Application of compounds targeting GPR183 in the preparation of drugs for treating acute myeloid leukemia

Abstract

The present application discloses the use of a compound targeting GPR183 in the preparation of a drug for treating acute myeloid leukemia. The compound is N-{6-[4-(2,4-dichlorobenzoyl)piperazin-1-yl]pyridin-3-yl}-N'-[4-(trifluoromethyl)phenyl]urea. The present application uses a computer virtual screening method to screen compounds effective against AML. Drug sensitivity experiments show that the compound has a killing effect on AML, and molecular docking analysis, RT-qPCR, and protein blotting prove that it can directly bind to GPR183, reduce the expression of GPR183 in AML cells, and inhibit AML cell proliferation. Therefore, the compound can be used as a compound targeting GPR183 in the preparation of a drug for treating acute myeloid leukemia.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a compound targeting GPR183 in the preparation of a drug for treating acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is the most common type of leukemia in adults, characterized by rapid progression and high heterogeneity. AML often develops in the elderly, with a median age of 68. Elderly patients have a poor prognosis, with a 5-year overall survival rate of less than 20%.

[0003] In related technologies, the chemotherapy regimen of cytarabine combined with anthracyclines is still the first-line treatment for AML, with an overall remission rate of about 70%, but the relapse and refractory rate exceeds 50%. Therefore, it is necessary to explore therapeutic targets and targeted drugs for highly heterogeneous AML.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the Invention

[0005] The technical task of the present application is to address the above shortcomings and provide an application of a compound targeting GPR183 in the preparation of a drug for treating acute myeloid leukemia. The present application uses a computer virtual screening method to screen compounds effective against AML. Drug sensitivity experiments show that N-{6-[4-(2,4-dichlorobenzoyl)piperazin-1-yl]pyridin-3-yl}-N'-[4-(trifluoromethyl)phenyl]urea has a killing effect on AML, and molecular docking analysis, RT-qPCR, and protein blotting have proven that it can directly bind to GPR183, reduce the expression of GPR183 in AML cells, and inhibit AML cell proliferation. Therefore, N-{6-[4-(2,4-dichlorobenzoyl)piperazin-1-yl]pyridin-3-yl}-N'-[4-(trifluoromethyl)phenyl]urea can be used as a compound targeting GPR183 in the preparation of drugs for treating acute myeloid leukemia.

[0006] To achieve the above objectives, this application provides the following technical solutions:

[0007] According to one aspect of the present application, a compound targeting GPR183 is provided for use in the preparation of a drug for treating acute myeloid leukemia, wherein the compound is N-{6-[4-(2,4-dichlorobenzoyl)piperazin-1-yl]pyridin-3-yl}-N'-[4-(trifluoromethyl)phenyl]urea, and the chemical structure is shown below:

[0008] .

[0009] In some embodiments, the compound targets the GPR183 gene.

[0010] In some embodiments, the compound has the effect of reducing GPR183 expression in acute myeloid leukemia cells.

[0011] In some embodiments, the compound has a killing effect on acute myeloid leukemia cell lines.

[0012] In some embodiments, the acute myeloid leukemia cell line is one of THP-1, MV4-11, and MOLM-13.

[0013] In some embodiments, the compound has a killing effect on primary cells from untreated acute myeloid leukemia patients.

[0014] In some embodiments, the compound has the effect of inhibiting acute myeloid leukemia cells.

[0015] In some embodiments, the acute myeloid leukemia cells are THP-1 or MOLM-13.

[0016] In some embodiments, the dosage form of the drug includes at least one of an injection, a tablet, a pill, and a granule.

[0017] Compared with the prior art, the advantages and positive effects of the present application are: the present application uses a computer virtual screening method to screen compounds effective against AML, and through drug sensitivity experiments, it is found that N-{6-[4-(2,4-dichlorobenzoyl)piperazin-1-yl]pyridin-3-yl}-N'-[4-(trifluoromethyl)phenyl]urea has a killing effect on AML, and through molecular docking analysis, RT-qPCR, and protein blotting, it is proved that it can directly bind to GPR183, reduce the expression of GPR183 in AML cells, and inhibit AML cell proliferation. Therefore, N-{6-[4-(2,4-dichlorobenzoyl)piperazin-1-yl]pyridin-3-yl}-N'-[4-(trifluoromethyl)phenyl]urea can be used as a compound targeting GPR183 in the preparation of drugs for the treatment of acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 Shown is a flow chart for screening compounds targeting GPR183 in Example 1 of the present application;

[0020] Figure 2 The molecular docking diagram and binding site diagram of GPG183 and N6 in Example 1 of the present application are shown;

[0021] Figure 3 The effect of different concentrations of N6 on GPR183 protein in AML cells in Example 1 of the present application is shown;

[0022] Figure 4 The inhibition fitting curves of N6 on different cell lines in Example 2 of the present application are shown;

[0023] Figure 5 The figure shows the inhibitory fitting curve of N6 on primary cells of untreated AML patients with different diagnoses in Example 3 of the present application;

[0024] Figure 6 Shown are the inhibitory fitting curves of different GPR183 inhibitors or antagonists on AML in Example 4 of the present application;

[0025] Figure 7The cell growth rate curve of AML cell line THP-1 at different concentrations of N6 in Example 5 of the present application is shown;

[0026] Figure 8 The cell growth rate curve of AML cell line MOLM-13 at different concentrations of N6 in Example 5 of the present application is shown. DETAILED DESCRIPTION

[0027] In order to more clearly understand the above-mentioned objects, features and advantages of the present application, the present application is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the embodiments of the present application and the features therein can be combined with each other without conflict.

[0028] N-{6-[4-(2,4-dichlorobenzoyl)piperazin-1-yl]pyridin-3-yl}-N'-[4-(trifluoromethyl)phenyl]urea (n-{6-[4-(2,4-dichlorobenzoyl)piperazin-1-yl]pyridin-3-yl}-n'-[4-(trifluoromethyl)phenyl]urea): This compound is referred to as N6 in this patent. N6 is from the ChemDiv library (official link: https: / / www.chemdiv.com / ), with an ID of V023-4607; a molecular weight of 538.36; a short organic molecular structure (Smiles formula) of C1CN(CCN1C(c1ccc(cc1[Cl])[Cl])=O)c1ccc(cn1)NC(Nc1ccc(cc1)C(F)(F)F)=O; the molecular formula is C 24 H 20 Cl2F3N5O2, chemical formula is .

[0029] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0030] Example 1: Virtual screening and verification of compounds capable of binding to GPR183 receptor protein

[0031] Virtual screening uses a mixed ligand-based and receptor-based strategy. Figure 1 The screening flow chart of the compound targeting GPR183 in Example 1 of the present application is shown. Figure 1As shown in the figure, approximately 1.5 million compounds from the ChemDiv compound library (official link: https: / / www.chemdiv.com / ) were used as a virtual screening library. All compounds were first desalted and freed of free ions. Three-dimensional shape similarity matching was then performed using a weighted Gaussian algorithm with the inverse agonist GSK682753A (PMID: 35537452). The top 50,000 compounds with the highest similarity scores were used for the next docking simulation.

[0032] Docking simulations were performed using Autodock Vina, using the crystal structure of GPR183 (PDB code: 7TUY) as the receptor and 50,000 similar compounds as the ligand. The binding pocket of the inverse agonist GSK682753A was used as the docking pocket. Before docking, the grid box center and dimensions were set according to the pocket. During docking, the receptor protein was rigid, while the ligand had a certain degree of flexibility. This semi-flexible docking calculation was performed using the Lamarckian genetic algorithm. The docking process searches for possible ligand conformations within the grid box and selects the one with the lowest binding energy as the optimal conformation. The top 10,000 compounds were ranked according to their docking scores and clustered using molecular fingerprint analysis. A fingerprint similarity cutoff of 0.7 was set (i.e., compounds with a fingerprint similarity greater than or equal to 0.7 were considered to belong to the same class). The highest-scoring compound for each class was selected as the representative molecule, resulting in a total of 3,939 compounds. The 3939 compounds were ranked according to the docking scores, and the top 1000 compounds that could potentially bind to the GPR183 receptor protein were finally obtained as hit molecules.

[0033] Based on the docking scores, the APIs of the top 17 compounds (Top 17) with the strongest docking scores among the 1000 hit molecules were purchased. The APIs were purchased from the ChemDiv compound library. The compound numbers, IDs, purchased masses, and molecular weights are shown in Table 1. Drug sensitivity experiments were performed to screen for compounds that were effective against AML cells.

[0034] Table 1 ChemDiv compound library information and computer virtual docking scores of the top 17 compounds

[0035]

[0036] The raw material of Top17 compound was dissolved in DMSO and prepared into a stock solution concentration of 50mM. The concentration was set at 50uM as the cell action concentration for the initial screening. The AML cell line THP-1 (ATCC, catalog number: TIB-202) was used as the drug screening carrier, and the plating concentration was 2x10 5The stock solution of Top17 compounds was plated in 24-well plates at 10 μM / ml. The stock solution was further diluted to a 10 mM working concentration, and 5 μl was added to each well. A control well was also set up. After 48 hours, the solution was pipetted and transferred to a 96-well plate at a rate of 100 μl / well. Three replicates were set up, and 20 μl of MTS was added to each well. After 4 hours, the absorbance was measured using a microplate reader, and the inhibitory rate of Top17 compounds against the AML cell line THP-1 was calculated. The results are shown in Table 2. At a concentration of 10 μM, only N6 of the Top17 compounds showed a killing effect on the AML cell line THP-1, indicating that it is an effective AML inhibitor.

[0037] Table 2 Inhibitory effect of Top17 compounds on AML cells

[0038]

[0039] Figure 2 The molecular docking diagram and binding site diagram of GPG183 and N6 in Example 1 of the present application are shown. The specific experimental steps are to download the molecular structure 3D file of N6 from the ChemDiv website (https: / / pubchem.ncbi.nlm.nih.gov / ). Download the crystal structure of GPR183 from the PDB website (https: / / www.rcsb.org / ), and select the PDB code of GPR183: 7TUY crystal structure. Further use Auto Dock4 software for molecular docking. The specific docking steps refer to the official website of autodock (https: / / autodock.scripps.edu / ), and finally use PyMol software to display the docking results and output images. The docking results are as follows. Figure 2 Figure 2 shows the three-dimensional binding pattern of compound N6 with the protein GPR183. Compound N6 is shown as yellow sticks, and the GPR183 backbone is shown in slate blue. Residues within the binding pocket are shown as slate blue sticks. Traditional hydrogen bonds, carbon-hydrogen bonds, halogen interactions, electrostatic interactions, π-π stacking interactions, and hydrophobic interactions between compound N6 and GPR183 are represented by dashed lines in green, light green, cyan, orange, dark pink, and pink, respectively. Direct binding between N6 and GPR183 occurs with a binding energy of -13.11.

[0040] Figure 3 The effect of different concentrations of N6 on GPR183 protein in AML cells in Example 1 of the present application is shown. The specific experimental process is to take the above-mentioned cultured AML cell line THP-1, count the cell concentration with a cell counter, and calculate the concentration of 2×10 5THP-1 cell solution was prepared at a concentration of cells / ml, and then plated on a 6-well cell culture plate, with 5ml of cell solution plated on each well. Set up a control (Control, without N6) and 4 N6 doping concentrations, namely 4, 6, 8, and 10uM. The laid 6-well plate was cultured in a constant temperature and humidity carbon dioxide incubator for 48 hours, and the cells in each well of the 6-well plate were collected into different 15ml centrifuge tubes. After centrifugation, the supernatant was discarded and the cell clumps were collected. The cells were then lysed using 1×PIRA cell lysis buffer (with protease inhibitors added in advance), and the protein concentration was measured by the BCA method. Then, protein electrophoresis, membrane transfer, skimmed milk powder blocking the protein membrane, incubation with 1 antibody (including the target protein GPR183 and the reference protein GAPDH), incubation with 2 antibodies, and protein expression was displayed by ECL development. The results are as follows. Figure 3 As shown in Figure 3, N6 can reduce the expression of GPR183 in AML cells.

[0041] Example 2: Cytotoxic effect of N6 on AML cell lines

[0042] A drug sensitivity assay was performed to analyze the cytotoxicity of different tumor cell lines to various concentrations of N6. The N6 concentration range was 0.5, 1, 2.5, 5, 10, 20, and 40 μM. The tumor cell lines used were the AML cell lines THP-1, MV4-11 (ATCC, Catalog No. CRL-9591), and MOLM-13 (DSMZ, Catalog No. ACC-554).

[0043] The specific experimental process is as follows: different cell lines were collected at 2×10 5 Plate the plate with 100 μg / ml of N6 in a 24-well plate. Add N6 to the plate using the above concentration gradient, and set up control wells. After 48 hours, transfer the plate to a 96-well plate. Add MTS and measure absorbance on a microplate reader 4 hours later. Calculate the half-maximal inhibitory concentration (IC50) of N6 for different cell lines.

[0044] The IC50 of N6 on different cell lines is shown in Table 3, and the inhibitory fitting curves of N6 on different cell lines are shown in Figure 4 As shown, the calculated IC50 for MOLM-13 cell line is 7.19uM, the IC50 for MV4-11 cell line is 8.06uM, and the IC50 for THP-1 cell line is 9.26uM, all of which have killing effects.

[0045] Table 3 IC50 of N6 against different cell lines

[0046]

[0047] Example 3: Cytotoxic effect of N6 on untreated primary AML cells

[0048] A drug sensitivity assay was used to analyze the cytotoxicity of different concentrations of N6 against untreated primary AML cells from patients with different diagnoses. N6 concentrations ranged from 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM. The primary AML cells used were derived from 13 AML patients. Inclusion criteria included patients with a confirmed diagnosis of AML and no prior treatment. Exclusion criteria included patients with other tumors. Patient information is detailed in Table 4.

[0049] Table 4 AML patient information

[0050]

[0051] The specific experimental process is that the steps for separating AML cells are as follows: take 5 ml of peripheral blood or bone marrow from a patient with a clear diagnosis of AML who has obtained informed consent. Take a 15 ml sterile centrifuge tube and add 5 ml of human peripheral blood lymphocyte separation fluid, use a pipette to add healthy human peripheral blood to the centrifuge tube, and centrifuge at 2000 rpm for 20 minutes. After the centrifugation is completed, take the white film layer at the contact surface between the upper layer of the lymphocyte separation fluid and the lower layer of the plasma layer, that is, the normal blood cell layer. Transfer the white film layer to a new 15 ml sterile centrifuge tube, wash the normal peripheral blood cells twice with sterile 1×PBS, resuspend the blood cells in IMDM culture containing 10% FBS, and count the cells with a cell counter. Press 1×10 6 N6 was added to the 24-well plate at a gradient of 1uM, 2.5uM, 5uM, 10uM, 20uM, 40uM, and 80uM, and a control well without drug was set up. After 48 hours, the plate was transferred to a 96-well plate. The absorbance value was measured by microplate reader 4 hours after adding MTS, and the IC50 of N6 on primary cells of untreated AML patients with different diagnoses was calculated. The inhibitory fitting curve of N6 on primary cells of untreated AML patients with different diagnoses is shown in the figure. Figure 5 (a) and Figure 5 As shown in (b), the IC50 values ​​of primary cells from different AML patients are shown in Table 5. The IC50 values ​​range from 0.48 to 11.74 μM, and all of them have cytocidal effects.

[0052] Table 5 IC50 of primary cells from untreated AML patients with different diagnoses

[0053]

[0054] Example 4: Inhibitory effects of different GPR183 inhibitors on AML.

[0055] The drug sensitivity test was performed to compare and analyze the four known and commercial GPR183 inhibitors or antagonists, GPR183 antagonist-1 (antagonist, purchased from MCE, product number HY-147222), GPR183 antagonist-2 (antagonist, purchased from MCE, product number HY-162011), ML-401 (antagonist, purchased from MCE, product number HY-116814), and GPR183-IN2 (inhibitor, purchased from MCE, product number HY-163796). The concentrations were set at 1uM, 5uM, 10uM, 25uM, 50uM, and 100uM. The results are shown in the figure. Figure 6 As shown, even at a concentration of 100 uM, the four GPR183 inhibitors or antagonists had a weak killing effect on the AML cell line THP-1 cells, and did not reach the half-inhibitory concentration. That is, the IC50 of the four known GPR183 inhibitors or antagonists against AML cells was greater than 100 uM, which was much higher than the IC50 of the AML cell line in Example 2 and the AML primary cells in Example 3. This shows that compared with existing GPR183 inhibitors or antagonists, N6 has a significant killing effect on AML cells.

[0056] Example 5: Growth inhibitory effect of N6 on AML cells

[0057] The growth inhibition experiment was used to analyze the growth inhibition effect of N6 on AML cells. AML cell lines THP-1 and MOLM-13 cells were cultured at 1×10 5 The concentration of N6 was plated in a 96-well plate at 6uM, 8uM, and 10uM, and a control well without drug was set. The absorbance was measured by microplate reader at 0, 24, 48, 72, and 96 hours. The growth curve was used to determine the inhibitory effect of N6 on the growth of AML cells. Figure 7-8 As shown, different concentrations of N6 have a significant growth inhibitory effect on AML cells.

[0058] Through the above specific embodiments, those skilled in the art can easily implement the present application. However, it should be understood that the present application is not limited to the above specific embodiments. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to implement different technical solutions.

Claims

1. Use of a compound targeting GPR183 in the preparation of a drug for treating acute myeloid leukemia, characterized in that: The compound is N-{6-[4-(2,4-dichlorobenzoyl)piperazin-1-yl]pyridin-3-yl}-N'-[4-(trifluoromethyl)phenyl]urea, and its chemical structure is shown below: 。 2. The use according to claim 1, characterized in that The compound has a killing effect on an acute myeloid leukemia cell line, and the acute myeloid leukemia cell line is one of THP-1, MV4-11, and MOLM-13.

3. The use according to claim 1, characterized in that The compound has the effect of inhibiting acute myeloid leukemia cells, and the acute myeloid leukemia cells are THP-1 and MOLM-13.

4. The use according to claim 1, characterized in that The dosage form of the drug includes at least one of injection, tablet, pill and granule.

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