Painless nerve growth factor-like peptide and its application

Abstract

The present application discloses a painless nerve growth factor-like peptide and its application. The amino acid sequence of the painless nerve growth factor-like peptide is shown in SEQ ID NO. 1-7. The painless nerve growth factor-like peptide has good biological activity, strong affinity for Trk A, good intracellular penetration, and dose-dependent cell growth promotion. In addition, the dosage for promoting cell growth is minimal. It does not cause pain hypersensitivity in animals and can overcome the pain reaction caused by NGF administration.

Description

Technical Field

[0001] The present application belongs to the field of synthetic biology technology, and in particular relates to a painless nerve growth factor-like peptide and its application. Background Art

[0002] Peripheral nerve injury (PNI) is primarily caused by falls from height, traffic accidents, cuts with sharp objects, limb ischemia and spasm, and other factors, resulting in neuronal damage and structural disruption. This damage can lead to motor, sensory, and autonomic dysfunction in patients' limbs, severely impacting their quality of life. Currently, microsurgery or autologous nerve transplantation are the primary treatments for this condition. In addition, exogenous administration of biomaterials, seed cells, and / or trophic factors has also been well-established in animal models of PNI repair. While these interventions can restore the structure and function of damaged peripheral nerves to varying degrees, patients can experience varying degrees of acute and chronic pain after surgery, manifesting as persistent, paroxysmal, or pulling pain in the surgically injured area, particularly during rest at night.

[0003] Nerve growth factor (NGF) is a secreted protein that promotes and nourishes nerves. It is widely expressed in various tissues and cells, including the brain, ganglia, skeletal muscle, glial cells, and Schwann cells. In vitro, NGF promotes the growth and development of sympathetic and sensory neurons. In vivo, exogenous NGF administration promotes early embryonic development and significantly improves axonal and myelin regeneration and motor function recovery after perineuronal infarction (PNI). NGF is also widely used in the treatment of hypoxic-ischemic encephalopathy, spinal cord injury, and diabetic peripheral neuropathy (DPN). However, after PNI, endogenous NGF expression levels are abnormally elevated. In a Phase III clinical trial for the treatment of DPN, high-dose NGF injections were associated with injection-site allodynia, accompanied by side effects such as myalgia and arthralgia. This hyperalgesia persisted for up to seven weeks in a dose-dependent manner. Therefore, addressing NGF-induced pain responses is a key scientific concern.

[0004] Currently developed painless NGF drugs all have drawbacks. For example, the preparation process for NGF-targeted monoclonal antibodies is complex and expensive, and long-term injections can be addictive and lead to neuropathy and joint damage. Small molecule compounds also lack target specificity, resulting in limited analgesic and nerve growth-promoting effects and even inducing cell apoptosis. NGF mutants are primarily obtained from patients in the Middle East with hereditary sensory and autonomic neuropathies, making their availability limited and preventing widespread application. Therefore, there is an urgent need for a simple, effective, and painless NGF peptide drug. Summary of the Invention

[0005] The embodiments of the present application provide a painless nerve growth factor-like peptide and its application. The painless nerve growth factor-like peptide of the present application has good biological activity, a strong affinity with Trk A, can penetrate well into cells and promote cell growth in a dose-dependent manner; in addition, the dosage for promoting cell growth is small, will not cause pain allergic reactions in animals, and can overcome the pain reaction caused by NGF when administered.

[0006] In a first aspect, the embodiments of the present application provide a painless nerve growth factor-like peptide, the amino acid sequence of the painless nerve growth factor-like peptide is shown in SEQ ID NO. 1-7.

[0007] Nerve growth factor (NGF) consists of a hairpin loop and an N-terminus. Loop I is the first hairpin loop region in the NGF molecule and is typically located near the N-terminus of the protein. Loop IV is another key hairpin loop region in the NGF molecule and is typically located near the C-terminus of the protein. The N-terminus of NGF refers to the region between the first amino acid residue of the protein chain and the first structural domain (usually Loop I).

[0008] TrkA (Tropomyosin receptor kinase A) is a high-affinity receptor for Nerve Growth Factor (NGF). The interaction between the two plays a critical role in the development and functional maintenance of the nervous system. The binding of NGF to TrkA is highly specific. NGF binds to the extracellular domain of TrkA through its domains (including Loop I and Loop IV), forming a stable complex. This binding triggers TrkA dimerization, the first step in signal transduction.

[0009] Crystallography and molecular structure simulations indicate that the hairpin Loop I, Loop IV, and N-term of the NGF-targeted monoclonal antibody are key regions that bind to Trk A and mediate various downstream intracellular signaling cascades. Among these three regions, studies have found that Loop I is exposed outward in the form of β-turn, and small molecule peptides with this sequence structure can increase the effect of nerve growth by 10 times. Therefore, this region exerts the greatest biological effect.

[0010] By modifying the length and amino acid mutations of the core sequence (TDIKGKEVM) of the Loop I sequence (TTATDIKGKEVMVL) that binds human NGF to Trk A, we designed small molecule peptides with different lengths and site mutations (e.g. Figure 1 Twelve linear peptides were designed and synthesized, purified, and identified through solid-phase synthesis, HPLC column chromatography, and mass spectrometry analysis. Fluorescence polarization (FP) assays, cell membrane permeation assays, and CCK-8 assays were used to screen for peptides with strong Trk A binding, good intracellular penetration, and high activity, ultimately identifying the most effective peptide.

[0011] In some embodiments, the amino acid sequence of the pain-free nerve growth factor-like peptide is shown in SEQ ID NO. 7.

[0012] Research has found that the painless nerve growth factor-like peptide with the amino acid sequence shown in SEQ ID NO. 7 has the strongest binding to Trk A, the best intracellular penetration and the highest activity.

[0013] In a second aspect, the embodiments of the present application provide a method for preparing a painless nerve growth factor-like peptide, wherein the preparation method is selected from any one of solid-phase synthesis, liquid-phase polypeptide synthesis, and genetic engineering.

[0014] In some embodiments, in the process of obtaining the pain-free nerve growth factor-like peptide by solid phase synthesis, the equivalent of each amino acid is between 2-5.

[0015] In some embodiments, a solid phase synthesis method is used, which can be used for small-scale synthesis of a small molecule polypeptide sequence as shown in any one of SEQ ID NOs. 1-7.

[0016] In a third aspect, an embodiment of the present application provides a nucleic acid molecule for large-scale encoding of an amino acid sequence as shown in any one of SEQ ID NOs. 1-7.

[0017] In a fourth aspect, an embodiment of the present application provides a recombinant plasmid, wherein the recombinant plasmid contains the nucleic acid molecule described in the third aspect.

[0018] In a fifth aspect, an embodiment of the present application provides a transformant, which comprises the nucleic acid molecule of the third aspect or the recombinant plasmid of the fourth aspect.

[0019] In some embodiments, the transformant is Escherichia coli.

[0020] In a sixth aspect, the present invention provides a drug comprising:

[0021] The pain-free nerve growth factor-like peptide of the first aspect; and

[0022] Pharmaceutically acceptable adjuvants.

[0023] Pharmaceutically acceptable adjuvants may be various buffers or various additives.

[0024] In some embodiments, the mass concentration of the pain-free nerve growth factor analog peptide in the drug is 5 ng / mL to 50 μg / mL.

[0025] In the seventh aspect, the embodiments of the present application provide an application of the painless nerve growth factor-like peptide of the first aspect, the nucleic acid molecule of the third aspect, the recombinant plasmid of the fourth aspect, or the transformant of the fifth aspect in the preparation of peripheral nerve injury drugs for analgesia.

[0026] The embodiments of the present application have at least the following beneficial effects:

[0027] The pain-free nerve growth factor-like peptide of the present application has excellent biological activity, strong affinity for Trk A, good intracellular penetration, and a dose-dependent cell growth promotion effect. Furthermore, the dosage required to promote cell growth is minimal. It does not induce pain hypersensitivity in animals, thus overcoming the pain response typically induced by NGF administration.

[0028] The painless nerve growth factor-like peptide prepared in the present application complies with the concept of green environmental protection. No environmentally unfriendly chemical reagents are used in the synthesis process, and the safety is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments of the present application. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0030] Figure 1The graph shows the binding strength of the small molecule peptides of the examples of the present application to Trk A.

[0031] Figure 2 The diagram shows the effect of cellular uptake of each FITC-linked peptide in the examples of the present application on the intracellular content.

[0032] Figure 3 A graph showing the effects of different small molecule peptides and cell survival rates according to the examples of the present application is shown.

[0033] FIG4 shows a diagram showing that NPL7 promotes nerve regeneration after PNI according to an example of the present application.

[0034] Figure 5 A graph showing the intensity of pain hypersensitivity caused by NPL7 in an embodiment of the present application when subjected to mechanical or thermal stimulation is shown. DETAILED DESCRIPTION

[0035] In order to make the invention purpose, technical solution and beneficial technical effect of this application clearer, this application is described in detail below with reference to specific embodiments. It should be understood that the embodiments described in this specification are only for explaining this application and are not intended to limit this application.

[0036] For simplicity, only some numerical ranges are explicitly disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and likewise, any upper limit can be combined with any other upper limit to form an unspecified range. In addition, although not explicitly stated, each point or individual value between the endpoints of a range is included in the range. Thus, each point or individual value can serve as its own lower limit or upper limit and be combined with any other point or individual value, or with other lower limits or upper limits, to form an unspecified range.

[0037] In the description of this article, it should be noted that, unless otherwise specified, “above” and “below” are inclusive of the number themselves, and “several” in “one or several” means two or more.

[0038] The above summary of the invention of this application is not intended to describe every disclosed embodiment or every implementation in this application. The following description more specifically illustrates exemplary embodiments. In many places throughout the application, guidance is provided through a series of examples, which can be used in various combinations. In each example, the enumeration is intended only as a representative group and should be interpreted as exhaustive.

[0039] The following examples describe the present disclosure in more detail. These examples are intended to be illustrative only, as various modifications and variations within the scope of the present disclosure will be apparent to those skilled in the art. Unless otherwise indicated, all percentages reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and used directly without further processing, and all instruments used in the examples are commercially available.

[0040] Example 1 Preparation and purification of painless nerve growth factor-like peptide

[0041] According to the mass of each pre-synthesized peptide, a certain loading degree of Rink Amide-MBHA Resin (Product No.: XW0243104183702, Jier Biochemical Co., Ltd., Shanghai, China) was weighed and transferred to a peptide receiving tube. Dimethylformamide (DMF) was added and nitrogen (N2) was introduced for 20 min. A 50% morpholine solution (morpholine:DMF = 1:1) was prepared and added to the peptide receiving tube. N2 was introduced for 30 min. min, repeat twice, alternating washing with dichloromethane (DCM) and DMF three times in between. Calculate the amount of each amino acid used at 2-5 equivalents, add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamine (DIPEA) at a molar ratio of 3.5:1, and thoroughly dissolve in an appropriate amount of DMF. Add the mixture to the peptide-seeding tube and react under N2 bubbling for 1.5-2 hours. Follow the above steps to cleave the corresponding amino acids of each peptide, according to their sequence. After the last amino acid is fused, concentrate the mixture with methanol for 5 minutes and thoroughly dry it with N2 bubbling.

[0042] The solid-phase synthesized peptide was separated from the resin using a shearing solution (trifluoroacetic acid (TFA): triisopropylsilane: purified water = 95:2.5:2.5). After carefully blowing off the TFA with N2, ether was added and ultracentrifuged for 5 minutes. After carefully removing the ether, 50% acetonitrile solution was added to the centrifuge tube containing the peptide. After the peptide was fully dissolved in acetonitrile, it was filtered using a 0.22 μm filter membrane. The high-performance liquid chromatography (HPLC) was started, the sample was injected, and the instrument parameters and time program were set. During the execution of the program by the instrument, samples with different peaks on the instrument at different time points were collected. The collected samples were identified using a mass spectrometer and the target peptide was confirmed based on the molecular weight of each peptide. The target peptide solution separated by HPLC was collected, and then the peptide solution was frozen using liquid nitrogen and placed in a freeze dryer to obtain purified peptide powder, which was stored at -20°C for long-term storage.

[0043] By these methods, amino acid sequences including linear polypeptides shown in SEQ ID No. 1-7 and 5 additional ones can be obtained, as shown in Table 1.

[0044] Table 1

[0045]

[0046] Example 2

[0047] Fluorescence polarization test (FP): According to the literature "Fluorescence Polarization Measurement System Using a Liquid Crystal Layer and an Image Sensor", the above 12 amino acid sequences were tested by fluorescence polarization test, and the following results were obtained: Figure 1 The results shown.

[0048] In the FP experiment Figure 1 The results show that NPL a The binding constants (Kd) of the 12 peptides of -NPL7 to Trk A were 88.3 nM, 87.5 nM, 107.0 nM, 182.4 nM, 146.4 nM, 97.0 nM, 350.8 nM, 585.4 nM, 135.2 nM, 136.6 nM, 299.4 nM and 24.0 nM, respectively. The Kd values ​​of each linear peptide to Trk A were all in the nM level, indicating that they bind strongly to Trk A. Among them, NPL7 has the smallest Kd value, but it is comparable to NPL a , NPL b , the numerical difference of NPL1 is smaller. In addition, NPL cThe Kd values ​​of NPL4 and NPL5 were also less than 150 nM, and further screening was required through membrane penetration and activity experiments.

[0049] Cell penetration assay (IF): According to the literature "Reversible stapling of unprotected peptides via chemoselective methionine bis-alkylation / dealkylation", the above 12 amino acid sequences were tested by fluorescence polarization assay, and the following results were obtained: Figure 2 The results shown.

[0050] In cell membrane penetration experiments Figure 2 The results showed that a certain concentration of linear peptides with FITC was added to the cell culture medium to a final concentration of 5 μM. After culturing PC12 cells for 4 hours, the cells were fixed and the nuclei were stained with DAPI. The intensity of green fluorescence emitted by the cells was observed under a laser confocal microscope. IF results showed that the amount of FITC linear peptides taken up by the cells varied. Compared with the normal group, the cells took up NPL a 、NPL d , NPL6, and NPL7 were relatively abundant, with values ​​of 165.28 ± 26.3%, 253.18 ± 36.5%, 247.16 ± 24.6%, and 355.16 ± 41.8%, respectively. Compared with other linear peptides, these four peptides emitted stronger fluorescence in PC12 cells. Therefore, these amino acid sequences can easily penetrate the membrane and then quickly penetrate into the cell to exert their drug effects.

[0051] Cell survival rate detection: The CCK-8 kit of Yisheng Biotechnology (Shanghai) Co., Ltd. was used to detect the cell survival rate of the above 12 amino acid sequences. Different linear peptides with gradient concentrations were added to PC12 cell culture medium (89% DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin / streptomycin) for 24 hours. The survival rate of cells in different groups was detected by CCK-8, and the following results were obtained: Figure 3 The results shown.

[0052] NPL c , NPL5 and NPL7 any group of cell survival ratio = NPL c , cell survival rate of any NPL5 and NPL7 groups / cell survival rate of NPL group.

[0053] Figure 3 The experimental results show that NPL c The cell growth-promoting effects of NPL5 and NPL7 increased with increasing doses, and their respective optimal administration concentrations were 400 μM, 200 μM and 100 μM.

[0054] In summary, the above analysis showed that NPL7 has a strong affinity for Trk A, can penetrate into cells well, and promotes cell growth in a dose-dependent manner; in addition, the dosage required to promote cell growth is minimal.

[0055] Animal model experiments:

[0056] A sciatic nerve compression model was established in adult SD rats and divided into three groups: PNI, NPL7, and NGF. The NGF group served as a positive control to compare with NPL7 in the evaluation of analgesia and efficacy, thereby demonstrating whether NPL7 could overcome the pain response induced by NGF while retaining its biological activity. Furthermore, to observe the morphology and function of the sciatic nerves of normal SD rats, only the sciatic nerves were exposed and designated the sham group. Different groups were administered different drugs. The NPL7 group received an in situ injection of 10 μL of NPL7 (8 ng / μL), while the NGF group received an in situ injection of 10 μL of NGF (8 ng / μL). Both groups received this drug treatment for 21 consecutive days. The PNI group received an in situ injection of normal saline for the same volume and duration. The animals were then sacrificed, and their sciatic nerve tissues were collected for pathological sectioning and staining.

[0057] Figure 4. NPL7 promotes nerve regeneration after PNI: Panel A shows H&E staining of nerves in each group; Panel B shows NF-200 and MBP fluorescence staining of nerves in each group, scale bar = 100 μm; Panel C shows the density of nerve fibers in each group; Panels D and E show the positive areas of nerves in each group stained with NF-200 and MBP. ** P < 0.01, *** P < 0.001, P > 0.05 (ns). The sciatic nerve tissues of the animals in each group were stained after sacrifice. The H&E staining results showed that the nerve fibers in the PNI group grew sparsely and had a low density. However, after treatment with NPL7 or NGF, the density of the newly formed nerve fibers increased significantly ( Figure 4 NF-200 and MBP staining results also showed that the number of axons and myelin sheaths in the PNI group was small and their arrangement was disordered, while NPL7 or NGF could significantly increase their number and arrange them in an orderly manner ( Figure 4 Figures B, D, and E show that NPL7 promotes nerve regeneration after PNI similarly to NGF. Therefore, NPL7 is as effective as NGF in promoting nerve fiber growth, axonal regeneration, and myelin sheath regeneration, indicating that the modified NPL7 still retains the biological activity of NGF.

[0058] Pain assessment: Low, medium and high doses of NPL7 or NGF (concentrations of 0.05 μg / kg, 0.5 μg / kg and 5 μg / kg, respectively) were injected into the soles of normal Balb / c mice, and the mechanical stimulation paw withdrawal threshold and paw withdrawal latency were tested using a Von Frey analgesia and hot plate apparatus before administration (marked as 0 min) and 15 min, 30 min, 45 min and 60 min after administration, respectively.

[0059] Figure 5 The results show that NPL7 at different concentrations does not cause pain hypersensitivity when mechanically or thermally stimulated. (AC) Von Frey fiber test: changes in mechanical withdrawal threshold over time after intraplantar injection of 0.05 µg / kg (low), 0.5 µg / kg (medium), and 5 µg / kg (high) doses of NPL7 or NGF in Balb / c mice; (DE) Exploration of the effects of different doses of NPL7 or NGF on the mechanical withdrawal threshold of mice; (FH) Hot plate test: changes in paw withdrawal latency over time after intraplantar injection of 0.05 µg / kg (low), 0.5 µg / kg (medium), and 5 µg / kg (high) doses of NPL7 or NGF in Balb / c mice; (IJ) Exploration of the effects of different doses of NPL7 or NGF on the paw withdrawal latency of mice. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0060] The experimental results showed that under the same dose conditions, after the injection of NGF, the mechanical withdrawal reflex threshold of mice gradually decreased over time, while the injection of NPL7 had little effect on the mechanical withdrawal reflex threshold of mice ( Figure 5 AC). In addition, the comparative analysis results of drug gradient concentrations showed that the mechanical withdrawal reflex threshold of mice did not change significantly with the increase of NPL7 injection dose, but the mechanical withdrawal reflex threshold of mice gradually decreased with the increase of NGF injection dose ( Figure 5 Similarly, the results of the hot plate test on the paw withdrawal latency of mice in different groups were consistent with the analysis trend of the Von Frey analgesia ( Figure 5 FJ). These results suggest that NPL7 injection does not induce hyperalgesia in animals, whereas NGF injection induces severe pain. Therefore, NPL7 can overcome the pain response induced by NGF administration.

[0061] The current type of disease being treated is peripheral nerve injury, and will subsequently be expanded to the treatment of neurological diseases such as spinal cord injury, craniocerebral trauma, neonatal hypoxic-ischemic encephalopathy, and neurodegenerative diseases.

Claims

1. A painless nerve growth factor-like peptide, characterized in that: The amino acid sequence of the painless nerve growth factor-like peptide is shown in SEQ ID NO.

7.

2. The method for preparing the painless nerve growth factor-like peptide according to claim 1, characterized in that: The preparation method is selected from any one of solid phase synthesis, liquid phase polypeptide synthesis and genetic engineering.

3. The method for preparing the painless nerve growth factor-like peptide according to claim 2, characterized in that: In the process of obtaining painless nerve growth factor-like peptides through solid phase synthesis, the equivalent of each amino acid is between 2 and 5.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule is used to encode the amino acid sequence shown in SEQ ID NO.

7.

5. A recombinant plasmid, characterized in that: The recombinant plasmid contains the nucleic acid molecule according to claim 4.

6. A transformant, characterized in that The transformant comprises the nucleic acid molecule according to claim 4 or the recombinant plasmid according to claim 5.

7. The transformant according to claim 6, characterized in that The transformant is Escherichia coli.

8. A drug, characterized in that include: The painless nerve growth factor-like peptide according to claim 1; as well as Pharmaceutically acceptable adjuvants.

9. The drug according to claim 8, characterized in that The mass concentration of the painless nerve growth factor-like peptide in the drug is 5 ng / mL to 50 μg / mL.

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