A method of screening for trpm8 antagonists

Abstract

The application relates to the field of biological medicines, and discloses a method for screening a TRPM8 antagonist, which comprises the following steps: step 1, contacting a candidate substance with a TRPM8 protein or a functional fragment thereof, wherein the TRPM8 protein or the functional fragment thereof at least contains 796D, 861Q and 862R amino acid residues; step 2, detecting whether the candidate substance is specifically combined with at least one residue in the TRPM8 protein or the functional fragment thereof; and step 3, if combined and at the same time can produce an allosteric effect on the protein, it can be preliminarily judged that the candidate substance is a TRPM8 antagonist. The application fills the gap of the existing technology about the TRPM8 binding mode and the screening method by disclosing the key residues and the allosteric binding pocket of the TRPM8 protein combined with the antagonist, and promotes the clinical application transformation of the TRPM8 antagonist in the fields of neuropathic pain, antitumor and bone repair.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, it relates to a method for screening TRPM8 antagonists. Background Technology

[0002] Transient Receptor Potential Melastatin 8 (TRPM8) is an important ion channel protein that plays a crucial role in sensing cold stimuli and regulating nerve conduction. In recent years, TRPM8 has become an important target for treating neuropathic pain, anti-tumor therapy, and promoting bone growth. Antagonists bind to TRPM8, blocking its ion channel function and thus alleviating symptoms of related diseases. Research on TRPM8 antagonists has broad clinical application prospects. Some compounds, such as PF-05105679 and AMG-333, as selective antagonists, have completed preliminary clinical trials. New antagonists have also emerged in recent years, and antagonists derived from natural products, such as Riparin II and Riluzole, have successfully become marketed drugs.

[0003] Although some TRPM8 antagonists have been reported in the prior art, the binding mode, key binding sites, and allosteric regulatory mechanisms between TRPM8 and its small molecule antagonists have not yet been fully disclosed. A high-throughput screening method based on specific binding sites is lacking in the current technology.

[0004] Chinese patent application CN119606948A first disclosed that levistilide A and its derivatives can be used as TRPM8 antagonists and in the preparation of drugs for analgesia, antitumor, menstrual regulation, and bone promotion. However, this application only protects the specific compound levistilide A and its derivatives, without revealing its specific binding site with TRPM8, nor providing a screening method based on the specific binding site.

[0005] Therefore, developing an antagonist screening platform based on the TRPM8 specific binding site to discover TRPM8 antagonists with novel structures, higher selectivity, and fewer side effects has important clinical value. Summary of the Invention

[0006] The purpose of this application is to provide a method for screening antagonists based on specific key residues of the TRPM8 protein, so as to achieve efficient and accurate screening of TRPM8 antagonists; at the same time, it provides antagonists that specifically bind to the allosteric binding pocket of the TRPM8 protein and their pharmaceutical compositions, providing new ideas and technical support for the development of TRPM8 targeted drugs.

[0007] To achieve the above-mentioned objectives, this application adopts the following technical solution:

[0008] In a first aspect, this application provides a method for screening TRPM8 antagonists, comprising the following steps: Step 1: Contact the candidate material with TRPM8 protein or its functional fragment, wherein the TRPM8 protein or its functional fragment contains at least 796D, 861Q and 862R amino acid residues; Step 2: Detect whether the candidate substance specifically binds to at least one residue in the TRPM8 protein or its functional fragment; Step 3: If the substance binds and simultaneously induces a structural effect in the protein, then the candidate substance can be preliminarily identified as a TRPM8 antagonist.

[0009] Furthermore, the TRPM8 protein or its functional fragment also contains one or more residues selected from 797L, 798W, 1000F, 864L, and 865I.

[0010] Furthermore, in step 2, cryo-electron microscopy is used to detect whether the candidate substance specifically binds to at least one residue in the TRPM8 protein or its functional fragment.

[0011] Secondly, this application provides a TRPM8 antagonist that specifically binds to an allosteric binding pocket of the TRPM8 protein, the allosteric binding pocket containing at least residues 796D, 861Q, and 862R.

[0012] Furthermore, the allosteric binding pocket also contains one or more residues selected from 797L, 798W, 1000F, 864L, and 865I.

[0013] Thirdly, this application provides a pharmaceutical composition characterized by comprising the aforementioned TRPM8 antagonist, and a pharmaceutically acceptable carrier or excipient.

[0014] In summary, this application has the following beneficial effects: This application reveals the role of angelica lactone A, an extract of the traditional Chinese medicine Ligusticum chuanxiong, as a novel natural product-derived antagonist of the cell membrane channel protein TRPM8. Angelica lactone A and its derivatives can specifically bind to TRPM8 (through direct observation), and the specific binding sites and key action locations of both have been identified. This provides new insights into its application in the treatment of related diseases.

[0015] Furthermore, this application fills the gap in the prior art regarding TRPM8 binding modes and screening methods by disclosing the key residues and allosteric binding pockets of TRPM8 protein binding to antagonists, and promotes the clinical application of TRPM8 antagonists in the fields of neuropathic pain, anti-tumor and bone repair. Attached Figure Description

[0016] Figure 1 The expression and purification reaction system of TRPM8 membrane protein and electron microscopy images observed at 200KV; Figure 2 : Two-dimensional image and three-dimensional model of the sample after TRPM8 loading (LA); Figure 3 3D modeling of angelica lactone A (LA) combined with TRPM8; Figure 4 Structural analysis of the complex of angelica lactone A (LA) and TRPM8; Figure 5 : After a point mutation at the TRPM8 binding site, the current map of the target small molecule (LA) is obtained; Figure 6 Table of TRPM8 channel current values ​​after point mutation. Detailed Implementation

[0017] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0018] The angelicin A described in this application has the structure shown in formula (I): Equation (I); CAS Number: 88182-33-6; Molecular formula: C 24 H 28 O4; Molecular weight: 380.48; Purity: Usually ≥98%; Appearance: White to off-white crystals; Melting point: 59-61°C; Optical rotation: -44.1° (c=0.301, chloroform).

[0019] Example 1 To investigate the interaction between angelica lactone A, an extract of the traditional Chinese medicine Ligusticum chuanxiong, and the cell membrane channel protein TRPM8 (Transient Receptor Potential Melastatin 8), cryo-electron microscopy was used for direct observation (the experiment was conducted by the School of Basic Medical Sciences, Zhejiang University), and manual patch-clamp current testing was used for reverse verification (the experiment was conducted by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences).

[0020] I. Eutectic precipitation (observed by cryo-electron microscopy) Methods: A high-concentration purified protein TRPM8 and target small molecule interaction sample was prepared, and two-dimensional image data of the sample were collected by cryo-electron microscopy. Figure 1 ), using computer algorithms for three-dimensional structure reconstruction ( Figure 2 ). Elucidating the conformation of small molecule-protein interaction complexes ( Figure 3 ,4).

[0021] (1) Sample preparation: Sample selection and purification: Obtain high-purity, high-concentration protein sample (TRPM8) solutions through biochemical methods (protein expression and purification). Ensure that the sample structure is intact, homogeneous, and free of impurities or aggregates.

[0022] Screen preparation: Select a suitable screen (copper screen) and perform plasma cleaning or chemical treatment to make the screen surface hydrophilic, facilitating uniform sample distribution. The cleaned screen must be stored in a dry, dust-free environment.

[0023] Sample loading: A small amount of sample solution (3 μL) is dropped onto the carrier grid, and the sample is formed into a uniform thin layer on the carrier grid through filter paper suction or automatic adsorption device.

[0024] Rapid freezing: The carrier mesh is quickly immersed in liquid ethane or an ethane-propane mixture, causing the water molecules in the sample to vitrify rapidly, forming an amorphous ice layer and fixing the sample structure.

[0025] Protein information: 0.75 mg / mL, 3.6 uM. Small molecule incubation: M8 (3.6 uM) + Levistolide A (200 uM), incubated on ice for 5 h; Grid information: Cu300 + 2 nm C (R1.2 / 1.3); (2) Data collection: Sample transfer: The frozen grid is transferred to the sample holder of the cryo-electron microscope via a cryo-transfer system and kept at a low temperature.

[0026] Parameter settings: Select acceleration voltage (200kV, 300kV).

[0027] Image acquisition: The sample is scanned using the electron beam of a cryo-electron microscope to acquire two-dimensional projection images.

[0028] (3) Three-dimensional reconstruction: Image processing: Preprocessing of acquired 2D images, including image alignment, contrast enhancement, and noise filtering. Classification and averaging of images using specialized software.

[0029] Angle determination: The spatial orientation and center parameters of each particle or tomographic image are determined by an algorithm to establish a mapping relationship between two-dimensional images and three-dimensional structures.

[0030] 3D Reconstruction: Using the projection slicing theorem, 2D image data is integrated and reconstructed into a 3D density map.

[0031] (4) Model building and analysis: Model building: Based on the 3D density map, use molecular modeling software to build an atomic model, fit the amino acid sequence or molecular structure to the density map, and determine the atomic positions and conformations.

[0032] Model optimization: The model is optimized using methods such as energy minimization and molecular dynamics simulations to improve its accuracy and rationality. Model quality is then evaluated.

[0033] Structural analysis: Perform functional analysis on the reconstructed three-dimensional structure.

[0034] II. Binding site verification test (manual patch clamp) Methods: Using the binding sites of both obtained from the above structural analysis, the binding current after point mutation at the corresponding site of TRPM8 was measured. Figure 5 ,6), reverse verification of the binding sites of the two (completed by the Institute of Materia Medica, Chinese Academy of Sciences).

[0035] The experimental method is as follows: The channel currents of wild-type and mutant hTRPM8 were measured using the electrophysiological whole-cell manual patch-clamp method.

[0036] Cell Culture and Processing: HEK293 cell lines were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cell Passaging: The old medium was removed and the cells were washed once with PBS. Then, 1 mL of 0.25% Trypsin-EDTA solution was added, and the cells were incubated at room temperature for 1 minute. When the cells detached from the bottom of the dish, 3 mL of pre-warmed complete medium (90% DMEM + 10% FBS) at 37°C was added. The cell suspension was gently pipetted to separate the aggregated cells. The cell suspension was transferred to sterile centrifuge tubes and centrifuged at 800 rpm for 3 minutes to collect the cells. Cells were seeded in T25 cell culture flasks at a 1:5 ratio for expansion or maintenance culture. Transient Transfection: 24 hours before transient transfection, HEK-293 cells at approximately 80% confluence were reseeded into 35 mm cell culture dishes, with a seeding density of 3 × 10⁶ cells per dish. 5 Cells. Prepare transient conversion reagents for each well according to the following volumes: 1) Add 7.5 L of Lipofectamine 3000 to 250 L of Opti-MEM medium, gently mix by pipetting, and incubate at room temperature for 5 min. 2) Add 4 g of TRPM8 plasmid, 0.4 g of GFP plasmid, and 7.5 L of 3000 to 250 L of Opti-MEM medium, gently mix by pipetting, and incubate at room temperature for 5 min. 3) Add component B to component A, gently mix by pipetting, and incubate at room temperature for 15 min to form a DNA-liposome mixture. 4) Add the DNA-liposome mixture to a 35 mm... 2 500 μL per well in cell culture dishes. Place in an incubator for further culture. 5) After 6 hours, change the medium. Aspirate all the culture medium from the 35 mm diameter cell culture dishes and add 2 mL / well of complete culture medium (90% DMEM + 10% FBS). Continue culturing for 18 hours before patch-clamp detection. On the day of the experiment, aspirate the cell culture medium, rinse once with extracellular fluid, add 0.25% Trypsin-EDTA (Invitrogen) solution, and digest for 1-2 minutes at room temperature. Aspirate the digestion solution, resuspend the cells in extracellular fluid, and transfer them to experimental dishes for electrophysiological recording.

[0037] Compound preparation: 1) Menthol preparation: On the day of testing, add 100 mM Menthol-DMSO stock solution to the extracellular fluid and dilute 1000 times to obtain a 100 μM Menthol solution. 2) Assay compound angelica lactone: Add 4 mg of the compound powder to 525.7 μL of DMSO to prepare a 20 mM stock solution. Then, add 15 μL of the 20 mM stock solution to 9985 μL of a 100 μM Menthol solution and dilute 666.67 times to obtain a 30 μM test concentration. 3) Compound AMTB preparation: On the day of testing, add 20 mM of the stock solution to a 100 μM Menthol solution and dilute 1000 times to obtain a 20 μM test concentration. 4) The DMSO content in the final test concentration should not exceed 0.5%, as this concentration of DMSO has no effect on the channel current.

[0038] Electrophysiological recording procedure: HEK293 cells transiently overexpressing the hTRPM8 channel were used to record the channel currents induced by the corresponding agonist at room temperature using whole-cell patch-clamp technique. Glass microelectrodes were fabricated from glass electrode blanks (BF150-86-10, Sutter) using a stretching device (P97, Sutter). The tip resistance after perfusion with electrode fluid was approximately 2-5 MΩ. The glass microelectrodes were inserted into the amplifier probe to connect to the patch-clamp amplifier. Clamping voltage and data recording were controlled and recorded by computer using pClamp10 software, with a sampling frequency of 10 kHz and a filtering frequency of 2 kHz. After obtaining whole-cell recordings, the cells were clamped at 0 mV, and a 300 ms ramp voltage was applied from -100 mV to +100 mV, with this voltage applied every 2 s to stimulate and induce the channel current. Before drug administration, the current amplitude was greater than 300 pA. Once the current activation was confirmed to be stable and without decay, the drug administration process began. Compounds at each test concentration were administered for 1 minute to reach steady state or for a maximum of 3 minutes, with at least 3 cells tested per concentration (n≥3). After the test of the test compound was completed, AMTB was administered to inhibit the hTRPM8 current.

[0039] Data Processing: Data analysis and processing were performed using pClamp 10, GraphPad Prism 8, and Excel software. The degree of inhibition of the TRP channel current (the current amplitude induced at +100mV) by the compound was calculated using the following formula: Inhibition% = [(IMenthol - IAMTB) – (ILA - IAMTB)] / (IMenthol - IAMTB) × 100% Where, Inhibition% represents the percentage of current inhibition by the compound; IMenthol represents the current amplitude induced by the agonist Menthol; IAMTB represents the current amplitude induced by Menthol in the presence of the IAMTB inhibitor AMTB; and ILA represents the current amplitude induced by Menthol in the presence of the test compound angelocinolone.

[0040] Quality control: The experimental data in the report meet the following quality control indicators: whole-cell sealing impedance >1GΩ; AMTB inhibitors can significantly inhibit Menthol-induced TRPM8 current.

[0041] In summary, this application reveals the role of angelica lactone A, an extract of the traditional Chinese medicine Ligusticum chuanxiong, as a novel natural product-derived antagonist of the cell membrane channel protein TRPM8. Angelica lactone A and its derivatives can specifically bind to TRPM8 (through direct observation), and the specific binding sites and key action locations of both have been identified. This provides new insights into its application in the treatment of related diseases.

[0042] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for screening TRPM8 antagonists, characterized in that, Includes the following steps: Step 1: Contact the candidate material with TRPM8 protein or its functional fragment, wherein the TRPM8 protein or its functional fragment contains at least 796D, 861Q and 862R amino acid residues; Step 2: Detect whether the candidate substance specifically binds to at least one residue in the TRPM8 protein or its functional fragment; Step 3: If the substance binds and simultaneously induces a structural effect in the protein, then the candidate substance can be preliminarily identified as a TRPM8 antagonist.

2. The method for screening TRPM8 antagonists according to claim 1, characterized in that, The TRPM8 protein or its functional fragments also contain one or more residues selected from 797L, 798W, 1000F, 864L, and 865I.

3. The method for screening TRPM8 antagonists according to claim 1, characterized in that, In step 2, cryo-electron microscopy is used to detect whether the candidate substance specifically binds to at least one residue in the TRPM8 protein or its functional fragment.

4. A TRPM8 antagonist, characterized in that, It specifically binds to the allosteric binding pocket of the TRPM8 protein, which contains at least residues 796D, 861Q, and 862R.

5. The TRPM8 antagonist according to claim 4, characterized in that, The allosteric binding pocket also contains one or more residues from 797L, 798W, 1000F, 864L, and 865I.

6. A pharmaceutical composition, characterized in that, It comprises the TRPM8 antagonist as described in claim 4 or 5, and a pharmaceutically acceptable carrier or excipient.

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