Screening method for a glucagon-like peptide-1 receptor agonist and use of screened compounds

Abstract

The application discloses a screening method of a glucagon-like peptide-1 receptor agonist and application of screened compounds, and belongs to the technical field of biological medicines. The screening method comprises the following steps: (1) preliminary preparation: establishing an active pocket of GLP-1R and establishing an SPR system; (2) establishing a compound library to be screened; (3) compound screening: after SPR affinity detection, molecular docking detection and pharmacodynamics verification are performed on the small-molecule compounds to be selected obtained in the step (2), the small-molecule compound Bafetinib which can be used as the glucagon-like peptide-1 receptor agonist is obtained.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for screening glucagon-like peptide-1 receptor agonists and the uses of the screened compounds. Background Technology

[0002] In recent years, the spread of metabolic diseases such as diabetes has made insulin and its replacement therapies the primary means of treating diabetes. However, while traditional treatments, which regulate blood sugar by increasing insulin secretion or supplementing with exogenous insulin, are effective, they often come with side effects such as hypoglycemia. Therefore, researchers have turned their attention to controlling blood sugar levels by modulating the glucagon-like peptide-1 (GLP-1) receptor mechanism. GLP-1 is a hormone secreted by the intestines that promotes insulin secretion, delays gastric emptying, and increases satiety. In recent years, GLP-1 receptor agonists (GLP-1RAs) have become an important direction in diabetes treatment, as they mimic GLP-1 to activate insulin secretion and lower blood sugar levels. Currently, several GLP-1RAs are available on the market, such as liraglutide, daraglutide, and sermeglutide. These drugs have achieved significant results in clinical treatment, especially in the treatment of type 2 diabetes, demonstrating good blood sugar lowering and weight loss effects. However, most of these drugs are injectable formulations, requiring patients to inject daily or weekly, which negatively impacts patient adherence and quality of life. Meanwhile, existing drugs also have side effects, such as nausea, vomiting, and pancreatitis, which limit their use in some patients. Therefore, developing new GLP-1RAs, especially compounds with good oral bioavailability and sustained activity, has become an urgent problem to be solved in the field of diabetes treatment.

[0003] With a deeper understanding of the potential of GLP-1RAs, research has gradually shifted from natural peptide compounds to synthetic small molecule compounds, which offer the advantage of oral administration. Although some potential GLP-1RAs have demonstrated efficacy in preclinical studies, challenges remain regarding bioavailability, selectivity, and side effects. Therefore, developing novel GLP-1RAs with better oral bioavailability, higher selectivity, and fewer side effects has become an important research direction for future diabetes treatment. In recent years, drug reuse has gradually gained widespread attention as a drug development strategy, mainly due to its advantages such as lower development risk, shorter development cycle, and lower development cost. These factors make drug reuse a promising drug development strategy.

[0004] Chinese patent application CN116891522A discloses a long-acting glucagon-like peptide-1 derivative, its preparation method, and its uses. The long-acting glucagon-like peptide derivative is as shown in formula (I), where the main chain X1 contains one and only one amino group of its side chain connected to an acyl group of the branch chain shown in formula (I) to form an amide. The long-acting glucagon-like peptide-1 derivative or its salts and pharmaceutical preparations are used in the treatment and / or prevention of type II diabetes, impaired glucose tolerance, type I diabetes, obesity-related diseases, metabolic syndrome, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, neurodegenerative diseases, etc. However, this patent does not address how to screen potential glucagon-like peptide-1 receptor agonists; therefore, further improvement is needed. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for discovering potential novel small molecule drugs as GLP-1R agonists.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] This invention proposes a method for screening glucagon-like peptide-1 receptor agonists, comprising the following steps:

[0008] (1) Preliminary preparation: Find the protein PDB number of the GLP-1R target agonist from the literature and establish the active pocket of GLP-1R; use CM5 chip 1 as reference pathway and CM5 chip 2 as detection pathway, and couple the sodium acetate solution of GLP-1R protein to the surface of SPR chip through cross-linking reaction to establish SPR system.

[0009] (2) Establish a library of compounds to be screened: From the T001 small molecule library, drug-like filtering is performed using Lipinski's "Rule of Five" & Veber's rules. Then, based on different docking methods of Libdock & CDOCKER, receptor-based simulated docking screening is performed. Based on the docking results, the top 5-10% of the candidate small molecule compounds are screened out.

[0010] (3) Compound screening: The candidate small molecule compounds obtained in (2) are subjected to SPR affinity detection, molecular docking detection and pharmacodynamic verification to obtain the final product.

[0011] SPR system: an active ingredient screening system based on surface plasmon resonance (SPR) biosensors.

[0012] Preferably, in (1), the final concentration of the sodium acetate solution is 40-60 μg / mL, and more preferably 50 μg / mL.

[0013] Preferably, in (2), drug-like filtration specifically screens and filters drugs from five aspects: absorption, distribution, metabolism, excretion, and toxicity.

[0014] Preferably, in (3), the SPR affinity detection is specifically performed as follows: the small molecule compound standard to be selected is dissolved in a PBS buffer solution containing 5% DMSO to prepare a gradient solution with a concentration of 6.25 to 200 μM, and the sample to be tested is injected into the SPR system at a rate of 30 μL / min.

[0015] Preferably, in the SPR system, the binding time and dissociation time of the test sample with GLP-1R protein are both set to 120s.

[0016] Preferably, in the SPR system, the analysis software built into the Biacore T200 system is used to process the data, calculate the affinity of the sample and obtain the affinity parameters. The model used is a 1:1 steady-state affinity model.

[0017] Preferably, in (3), the molecular docking detection is as follows: the crystal structure of the GLP-1R protein is downloaded from the Protein Data Bank (PDB), with PDB ID 6X1A; the chemical structures of all candidate small molecule compounds are optimized using Discovery Studio 3.0 (DS 3.0) software and saved in mol2 format; molecular docking is performed using the CDOCKER module, and the optimal conformation is evaluated based on the CDOCKER binding energy; the docking results are visualized using PYMOL software.

[0018] Preferably, in (3), the pharmacodynamic verification specifically involves detecting the change in the protein content of GLP-1R by Western blotting of small molecule compounds, and setting up a blank control group and a positive drug control group for comparison and verification.

[0019] Preferably, the drug in the positive drug control group is the GLP-1R agonist EX4.

[0020] The present invention also proposes the use of the small molecule compound Bafetinib obtained by the above screening method as a glucagon-like peptide-1 receptor agonist.

[0021] Small molecule compound Bafetinib: CAS number: 859212-16-1.

[0022] A third aspect of the present invention provides a medicament for a glucagon-like peptide-1 receptor agonist, the main component of which includes the compound Bafetinib.

[0023] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0024] "Pharmaceutical acceptable" means non-toxic material that does not diminish the active ingredient. Such pharmaceutically acceptable buffers, carriers or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, edited by A. R. Gennaro, Mack Publishing Company (1990) and hand book of Pharmaceutical Excipients, 3rd edition, edited by A. Kibbe, Pharmaceutical Press (2000)).

[0025] Preferably, the pharmaceutically acceptable carrier includes, but is not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0026] Preferably, the drug is formulated into a pharmaceutically acceptable dosage form.

[0027] Preferably, the dosage form includes tablets, capsules, granules, pills, drop pills, syrups, powders, granules, suppositories, drops, emulsions, injections, solutions, or suspensions.

[0028] The beneficial effects of this invention are as follows:

[0029] 1. This invention provides a screening method for glucagon-like peptide-1 receptor agonists. Through rigorous scientific screening procedures, small molecule compounds that can be used as glucagon-like peptide-1 receptor agonists are obtained, significantly saving research and development time and costs. A small molecule compound, Bafetinib, was obtained through this screening method.

[0030] 2. This invention utilizes drug repurposing technology to use known and clinically validated small molecules to treat other diseases. Because there is already clinical data to support it, drug repurposing can avoid complex safety assessments, significantly accelerate the clinical validation process for new indications, shorten the time to market, and significantly save research and development time and costs. It can be used in combination with existing treatment regimens to enhance efficacy or reduce side effects, and plays an important role in combating drug resistance, thereby improving clinical outcomes.

[0031] 3. Reusing clinically validated drugs reduces the risk of failure in clinical trials due to safety and toxicity issues, thus lowering R&D risks; it reveals the mechanisms of new drugs, promotes the discovery of potential links between diseases, drives the development of personalized treatment plans, and promotes medical innovation; it also reduces R&D costs and patient medical expenses, providing patients with more cost-effective treatment options. Attached Figure Description

[0032] Figure 1 The structural formula of Bafetinib (CAS No.: 859212-16-1), a small molecule compound screened in Example 1 of this invention;

[0033] Figure 2 The graph shows the SPR affinity results of Bafetinib at 6.25, 12.5, 25, 50, 100, and 200 μM in Example 1 of this invention.

[0034] Figure 3 This is a 2D interaction diagram between the small molecule compound Bafetinib and the active site of protein GLP-1R in Example 1 of this invention;

[0035] Figure 4 This is a 3D interaction diagram between the small molecule compound Bafetinib and the active site of protein GLP-1R in Example 1 of this invention;

[0036] Figure 5 The images show the Western blotting and statistical diagrams of HT-22 cells using the small molecule compound Bafetinib and the positive control drug EX4 in Example 1 of this invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0039] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0040] Example 1:

[0041] A method for screening glucagon-like peptide-1 receptor agonists, comprising the following steps:

[0042] (1) Preliminary preparation: Find the protein PDB number of the GLP-1R target agonist from the literature and establish the active pocket of GLP-1R; use CM5 chip 1 as reference pathway and CM5 chip 2 as detection pathway, and couple the sodium acetate solution of GLP-1R protein to the surface of SPR chip through cross-linking reaction to establish SPR system.

[0043] (2) Establishing a library of compounds to be screened: From the T001 small molecule library, drug-like filtering was performed using Lipinski's "Rule of Five" & Veber's rules to predict safety based on five aspects: absorption, distribution, metabolism, excretion, and toxicity; then, receptor-based simulated docking screening was performed according to different docking methods of Libdock & CDOCKER, and the top 5-10% of candidate small molecule compounds were screened based on the docking results.

[0044] (3) Compound screening: The candidate small molecule compounds obtained in (2) are subjected to SPR affinity testing, molecular docking testing, and pharmacodynamic verification to finally obtain the corresponding small molecule drugs. The SPR process is as follows:

[0045] 1. Prepare the buffer system: Measure 20ml of 10x PBS-P+buffer and 180ml of deionized water, mix well and put into a buffer bottle;

[0046] 2. Configure the reference path and detection path for the CM5 chip;

[0047] 3. Dilute 10×PBS-P+pH 7.4 10 times to prepare 200ml of running buffer;

[0048] 4. The protein was diluted to 10 μg / ml with sodium acetate at pH 5.5, 5.0, 4.5, and 4.0, respectively, with 100 μl prepared for each. Through pre-enrichment experiments, pH 4.0 was determined as the optimal coupling condition. Therefore, the ligand solution was diluted to 10 μg / ml with sodium acetate at pH 4.0, and 200 μl was prepared for the formal coupling operation.

[0049] 5. Prepare running buffer and solvent calibration curve: For small molecule samples, use PBS-P+ containing 5% DMSO as the running buffer. Take 52.5 ml of 10×PBS-P and dilute it with deionized water to 500 ml to prepare 1.05x PBS-P. Add DMSO according to the proportions in the table below to prepare 5% DMSO running buffer and solvent calibration stock solution;

[0050]

[0051] Table 1

[0052] Prepare a 5% DMSO concentration calibration curve by mixing 4.5% and 5.8% mother liquor according to the table below;

[0053]

[0054] Table 2

[0055] 6. Small molecule sample preparation: Dilute the 10mM small molecule stock solution 20-fold with 1.05x PBS-P to obtain 500μM of the small molecule in 5% DMSO. Then, dilute the analyte concentration to 200μM with the prepared 5% DMSO running buffer as the maximum injection concentration. Perform five downward halvings at concentration gradients, such as 100μM, 50μM, 25μM, 12.5μM, and 6.25μM. Set a repeat concentration at intervals, adding a 0 concentration each time.

[0056] 7. Prepare and place the sample according to its location, and seal the rubber bottle cap to prevent evaporation. Set up the program and save the method and data.

[0057] Pharmacodynamic validation: The study was divided into a blank group, a small molecule group, and a positive control group (GLP-1R agonist EX4). Changes in GLP-1R protein content were detected by Western blotting.

[0058] High protein levels indicate that the agonist is exerting its effect.

[0059] The structural formula of the small molecule compound Bafetinib obtained by screening is as follows: Figure 1 As shown.

[0060]

[0061] Table 3

[0062]

[0063] Table 4

[0064] from Figure 2As can be seen, with increasing concentration of the small molecule ligand Bafetinib, its binding signal (response value) with the immobilized protein receptor GLP-1R exhibits a dose-dependent increase, indicating a specific and reversible interaction between the two. The binding signal intensity is positively correlated with ligand concentration, which is consistent with the thermodynamic and kinetic laws of biomolecular interactions.

[0065] from Figure 3 The figure shows the 2D interaction between the small molecule compound Bafetinib and the active site of the protein GLP-1R. Leu32, Trp33, Ser206, Leu217, Phe230, and Arg380, present in the active site cavity of GLP-1R, are key amino acids in the docking process. The figure also shows that the tight binding of GLP-1R to Bafetinib is mainly due to the combined effects of hydrogen bonding, hydrophobicity, π-stacking, and electrostatic forces, with hydrogen bonding being the dominant force.

[0066] from Figure 4 As can be seen, the small molecule compound Bafetinib interacts 3D with the active site of the protein GLP-1R.

[0067] Table 3 shows the core parameters for quantifying the binding strength between the small molecule compound Bafetinib and the protein GLP-1R in molecular docking. These parameters can be used to systematically evaluate the binding potential of small molecule compounds and proteins, providing a theoretical basis for drug design.

[0068] Table 4 shows the kinetic data, including the affinity, kinetic properties, and data fitting quality, of the intermolecular interaction between the small molecule compound Bafetinib and the positive control EX4. A 1:1 Langmuir model was used for fitting, obtaining parameters such as KD, ka, and kd. The fitting quality was evaluated using χ² (χ²). 2 <5RU 2 ).

[0069] from Figure 5 As can be seen from the Western blotting and statistical diagrams of HT-22 cells stimulated by the small molecule compound Bafetinib and the positive control drug EX4, both Bafetinib and EX4 can reverse the trend of decreasing GLP-1R protein content in the MOD group.

[0070] Therefore, the small molecule compound Bafetinib obtained through screening can be used as a glucagon-like peptide-1 receptor agonist.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of compound Bafetinib in the preparation of drugs for glucagon-like peptide-1 receptor agonists.

2. The use according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

3. The use according to claim 2, characterized in that, Pharmaceutically acceptable carriers include diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, or disintegrants.

4. The use according to claim 3, characterized in that, The pharmaceutically acceptable carrier is a diluent.

5. The use according to claim 3, characterized in that, The pharmaceutically acceptable carrier is an adhesive.

6. The use according to claim 3, characterized in that, The pharmaceutically acceptable carrier is a surfactant.

7. The use according to claim 1, characterized in that, The drug is formulated into a pharmaceutically acceptable dosage form.

8. The use according to claim 7, characterized in that, The dosage form is a tablet.

9. The use according to claim 7, characterized in that, The dosage form is capsules.

10. The use according to claim 7, characterized in that, The dosage form is granules.

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