Application of (+)-ABX-CL compound in preparation of endothelin receptor inhibitor

Abstract

The invention discloses application of a (+)-ABX-CL compound in preparation of an endothelin receptor inhibitor in the field of pharmaceutical application. Basic research is carried out for finding effective medicines for treating diseases such as tumors, heart failure, atherosclerosis, cerebral vasospasm and the like, a new lead compound is found through in-vivo and in-vitro screening, and a basis is provided for finding new medicines.

Description

Technical Field

[0001] The invention belongs to the field of drug use, and particularly relates to the use of a (+)-ABX-CL compound in the preparation of an endothelin receptor inhibitor. Background Art

[0002] Endothelin (ET) is one of the dozens of bioactive substances synthesized and released by endothelial cells. It is composed of 21 amino acid residues and contains two disulfide bonds in the molecule. It is the most powerful vasoconstrictor active peptide known so far. It was isolated and purified from the supernatant of porcine aortic endothelial cells by Japanese scholars Yanagisawa et al. in 1988. ET has four isomers, namely ET-1, ET-2, ET-3, and ET-4, all of which are polypeptides containing 21 amino acids. Among them, ET-1 is the most important form. ET-1 is mainly expressed in endothelial cells and exerts its effects through ET receptors (ETR) and its receptors ETRA and ETRB.

[0003] ET and ET receptors are widely distributed in the human body, have strong biological effects, and are involved in the pathological processes of various cardiovascular and cerebrovascular diseases such as tumors, heart failure, atherosclerosis, and cerebral vasospasm. At present, the study of ET receptor antagonists has become a hot topic in this field. Endothelin receptor antagonists can be used to treat diseases such as tumors, heart failure, atherosclerosis, and cerebral vasospasm. It is believed that the synthesis of endothelin receptor antagonists may be the simplest and most promising method for finding therapeutic drugs. Endothelin receptor antagonists can be divided into selective receptor antagonists and non-selective receptor antagonists according to the different receptor selectivity. Therefore, actively exploring and developing drugs that affect the function of endothelin - endothelin receptor antagonists for disease prevention and treatment is one of the important topics in this field. Summary of the invention

[0004] The present invention intends to provide the use of a (+)-ABX-CL compound in the preparation of an endothelin receptor inhibitor. The structural formula of the (+)-ABX-CL compound is shown below:

[0005]

[0006] In this study, the compound (+)-ABX-CL was isolated and purified from Streptomyces, and its anti-tumor activity has been approved for invention patents, with patent number ZL202210922428.6. Theoretical and experimental studies were used to evaluate the antagonistic effect of the compound on endothelin receptors, and to find lead compounds for the treatment of diseases such as tumors, heart failure, atherosclerosis and cerebral vasospasm. The molecular docking method was used to study and evaluate the binding freedom and selectivity of the compound to the ETR receptor to determine the activity of the compound; the LSPR technique was used to determine the affinity constants of ETRA and (+)-ABX-CL, and the MST technique was used to determine the affinity constants of ETRB and (+)-ABX-CL. In the experiment, the endothelin-induced mouse death model was used to screen the antagonistic effect of the compound on ETR to screen the activity of the compound, conduct basic research for the search for effective drugs for the treatment of these diseases, find new lead compounds through in vivo and in vitro screening, provide an experimental basis for the search for new drugs, and ultimately provide a new treatment approach for the treatment of patients with clinical cardiovascular and cerebrovascular diseases.

[0007] In addition, experiments have shown that the compound (+)-ABX-CL has anti-lung cancer effects, and that it is through the compound (+)-ABX-CL that it blocks the endothelin axis conduction pathway (PI3K / AKT, MAPK), thereby inhibiting the occurrence and development of tumors, providing hope for finding new targets for inhibiting tumor invasion. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Schematic diagram of the Ligand dilution method;

[0009] Figure 2 is a three-dimensional diagram of the docking of (+)-ABX-CL compound and endothelin receptor A;

[0010] Figure 3 It is a three-dimensional magnified image of the docking position of (+)-ABX-CL compound and endothelin receptor A;

[0011] Figure 4 is the two-dimensional diagram of the docking of (+)-ABX-CL compound and endothelin receptor A;

[0012] Figure 5 It is a three-dimensional magnified image of the amino acid binding interface between the (+)-ABX-CL compound and endothelin receptor A;

[0013] Figure 6 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between the (+)-ABX-CL compound and endothelin receptor A;

[0014] Figure 7 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between the (+)-ABX-CL compound and endothelin receptor A;

[0015] Figure 8 is a three-dimensional diagram of the docking of (+)-ABX-CL compound and endothelin receptor B;

[0016] Fig. 9 It is a three-dimensional magnified image of the docking position of (+)-ABX-CL compound and endothelin receptor B;

[0017] Fig.10 is the two-dimensional diagram of the docking of (+)-ABX-CL compound and endothelin receptor B;

[0018] Fig.11 It is a three-dimensional magnified image of the amino acid binding interface between the (+)-ABX-CL compound and endothelin receptor B;

[0019] Fig.12 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between the (+)-ABX-CL compound and endothelin receptor B;

[0020] Fig.13 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between (+)-ABX-CL compound and endothelin receptor B;

[0021] Fig.14 This is a three-dimensional diagram of the docking of Ambrisentan and endothelin receptor A;

[0022] Fig.15 This is a three-dimensional magnified image of the docking position of Ambrisentan and endothelin receptor A;

[0023] Fig.16 This is a two-dimensional diagram of the docking of ambrisentan and endothelin receptor A;

[0024] Fig.17 It is a three-dimensional magnified image of the amino acid binding interface between ambrisentan and endothelin receptor A;

[0025] Fig.18 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between ambrisentan and endothelin receptor A;

[0026] Fig.19 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between ambrisentan and endothelin receptor A;

[0027] Fig. 20 This is a three-dimensional diagram of the docking of ambrisentan and endothelin receptor B;

[0028] Fig.21 This is a three-dimensional magnified image of the docking position of ambrisentan and endothelin receptor B;

[0029] Fig. 22 This is a two-dimensional diagram of the docking of ambrisentan and endothelin receptor B;

[0030] Fig.23 This is a three-dimensional magnified image of the amino acid binding interface between ambrisentan and endothelin receptor B;

[0031] Fig.24 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between ambrisentan and endothelin receptor B;

[0032] Fig.25 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between ambrisentan and endothelin receptor B;

[0033] Fig.26 This is a three-dimensional diagram of the docking of bosentan and endothelin receptor A;

[0034] Fig. 27 This is a three-dimensional magnified image of the docking position of bosentan and endothelin receptor A;

[0035] Fig.28 This is the two-dimensional diagram of the docking of bosentan and endothelin receptor A;

[0036] Fig.29 It is a three-dimensional magnified image of the amino acid binding interface between bosentan and endothelin receptor A;

[0037] Fig.30 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between bosentan and endothelin receptor A;

[0038] Fig.31 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between bosentan and endothelin receptor A;

[0039] Fig.32 This is a three-dimensional diagram of the docking of bosentan and endothelin receptor B;

[0040] Fig.33 This is a three-dimensional magnified image of the docking position of bosentan and endothelin receptor B;

[0041] Fig.34 This is the two-dimensional diagram of the docking of bosentan and endothelin receptor B;

[0042] Fig.35 It is a three-dimensional magnified image of the amino acid binding interface between bosentan and endothelin receptor B;

[0043] Fig.36 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between bosentan and endothelin receptor B;

[0044] Fig.37 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between bosentan and endothelin receptor B;

[0045] Fig.38is the sensorgram of the interaction between ETRA and (+)-ABX-CL;

[0046] Fig.39 It is the time-varying diagram of the interaction between ETRB and (+)-ABX-CL (MST fluorescence diagram);

[0047] Fig.40 is the S-curve diagram of the interaction between ETRB and (+)-ABX-CL (MST S-curve diagram);

[0048] Fig.41 This is a comparison chart of the antagonistic effects of each control group on ET-1;

[0049] Fig.42 It is a volcano map of genes that are up-regulated or down-regulated after drug action in cells;

[0050] Fig.43 KEGG classification diagram of differentially expressed genes;

[0051] Fig.44 GO classification diagram of differentially expressed genes (biological process);

[0052] Fig.45 is the GO classification map of differentially expressed genes (cellular components);

[0053] Fig.46 GO classification diagram of differentially expressed genes (molecular function);

[0054] Fig.47 is the expression of endothelin ET-1 in cells;

[0055] Fig.48 is the expression of endothelin receptor ETRA after the action of (+)-ABX-CL compound;

[0056] Fig.49 This is a clone formation experiment after the (+)-ABX-CL compound acts on cells;

[0057] Fig.50 The effect of (+)-ABX-CL compound on signaling pathway. DETAILED DESCRIPTION

[0058] The following is further described in detail through specific implementation methods:

[0059] Activity verification of (+)-ABX-CL compounds

[0060] 1. Molecular docking technology

[0061] 1. Endothelin receptors A and B were selected as positive receptors, and bosentan and ambrisentan were selected as positive ligands. The molecular docking program of AutoDock Vina (Vina, version 1.1.2) was used, and the docking pocket parameters were: center_x = 18.582, center_y = 2.225, center_z = 1.53, size_x = 126, size_y = 126, size_z = 126, energy_range = 4, exhaustiveness = 8, num_modes = 9. The binding free energy in the docking results was used to determine the binding free ability and selectivity of the compound to the endothelin receptor and predict the activity of the compound.

[0062] 2. The affinity constants of ETRA and (+)-ABX-CL were determined by SPR method.

[0063] 1. Ligand Immobilization

[0064] First, the chip was activated with an activator, 400 mM EDC and 100 mM NHS were mixed immediately before injection, and the chip was activated at a flow rate of 20 μL / min for 240 s. Secondly, the endothelin receptor protein ETRA was diluted to 100 μL / mL in a fixed buffer (10 mM sodium acetate, pH 4.0) and injected into the sample channel at a flow rate of 20 μL / min. Finally, the chip was inactivated with a blocking buffer (1 M ethanolamine hydrochloride) at a flow rate of 20 μL / min for 240 s to inactivate unbound proteins.

[0065] 2. Run the drug analysis with the multi-cycle method

[0066] (+)-ABX-CL was diluted to 6 concentrations (40, 20, 10, 5, 2.5 and 0 μM) with the same analyte buffer (1×PBS (pH 7.4) + 0.005% Tween 20 + 1% DMSO). (+)-ABX-CL was injected into the sample channel at a flow rate of 20 μL / min, with a binding period of 240 s and a dissociation period of 360 s. Both the binding and dissociation processes were carried out in the analyte buffer. Six cycles were repeated in ascending order according to the analyte concentration. After each cycle of interaction analysis, the sensor chip surface should be injected with regeneration buffer (10 mM glycine-HCl, pH 1.5) as the injection buffer at a flow rate of 150 μL / min for 10 s to fully regenerate the analyte, and then the injection and regeneration steps of the analyte (+)-ABX-CL in the next concentration cycle need to be repeated.

[0067] 3. The binding status of the interaction between ETRB and ABXCL was determined by MST.

[0068] Use RED-tris-NHS second-generation dye to label the protein. Before labeling, dilute the protein sample to about 10uM.

[0069] 1. Protein labeling steps

[0070] (1) Take a new, clean 1.5 mL centrifuge tube, add 7 μL of the newly prepared RED-NHS second-generation dye and 7 μL of NHS labeling buffer, and mix them evenly with a pipette to obtain a dye solution with a final concentration of 300 μM.

[0071] (2) Take another clean 1.5mL centrifuge tube and add 90μL of the prepared protein sample (concentration 10μM). Take 10μL of the above 300μM dye solution and add it to the sample. Use a pipette to gently pipette several times to mix evenly to obtain 100μL dye-protein solution. At this time, the dye concentration in the solution is three times the protein concentration.

[0072] (3) Incubate in dark place at room temperature for 30 min.

[0073] (4) While incubating, equilibrate the B-column.

[0074] (5) Purify the labeled protein and remove the unreacted dye to obtain the final experimental protein.

[0075] 2. Affinity detection

[0076] (1) Add 10 μL of buffer to each of PCR tubes 2-16;

[0077] (2) Prepare 25 μL of Ligand. Avoid introducing buffer differences during dilution.

[0078] (3) Add 20 μL of Ligand to PCR tube No. 1;

[0079] (4) Take 10 μL from tube 1 and add it to tube 2. Mix by pipetting repeatedly. Then take 10 μL and add it to tube 3. Then, dilute tubes 4-16 in the same way. Finally, discard the excess 10 μL Ligand from tube 16. The dilution method is as shown in the attached figure. Figure 1 shown.

[0080] (1) Add 10 μL of Target to each of tubes 1-16 and mix thoroughly.

[0081] (2) The sample is sucked up with a capillary tube and then tested on a NanoTemper, Monolith NT.115 instrument.

[0082] 3. Sample Dilution

[0083] Target was diluted with PBST (1×PBS (pH 7.4) + 0.05% Tween 20). Ligand was diluted with 1% DMSO PBST (1×PBS (pH 7.4) + 0.05% Tween 20 + 1% DMSO).

[0084] IV. Screening of the antagonistic effect of compound (+)-ABX-CL on ETR using the endothelin-induced mouse death model (in vivo screening of compound activity)

[0085] 1. Experimental Materials

[0086] The positive control drugs included bosentan (McLean, CAS: 147536-97-8) and ambrisentan (McLean, CAS: 177036-94-1);

[0087] Endothelin-1 (McLean, CAS: 117399-94-7);

[0088] PEG300 (Yuanye Biotechnology, CAS: 25322-68-3);

[0089] Animals: Healthy male mice, SPF grade, weighing 22-25 g, provided by the Animal Center of Zunyi Medical University, license number: SCXK(Guizhou)2021-0002.

[0090] 2. Experimental methods

[0091] ①Solution preparation

[0092] ET-1: Prepare with sterile saline and adjust the concentration to 1.0×10 -6 mol / L is 1nmol / mL;

[0093] Positive drugs and compounds: accurately weigh a certain amount of each compound using an electronic balance, dissolve it in 70% PEG300, and adjust the drug concentration to 10 mg / mL.

[0094] ② Animal grouping: The animals were randomly divided into a positive drug group, a compound group and a blank control group, with 6 animals in each group, for a total of 30 animals.

[0095] ③Dosage regimen: The positive drug group was given intragastric administration at a dose of 100 mg / kg.

[0096] The compound group was intragastrically administered at a dose of 50 mg / kg.

[0097] The blank control group was intragastrically administered with 10 ml / kg 70% PEG300.

[0098] ET-1 10 nmol / kg was injected into the tail vein 4 hours after administration. The conditions of mice in each group after injection were observed, the number of mice that died in each group within 30 minutes was recorded, and the mouse mortality rate (%) was calculated.

[0099] Experimental Results

[0100] 1. Molecular docking results: Molecular docking prediction results show that compound (+)-ABX-CL has strong binding freedom to endothelin receptor A and endothelin receptor B. Compound (+)-ABX-CL may have multi-target effects and its effect is non-selective.

[0101] The docking results of (+)-ABX-CL and endothelin receptor A molecules: The docking results include 9 conformations, and their binding free energies are shown in the following table:

[0102] Sorting Binding free energy (kcal / mol) 1 -7.972 2 -7.782 3 -7.753 4 -7.149 5 -7.074 6 -6.927 7 -6.775 8 -6.652 9 -6.498

[0103] Since the binding free energy is less than 0 and less than -5, it can be seen that this docking can be spontaneous and stable. The first conformation is selected for binding site and force analysis. Figure 2 is a three-dimensional diagram of the docking of (+)-ABX-CL compound and endothelin receptor A; Figure 3 This is a three-dimensional enlarged image of the docking position of the (+)-ABX-CL compound and the endothelin receptor A, where the colored ribbon structure is the protein endothelin receptor A, and the red stick structure is the compound (+)-ABX-CL. Figure 4 This is a two-dimensional docking diagram of the (+)-ABX-CL compound and endothelin receptor A, which shows the name information of the amino acid residues at the docking sites of the two and the type of interaction force. It can be seen that the compound (+)-ABX-CL binds to the glutamate (GLU342) and threonine (THR76) of the protein (endothelin receptor A) in the form of hydrogen bonds; and interacts with leucine (LEU348) and leucine (LEU349) in the form of Alkyl and Pi-Alkyl bonds, respectively. Figure 5 This is a three-dimensional enlarged view of the amino acid binding interface between the (+)-ABX-CL compound and the endothelin receptor A, showing the spatial position distribution and bond length information of each amino acid residue. They are 2.1A (GLU342) and 2.5A (THR76), 4.9A (LEU348), 4.7A and 5.1 (LEU349). Figure 6 The hydrogen bond distribution diagram at the amino acid binding interface between (+)-ABX-CL compound and endothelin receptor A is shown in FIG. Figure 7 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between the (+)-ABX-CL compound and endothelin receptor A.

[0104] Docking results of (+)-ABX-CL and endothelin receptor B molecules:

[0105] The docking results of compound (+)-ABX-CL and endothelin receptor B molecule: The docking results include 9 conformations, and their binding free energies are shown in the following table:

[0106] Sorting Binding free energy (kcal / mol) 1 -9.001 2 -8.421 3 -8.286 4 -7.658 5 -7.547 6 -7.569 7 -7.364 8 -7.310 9 -7.258

[0107] Since the binding free energy is less than 0 and less than -5, it can be seen that this docking can be spontaneous and stable. The first conformation is selected for binding site and force analysis. Figure 8 is a three-dimensional diagram of the docking of (+)-ABX-CL compound and endothelin receptor B; Fig. 9 This is a three-dimensional enlarged image of the docking position of the (+)-ABX-CL compound and the endothelin receptor B, in which the colored ribbon structure is the protein endothelin receptor B, and the red stick structure is the compound (+)-ABX-CL; Fig.10 The two-dimensional docking diagram of (+)-ABX-CL compound and endothelin receptor B shows the name information of the amino acid residues at the docking sites and the types of interaction forces. It can be seen that the compound (+)-ABX-CL interacts with the proline (PRO93), proline (PRO88), lysine (LYS97), isoleucine (ILE94) and tyrosine (TYR247) of the protein (endothelin receptor B) in the form of Alkyl and Pi-Alkyl bonds, respectively. Fig.11 This is a three-dimensional magnified image of the amino acid binding interface between the (+)-ABX-CL compound and endothelin receptor B, showing the spatial position distribution and bond length information of each amino acid residue; Fig.12 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between the (+)-ABX-CL compound and endothelin receptor B; Fig.13 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between the (+)-ABX-CL compound and endothelin receptor B.

[0108] The docking results of Ambrisentan and endothelin receptor A molecules: The docking results include 9 conformations, and their binding free energies are shown in the following table:

[0109] Sorting Binding free energy (kcal / mol) 1 -7.058 2 -6.827 3 -6.748 4 -6.591 5 -6.534 6 -6.515 7 -6.435 8 -6.433 9 -6.409

[0110] Since the binding free energy is less than 0 and less than -5, it can be seen that this docking can be spontaneous and stable. The first conformation is selected for binding site and force analysis. Fig.14 This is a three-dimensional diagram of the docking of Ambrisentan and endothelin receptor A; Fig.15 This is a three-dimensional magnified image of the docking position of ambrisentan and endothelin receptor A, in which the colored ribbon structure is the protein endothelin receptor A, and the blue stick structure is the compound ambrisentan; Fig.16This is a two-dimensional docking diagram of ambrisentan and endothelin receptor A, showing the name information and interaction type of the amino acid residues at the docking sites of the two. It can be seen that ambrisentan binds to the lysine (LYS74) of the endothelin receptor A in the form of hydrogen bonds and dioxygen bonds; interacts with leucine (LEU349) in the form of Pi-Sigma bonds; and interacts with isoleucine (ILE75) in the form of Pi-Alkyl bonds; Fig.17 This is a three-dimensional magnified image of the amino acid binding interface between ambrisentan and endothelin receptor A, showing the spatial position distribution and bond length information of each amino acid residue, which are 2.5A and 3.0A (LYS74), 3.6A and 4.9A (LEU349), and 4.8 (ILE75). Fig.18 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between ambrisentan and endothelin receptor A; Fig.19 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between ambrisentan and endothelin receptor A.

[0111] The docking results of Ambrisentan and endothelin receptor B molecules: The docking results include 9 conformations, and their binding free energies are shown in the following table

[0112] Sorting Binding free energy (kcal / mol) 1 -7.258 2 -7.199 3 -6.99 4 -6.945 5 -6.599 6 -6.567 7 -6.513 8 -6.472 9 -6.417

[0113] Since the binding free energy is less than 0 and less than -5, it can be seen that this docking can be spontaneous and stable. The first conformation is selected for binding site and force analysis. Fig. 20 It is a three-dimensional diagram of the docking of Ambrisentan and endothelin receptor B (protein 5X93);

[0114] Fig.21 This is a three-dimensional magnified image of the docking position of ambrisentan and endothelin receptor B, in which the colored ribbon structure is protein 5X93 and the blue stick structure is the compound ambrisentan; Fig. 22 This is a two-dimensional docking diagram of Ambrisentan and endothelin receptor B, showing the name information and interaction type of the amino acid residues at the docking sites of the two. It can be seen that the compound binds to the arginine (ARG201) of the protein in the form of Pi-Cation bonds; interacts with proline (PRO215), lysine (LYS210) and arginine (ARG208) in the form of Alkyl and Pi-Alkyl bonds, respectively, and binds to aspartic acid (ASP198) in the form of OO bonds; Fig.23 This is a three-dimensional enlarged view of the amino acid binding interface between Ambrisentan and endothelin receptor B, showing the spatial position distribution and bond length information of each amino acid residue. They are 3.6A (ARG201), 5.5A and 4.3A (PRO215), 5A (LYS210) and 4.7A and 4A (ARG208), 2.83 (ASP198); Fig.24This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between ambrisentan and endothelin receptor B; Fig.25 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between ambrisentan and endothelin receptor B.

[0115] The docking results of bosentan and endothelin receptor A molecules: The docking results include 9 conformations, and their binding free energies are shown in the following table:

[0116]

[0117]

[0118] Since the binding free energy is less than 0 and less than -5, it can be seen that this docking can be spontaneous and stable. The first conformation is selected for binding site and force analysis. Fig.26 This is a three-dimensional diagram of the docking of bosentan and endothelin receptor A; Fig. 27 This is a three-dimensional magnified image of the docking position of Bosentan and endothelin receptor A, where the colored ribbon structure is the protein endothelin receptor A, and the blue stick structure is the compound Bosentan; Fig.28 The two-dimensional docking diagram of Bosentan and endothelin receptor A shows the name information of the amino acid residues at the docking sites and the types of interaction forces. It can be seen that Bosentan binds to the phenylalanine (PHE139) and cysteine ​​(CYS88) of endothelin receptor A in the form of hydrogen bonds; interacts with phenylalanine (PHE91) in the form of Pi-Sigma bonds; and interacts with proline (PRO135), leucine (LEU134), valine (VAL138), leucine (LEU142) and alanine (ALA13) in the form of Pi-Alkyl bonds. Fig.29 This is a three-dimensional magnified image of the amino acid binding interface between bosentan and endothelin receptor A, showing the spatial position distribution and bond length information of each amino acid residue, which are 2.7A (PHE139), 5.1A, 2.9A, 4.9A (CYS88), 3.9A (PHE91), 4.6A (PRO135), 5.5A (LEU134), 4.6A (VAL138), 5.2A (LEU142) and 4.3A (ALA13); Fig.30 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between bosentan and endothelin receptor A; Fig.31 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between bosentan and endothelin receptor A.

[0119] The docking results of bosentan and endothelin receptor B molecules: The docking results include 9 conformations, and their binding free energies are shown in the following table:

[0120] Sorting Binding free energy (kcal / mol) 1 -9.637 2 -8.217 3 -7.888 4 -7.785 5 -7.547 6 -7.464 7 -6.956 8 -6.743 9 -6.643

[0121] Since the binding free energy is less than 0 and less than -5, it can be seen that this docking can be spontaneous and stable. The first conformation is selected for binding site and force analysis. Fig.32 This is a three-dimensional diagram of the docking of bosentan and endothelin receptor B; Fig.33 This is a three-dimensional magnified image of the docking position of bosentan and endothelin receptor B, in which the colored ribbon structure is protein 5X93 and the golden stick structure is the compound bosentan; Fig.34 The two-dimensional docking diagram of Bosentan and endothelin receptor B shows the name information and interaction type of the amino acid residues at the docking sites of the two. It can be seen that Bosentan binds to glutamine (GLN181), arginine (ARG343) and lysine (LYS182) of endothelin receptor B in the form of hydrogen bonds; and interacts with leucine (LEU277), proline (PRO178), valine (VAL177) and tryptophan (TRP167) in the form of Alkyl and Pi-Alkyl bonds, respectively. Fig.35 This is a three-dimensional magnified image of the amino acid binding interface between bosentan and endothelin receptor B, showing the spatial position distribution and bond length information of each amino acid residue, which are 2.2A (GLN181), 2.3A (ARG343) and 2.3A (LYS182) in the form of hydrogen bonds; and 4.3A (LEU277) 5.4A (PRO178), 5.0A (VAL177) and 5.1 (TRP167); Fig.36 This is a diagram showing the hydrogen bond distribution at the amino acid binding interface between bosentan and endothelin receptor B; Fig.37 This is a diagram showing the distribution of hydrophobic forces at the amino acid binding interface between bosentan and endothelin receptor B.

[0122] 2. LSPR molecular interaction results:

[0123] Affinity refers to how strongly a single molecule binds to its ligand and is usually measured and reported as the equilibrium dissociation constant (KD), which can be used to assess and rank the strength of an interaction between two molecules. At equilibrium, the rate at which a molecule-ligand complex forms is equal to the rate at which it dissociates into its components [single molecule] + [ligand]. Determining the reaction rate constant defines the equilibrium or affinity constant (1 / KD). In short, the smaller the KD value, the greater the affinity of the antibody for its target. Experimentally measured dissociation and association rates (kd / ka) are used to calculate KD values. KD values ​​are the ratio of the rate at which a single molecule dissociates from its ligand (kd) to the rate at which the molecule binds to the ligand (ka).

[0124] Binding Reaction (ka) - This reaction is designed to calculate the "on rate" (ka), a constant that characterizes how quickly an antibody binds to its target.

[0125] Dissociation Reaction (kd) - This reaction is designed to calculate the "dissociation rate" (kd), a constant that characterizes how quickly an antibody dissociates from its target.

[0126] A flat slope = slow dissociation rate / strong binding. A sharp downward trend = fast dissociation rate / weak binding.

[0127] (1) Kinetic affinity: ETRA & (+)-ABX-CL

[0128] Kinetic and affinity parameters

[0129]

[0130] From the attached Fig.38 From the “sensor diagram of the interaction between ETRA and (+)-ABX-CL”, it can be seen that the drug and the ligand begin to slowly bind from about 35 seconds into the reaction and remain in a continuous binding state for about 200 seconds. After 240 seconds, when the program begins to dissociate, the drug and the ligand also begin to slowly dissociate. This indicates that the drug and the ligand protein can bind stably, and as the drug concentration increases, the response signal of the drug and the ligand protein increases accordingly.

[0131] In summary, the affinity constant of ETRA immobilized on the COOH chip binding to (+)-ABX-CL was 16.2 μM as determined by LSPR;

[0132] 3. Binding test results of ETRB and ABXCL

[0133] Attached Fig.39 Figure 39 is the MST fluorescence image. It can be seen from Appendix 39 that there is no adsorption and aggregation phenomenon when ETRB binds to (+)-ABX-CL. Fig.40 The MST S curve is shown in the attached figure. Fig.40 It can be seen that a complete S curve can be formed when the concentration of (+)-ABX-CL is in the range of 100 μM-3.05 nM. As the concentration decreases, the relative fluorescence value gradually decreases to reach equilibrium, and the response amplitude value is 2.6. From the above, it can be seen that the fluorescently labeled ETRB protein binds to ABXCL with an affinity of 1.22 uM.

[0134]

[0135] In summary, the KD value of ETRB and ABXCL was 1.22 μM.

[0136] 4. Antagonistic effect of compound (+)-ABX-CL on ETR (in vivo screening of compound activity)

[0137] The comparison of the mortality rate of mice in each group is shown in the attached Fig.41 , Fig.41 In the table below, ABX-CL refers to the compound (+)-ABX-CL; the antagonistic effects of the compounds on ET-1 are shown in the following table:

[0138] Antagonistic effect of compound (+)-ABX-CL on ET-1

[0139]

[0140] Note: The experimental results show that the mortality rate of mice in the blank group was 66.7%, and the mortality rate of mice in the bosentan group was 16.7%. Bosentan administered by gavage can effectively counteract the rapid death of mice caused by large-dose intravenous injection of ET-1, and bosentan has a strong antagonistic effect on ET-1. Ambrisentan can also effectively counteract the rapid death of mice caused by large-dose intravenous injection, with a mortality rate of 33.3%. The compound (+)-ABX-CL can also effectively counteract the rapid death of mice caused by large-dose intravenous injection, with a mortality rate of 16.7%.

[0141] In summary, large doses can cause rapid death in animals, which may be related to acute and extreme ischemia of the heart caused by arrhythmia and vasoconstriction. Endothelin receptor antagonists can effectively counteract the rapid death of animals caused by. Through theoretical and experimental research on compounds, we found that it not only has a strong binding freedom ability to in theory, but also has a good antagonistic effect on vasoconstriction in experiments (compound (+)-ABX-CL can also effectively counteract the rapid death of mice caused by large-dose intravenous injection), especially in in vivo and in vitro efficacy experiments. It has a strong effect. Therefore, the combination of theory and experiment in this application has achieved good results. Basic research is carried out to find effective drugs for the treatment of diseases such as tumors, heart failure, atherosclerosis and cerebral vasospasm, and new lead compounds are found through in vivo and in vitro screening to provide a basis for finding new drugs.

[0142] 5. Study on the mechanism of compound (+)-ABX-CL as a dual endothelin receptor antagonist

[0143] (1) Transcriptome sequencing analysis

[0144] RNA sequencing analysis was performed on lung cancer cells H1299. The results showed that most genes in the blank group were changed compared with the (+)-ABX-CL treatment group, including 788 up-regulated genes and 1729 down-regulated genes (see Appendix Fig.42 ), and the expression of endothelin receptor EDNRA decreased significantly, and the drug bound to the endothelin receptor, which was consistent with the results of molecular docking and molecular interaction. Further, the KEGG annotation results of differentially expressed genes were classified according to the pathway types in KEGG ( Fig.43), such as Wnt, TNF, Hippo, MAPK and PI3K / AKT signaling pathways. Among them, PI3K / AKT and MAPK signaling pathways accounted for the highest proportion. GO analysis mainly describes the biological process of genes ( Fig.44 ), cellular components ( Fig.45 ) and molecular function ( Fig.46 ), GO analysis revealed that it affected multiple biological pathways related to cancer.

[0145] Understanding the role and function of genes in organisms can reveal the patterns of gene expression and biological processes. It can also compare metabolic and regulatory networks under different conditions, reveal the mechanisms of biological phenomena, and provide clues for discovering new drug targets and treatment strategies.

[0146] (2) Fluorescence quantitative PCR analysis of endothelin and endothelin receptor expression

[0147] Lung cancer cells SK-MES-1, H1299, and PC9 were cultured, RNA was extracted, and fluorescence quantitative PCR was used to detect the expression of endothelin ET-1 and endothelin receptor ETRA. It was found that both were highly expressed ( Fig.47 ), and after compound intervention, the expression of ETRA in 1299 cells decreased, which is consistent with the transcriptome sequencing results ( Fig.48 ).

[0148] (3) Compound (+)-ABX-CL acts as an endothelin receptor inhibitor through the PI3K / AKT and MAPK pathways

[0149] The compound was found to have good anti-tumor activity in vitro ( Fig.49 ), WB experiments also confirmed that the compound can inhibit the PI3K / AKT signaling pathway ( Fig.50 ), therefore, in summary, we speculate that the (+)-ABX-CL compound may act as a dual endothelin receptor antagonist, blocking the endothelin axis conduction pathway (PI3K / AKT, MAPK), thereby inhibiting tumor growth.

[0150] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Use of a (+)-ABX-CL compound in the preparation of an endothelin receptor inhibitor, characterized in that: The structural formula of the (+)-ABX-CL compound is shown below:

2. Use of the endothelin receptor inhibitor according to claim 1 in treating lung cancer.

3. Use of an endothelin receptor inhibitor in anti-lung cancer according to claim 2, wherein the anti-tumor effect is to block the PI3K / AKT and / or MAPK conduction pathway of the endothelin axis through the endothelin receptor inhibitor, thereby inhibiting the occurrence and development of tumors.

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