A polypeptide agonist targeting the allosteric site of cannabinoid receptor type 2 and uses thereof

Abstract

The application discloses a polypeptide agonist targeting a cannabinoid type 2 receptor allosteric site and application thereof, an amino acid sequence of the polypeptide agonist Pep-5 is SEQ ID NO.1: RWRWYYNRKKVT, the application is based on development of a polypeptide allosteric regulator of an extracellular domain site J site, flexible docking is carried out on a plurality of candidate polypeptides by using HPEPDOCK technology, and polypeptide activity is verified based on a cAMP detection method of LC-MS / MS, and the result shows that the inhibiting effect of the polypeptide agonist Pep-5 on cAMP in CHO-K1 cells transfected with a CB2 receptor plasmid is typical S type dose dependence, and the EC50 value is 7.306 μM, the polypeptide agonist can agonize the CB2 receptor in a low micromolar concentration range, and can be used for developing a high selectivity, metabolically stable anti-inflammatory analgesic candidate drug.

Description

Technical Field

[0001] This invention relates to a polypeptide agonist, particularly to a polypeptide agonist that targets the allosteric site of the cannabinoid type 2 receptor, and also to the application of the above agonist in CB2 receptor agonists, anti-inflammatory analgesics, or neurological disease drugs. Background Technology

[0002] Cannabinoid receptor type 2 (CB2), a key member of the endocannabinoid system (ECS), is widely expressed in the peripheral and immune systems and is a core target for regulating acute and chronic pain, especially neuropathic and inflammatory pain. Through the CB2 receptor-mediated signaling pathway, the ECS can effectively inhibit pain signal transmission, reduce neuroinflammation, and decrease immune cell activation, thereby producing analgesic effects. Unlike the CB1 (cannabinoid receptor type 1) receptor, which is mainly distributed in the central nervous system and is prone to causing psychogenic side effects, activation of the CB2 receptor does not produce addictive or sedative central nervous system side effects. This makes it an ideal target for developing novel, non-addictive, and highly safe analgesics. Recent studies have confirmed that CB2 agonists can inhibit microglial activation and regulate the ERK / p38-MAPK pathway, alleviating the pathological progression of Alzheimer's and Parkinson's diseases; they can also inhibit peripheral neuronal sensitization and the release of inflammatory mediators, providing a new non-addictive analgesic strategy; and they have shown regulatory effects in bone metabolism, liver fibrosis, and intestinal inflammation.

[0003] Existing CB2 small molecule agonists can be mainly divided into the following categories: First, plant-derived cannabinoids and their derivatives, such as cannabidiol, but these molecules usually have limited selectivity for CB2 and are prone to cross-reaction with CB1 receptors, leading to the risk of central nervous system side effects; Second, synthetic small molecule agonists, including indole, pyrazole, quinoline and other skeletal structures. Although some molecules with better selectivity have been obtained in recent years through medicinal chemistry optimization, they still generally have physicochemical problems such as excessive lipophilicity, low oral bioavailability and poor metabolic stability, making it difficult to meet the requirements of clinical translation; Third, conformation-restricted agonists that have emerged in recent years, which improve selectivity through structural rigidification strategies, but still mainly act on the orthomeric sites of the receptor.

[0004] Despite the progress made in the aforementioned research, existing CB2 agonists still face three major shortcomings: First, the problem of insufficient selectivity has not been fundamentally solved, with most molecules still exhibiting varying degrees of cross-activity with CB1, and the risk of central nervous system side effects remains; second, ortho-associated site agonists generally carry the risk of receptor desensitization, and the decline in efficacy after long-term use is a key bottleneck restricting their clinical application; third, clinical translation is progressing slowly, and the few CB2 agonists that have entered clinical trials have limited efficacy or have failed to reach the treatment endpoint.

[0005] Against this backdrop, allosteric regulation strategies offer a novel approach to overcoming the aforementioned bottlenecks due to their unique mechanistic advantages—selectively enhancing or modulating endogenous ligand signal transduction efficiency by binding to allosteric sites distal to the receptor. Compared to ortho-agonists, allosteric modulators can achieve more precise receptor function regulation by modulating the activity of the endocannabinoid system, theoretically possessing higher subtype selectivity, lower desensitization risk, and superior safety. Among the many types of allosteric modulators, peptide molecules have attracted widespread attention in the field of GPCR-targeted allosteric regulation due to their unique advantages such as high selectivity, low immunogenicity, low toxicity, and strong structural designability. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a polypeptide agonist that targets the allosteric site of the cannabinoid type 2 receptor, and also to provide the application of the above agonist.

[0007] Technical solution: The amino acid sequence of the peptide agonist Pep-5, which targets the allosteric site of the cannabinoid type 2 receptor, is SEQ ID NO.1: RWRWYYNRKKVT; it is designed using allosteric site molecular docking technology to specifically target the CB2 receptor and avoid cross-reaction with the CB1 receptor.

[0008] The peptide agonist also includes corresponding modifications to the peptide sequence Pep-5, with the Pep-5 sequence as the core. The modifying materials include nanomaterials, fluorescent materials, enzymes, and biotin. The modifications are selected from natural or non-natural amino acid substitutions, deletions, insertions, and terminal amidation, PEGylation, lipidation, or cyclization.

[0009] The aforementioned peptide agonists targeting the allosteric sites of cannabinoid type 2 receptors can be used in the preparation of CB2 receptor agonists, anti-inflammatory and analgesic drugs, or drugs for treating neurological diseases.

[0010] The present invention also discloses a pharmaceutical composition comprising a polypeptide Pep-5 targeting the allosteric site of cannabinoid type 2 receptor, and a pharmaceutically acceptable carrier or excipient.

[0011] The above-mentioned pharmaceutical composition can be used in the preparation of CB2 receptor agonists, anti-inflammatory and analgesic drugs, or drugs for treating neurological diseases.

[0012] Invention Principle: This invention utilizes computer-aided design to focus on the rational design and virtual screening of CB2 receptor allosteric modulators. By integrating structural biology and computational simulation techniques, it systematically explores a development strategy for peptide allosteric modulators based on the extracellular domain site J. Structural feature analysis shows that the peptide sequence RWRWYYNRKKVT (named Pep-5), obtained through molecular docking technology, exhibits unique structural characteristics, enabling it to precisely target the allosteric site J of the CB2 receptor. Computer simulations predict that this sequence has a tendency to form an amphiphilic α-helix: its N-terminus is rich in arginine (R) and tryptophan (W) residues, which can form a cation-π interaction network, facilitating electrostatic recognition with acid-rich regions (such as D24 and E181) in the allosteric site J of the CB2 receptor; the YY motif in the middle of the sequence forms a stable hydrophobic interaction with hydrophobic residues (such as P178 and L185) in site J through aromatic ring stacking—a feature highly consistent with the key role of the aromaticity of tyrosine residues in cannabinoid receptors in ligand recognition; the KKVT fragment at the C-terminus can further anchor polar residues (such as N188 and T272) at the top of site J through a hydrogen bonding network. This structural feature of multiple non-covalent interactions allows Pep-5 to specifically embed into the allosteric site J of the CB2 receptor with high affinity, precisely regulating receptor activity in terms of spatial conformation, while effectively avoiding cross-reaction with the CB1 receptor.

[0013] Nine candidate peptides were flexibly docked using HPEPDOCK technology, allowing for main chain fine-tuning and side chain rotation, to screen for highly active sequences. Pep-5, due to its unique structural features, achieved a docking score of -210, significantly superior to other candidate molecules (score range -165 to -190), demonstrating its structural complementarity with the CB2 allosteric site. To accurately assess regulatory activity, a cAMP detection method based on LC-MS / MS was developed. Results showed that Pep-5 can efficiently regulate the CB2 receptor through an allosteric mechanism, with an EC50 value of 7.306 μM. Compared with existing CB2-targeting peptides (such as vodo-C1 EC50), this demonstrates superior activity. 50At a concentration of 7.8 μM, the Pep-5 of this invention achieves a leading level of agonistic activity among similar molecules, while possessing the dual advantages of high selectivity (derived from allosteric site targeting) and good structural designability (derived from linear polypeptide sequences). More importantly, the dense distribution of basic amino acids in Pep-5 (containing 4 arginines and 2 lysines) endows it with good water solubility and cell membrane penetration ability, laying a physicochemical foundation for its subsequent cellular and in vivo pharmacodynamic studies. In summary, this invention, through structural feature-driven rational design, achieves precise targeting of the CB2 receptor allosteric site and obtains a clear agonistic effect in the low micromolar concentration range. This non-obvious technical effect fully demonstrates the inventiveness and progress of this application.

[0014] Furthermore, this invention provides an LC-MS / MS method for detecting cAMP to evaluate the regulatory activity of the agonist peptide. By optimizing chromatographic conditions (0.1% formic acid-water-acetonitrile, column temperature 40°C) and MRM mode, linear detection of 2.5–1000 nM (R² > 0.99) is achieved, and the single-sample analysis time is reduced to 5 minutes. In CHO-K1 cell lysates, the matrix effect is controlled at 92.96–103.15% (RSD ≤ 19.41%), and the recovery rate at a low concentration of 7.5 nM is 62.31 ± 8.47%, meeting the requirements for trace detection. Compared to traditional immunoassays (dynamic range of 2 orders of magnitude), this method extends the detection range to 3 orders of magnitude and is not affected by antibody cross-reactivity.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) Based on the allosteric characteristics of the CB2 allosteric site J, the present invention uses HPEPDOCK technology to perform flexible docking on several candidate peptides and screens out the high-activity sequence of the present invention. The docking score of peptide Pep-5 reaches -210, which is better than other candidate molecules; (2) Based on the cAMP detection method of LC-MS / MS to evaluate the regulatory activity, the Pep-5 peptide agonist of the present invention shows a typical S-type dose-dependent inhibitory effect on cAMP in CHO-K1 cells transfected with CB2 receptor plasmid. The EC50 value is 7.306 μM, indicating that the peptide agonist can efficiently and specifically agonize the CB2 receptor in the low micromolar concentration range, thereby significantly inhibiting the intracellular cAMP level. The high efficiency of CB2 agonist activity shown by the peptide of the present invention strongly predicts that it has good analgesic application potential and can be used as a lead compound for the development of new analgesics. Attached Figure Description

[0016] Figure 1 This is a diagram illustrating the interaction between the polypeptide agonist and the CB2 receptor of this invention.

[0017] Figure 2This is a curve showing the concentration-dependent inhibitory effect of the polypeptide agonist of the present invention on intracellular cAMP and the fitted EC50 value. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings. All materials and reagents used in the present invention are purchased from commercial products.

[0019] Example 1

[0020] The crystal structure of the human CB2 receptor (active state PDB ID: 8gus, resolution 2.84 Å) was obtained from the PDB database. The original file was preprocessed using the PDBfixer tool, specifically by adding missing heavy atoms and hydrogen atoms, constructing missing ring regions, converting non-standard residues to standard amino acid forms, and deleting heterogeneous molecules irrelevant to binding (such as water molecules, crystal buffer ions, and irrelevant ligands). Based on literature reports, the CB2 receptor allosteric site J consists of residues at the top of transmembrane regions 3-7 (M 22, D 24, Y 25, P 178, C 179, S 180, E 181, L 185, N 188, T 272). The coordinates of these residues were extracted using PyMOL's select command to generate the binding site file. The peptide ligand was input in FASTA format. Using the HPEPDOCK molecular docking tool, the MODPEP program was set to generate 1000 peptide conformations for the peptide, with 100 peptide binding modes output. The binding site was specified as the allosteric site J, thus improving the accuracy of the prediction model. The HPEPDOCK default composite scoring function was used, and the binding energy term included van der Waals forces, electrostatic potential, and desolvation effect. By default, the server performed global flexible peptide docking, and the preferred peptide sequence was RWRWYYNRKKVT, with a molecular docking score of -210.

[0021] Example 2

[0022] To evaluate the allosteric regulatory effect of the peptide on the CB2 receptor, a basal medium containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 50 μM forscolin (FSK) was used as a diluent to serially dilute a 5 mg / ml peptide stock solution to obtain drug solutions with concentration gradients of 200 μM, 50 μM, 20 μM, 5 μM, 3 μM, and 1 μM. CHO-K1 cells transfected with the CB2 receptor plasmid were seeded in 24-well plates, and the original medium was discarded before drug administration. 500 μl of the corresponding concentration of drug solution was added to each well, with three replicates for each concentration. Three blank control wells (containing only IBMX-FSK medium) were also included. After drug administration, the cells were incubated at 37°C and 5% CO2 for 2 hours. After discarding the drug solution, 200 μl of pre-chilled cell lysis buffer (80% acetonitrile) was quickly added, and the cells were mixed by pipetting and lysed at room temperature for 3 minutes. The solution was filtered through a 0.22 μm polyethersulfone (PES) membrane, and the filtrate was collected for LC-MS / MS detection.

[0023] LC-MS / MS detection was performed using a Thermo BDS HYPERSIL C8 column (100 × 4.6 mm, 2.4 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution, with a mobile phase ratio of 60:40. The column temperature was 40℃, the flow rate was 0.4 mL / min, the injection chamber temperature was 4℃, and the injection volume was 2 μL. The mass spectrometry conditions were: ESI electrospray ionization source (positive ion mode), drying gas temperature 300℃, flow rate 5 L / min, nebulizer gas pressure 45 psi, sheath gas temperature 250℃, flow rate 11.0 L / min, capillary voltage 3500 V, and nozzle voltage 500 V. The scan was performed in multiple reaction monitoring (MRM) mode, monitoring ion pairs m / z 330.2→136.1 (collision energy 30 eV), and the target analyte retention time was 2.401 min.

[0024] Results of in vitro functional experiments as follows Figure 2 As shown, Pep-5 exhibits a typical S-shaped dose-dependent curve for cAMP inhibition mediated by the CB2 receptor. Nonlinear regression fitting yielded an EC50 value of 7.306 μM (Hillslope = 1.184), indicating that the binding of Pep-5 to the CB2 receptor conforms to a single-point action model, with no significant synergistic or antagonistic effects. Figure 1 Molecular docking results and Figure 2 Functional activity data show that: Figure 1 The revealed multi-non-covalent interaction network (cation-π, aromatic ring stacking, hydrogen bonding) enables Pep-5 to achieve a high docking score of -210 and to stably bind to the CB2 allosteric site J with high affinity. Figure 2A clear dose-dependent agonistic effect was observed. Compared with other CB2-targeting peptides reported in the literature (such as vodo-C1 EC50≈8 μM), the agonistic activity of the peptides of this invention reaches the forefront of similar molecules.

[0025] Therefore, this invention develops a polypeptide allosteric regulator based on the J site of the extracellular domain. Several candidate polypeptides were flexibly docked using HPEPDOCK technology to obtain the polypeptide agonist Pep-5. The polypeptide activity was verified using LC-MS / MS cAMP detection. The results showed that the Pep-5 polypeptide agonist exhibited a typical S-type dose-dependent inhibitory effect on cAMP in CHO-K1 cells transfected with the CB2 receptor plasmid, with an EC50 value of 7.306 μM. This polypeptide agonist activates the CB2 receptor even at low micromolar concentrations. The polypeptide of this invention can serve as a potent candidate molecule for further in-depth analgesic pharmacodynamic evaluation and the development of highly selective, metabolically stable anti-inflammatory analgesic candidate drugs.

Claims

1. A polypeptide agonist targeting the allosteric site of cannabinoid type 2 receptor, characterized in that, The amino acid sequence of the peptide agonist Pep-5 is SEQ ID NO.1: RWRWYYNRKKVT.

2. The polypeptide agonist targeting the allosteric site of cannabinoid type 2 receptor according to claim 1, characterized in that, The peptide agonist comprises a peptide sequence Pep-5 as the core, and corresponding modifications to the peptide sequence Pep-5.

3. The polypeptide agonist targeting the allosteric site of cannabinoid type 2 receptor according to claim 2, characterized in that, The modified materials include nanomaterials, fluorescent materials, enzymes, and biotin; the modifications are selected from natural or non-natural amino acid substitutions, deletions, insertions, and terminal amidation, PEGylation, lipidation, or cyclization.

4. The use of the polypeptide agonist targeting the cannabinoid type 2 receptor allosteric site as described in claim 1 in the preparation of CB2 receptor agonists.

5. The use of the polypeptide agonist targeting the cannabinoid type 2 receptor allosteric site as described in claim 1 in the preparation of an anti-inflammatory and analgesic drug.

6. The use of the polypeptide agonist targeting the cannabinoid type 2 receptor allosteric site as described in claim 1 in the preparation of a medicament for treating neurological diseases.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the peptide Pep-5, which targets the allosteric site of the cannabinoid type 2 receptor as described in claim 1, and a pharmaceutically acceptable carrier or excipient.

8. Use of the pharmaceutical composition of claim 7 in the preparation of a CB2 receptor agonist.

9. Use of the pharmaceutical composition of claim 7 in the preparation of an anti-inflammatory and analgesic drug.

10. Use of the pharmaceutical composition of claim 7 in the preparation of a medicament for treating neurological diseases.

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