications, the conductive polypeptide of the present invention can be used as a targeting polypeptide for the repair and treatment of nerve damage.

[0066] In summary, the invention includes, but is not limited to, the above embodiments. Any equivalent substitutions or partial improvements made under the spirit and principles of this invention shall be considered to be within the protection scope of this invention.

Claims

1. An electrically conductive polypeptide targeting NgRl, characterized in that: The general formula of the amino acid sequence of the polypeptide is X1YYX2RYE, where X1 and X2 are W, A, or F, respectively.

2. The conductive polypeptide targeting NgR1 according to claim 1, characterized in that: The amino acid sequence is WYYWRYE, AYYWRYE, AYYARYE, or FYYFRYE.

3. The conductive polypeptide targeting NgR1 according to claim 1, characterized in that: The amino acid sequence of the conductive polypeptide is WYYWRYE.

4. The conductive polypeptide targeting NgR1 according to claim 1, characterized in that: The structural formula of the conductive polypeptide is: 。 5. A nucleic acid comprising a nucleotide sequence encoding the conductive polypeptide of any one of claims 1 to 4.

6. A biomaterial comprising the nucleic acid according to any one of claims 1 to 4, characterized in that: The biological materials include expression cassettes, vectors, transposons, host cells, or transgenic cell lines.

7. A divalent or multivalent form, characterized in that: It is assembled from the conductive polypeptide described in any one of claims 1 to 4; Preferably, the divalent or multivalent is formed by covalently linking molecules, or by mixing with a polymer and non-covalently linking them; more preferably, the polymer is polyethylene glycol.

8. A nanofiber, characterized in that: It is formed by the self-assembly of the divalent or multivalent as described in any one of claims 1 to 4 under physiological conditions.

9. A coupling, characterized in that: The invention comprises the conductive polypeptide and carrier according to any one of claims 1 to 4, wherein the carrier is one or more of nanomaterials, liposomes, polymers and oily compounds.

10. The use of the conductive polypeptide of any one of claims 1 to 4, the nucleic acid of claim 5, the biomaterial of claim 6, the divalent or multivalent of claim 7, the nanofiber of claim 8, and the conjugate of claim 9 in the preparation of products for the prevention and / or treatment of nerve damage or neurological dysfunction.

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