Application of compound DB07268 in preparation of RAGAP1 inhibitor

Abstract

The invention relates to an application of a compound DB07268 in preparation of a RAGAP1 inhibitor. Candidate compounds possibly interacting with RACGAP1 protein are screened out through large-scale virtual docking and CCK8 experiments and verified through SPR experiments, the compound DB07268 with the highest affinity with RACGAP1 is further screened out, and finally the acting effect of the compound DB07268 is verified through cell experiments and molecular experiments. Compared with the prior art, the invention discovers that DB07268 can be used as the RACGAP1 inhibitor for targeted therapy of RACGAP1 related diseases, such as various tumors, diabetic nephropathy, acute kidney injury, congenital heart disease, congenital erythropoiesis abnormality anemia, virus infectious diseases and the like.

Description

Technical Field

[0001] The present invention relates to the field of computer-aided drug design, and in particular to application of a compound DB07268 in the preparation of a RACGAP1 inhibitor. Background Art

[0002] Rac GTPase-activating protein 1 (RACGAP1) is a member of the guanine triphosphatase (GTPase) activating protein family and regulates the conversion of Rho GTPase from guanine triphosphate-bound to guanine diphosphate-bound forms. RACGAP1 is involved in a variety of cellular processes, including cytokinesis, differentiation, and migration, and controls cytokinesis by forming the central spindle protein complex and mediating the Rho-dependent signaling pathway required for the assembly of the actomyosin contractile ring.

[0003] Studies have found that RACGAP1 is abnormally expressed in many tumors, including liver cancer, gastric cancer, cervical cancer, and high-grade meningioma, and participates in the occurrence and development of tumors by regulating the conduction of multiple signal pathways such as the cell cycle.

[0004] Previous studies on RACGAP1 were all conducted by knocking down / overexpressing RACGAP1. Currently, there are no targeted inhibitors that directly act on RACGAP1, which greatly limits its clinical transformation and application value.

[0005] The chemical formula of DB07268 is C 17 H 15 N 5 O 2 , DB07268 structural formula is as follows Figure 1 In 2007, Mei Liu et al. synthesized DB07268 and verified it as an effective JNK1 inhibitor (Liu M, et al., Discovery of a new class of 4-anilinopyrimidines as potent c-Jun N-terminal kinase inhibitors: Synthesis and SAR studies. Bioorg Med Chem Lett, 2007. 17 (3): 668-72.). However, no studies have shown the relationship between DB07268 and RACGAP1. Summary of the invention

[0006] The purpose of the present invention is to provide an application of a compound DB07268 in the preparation of a RACGAP1 inhibitor. The present invention screens candidate compounds that may interact with the RACGAP1 protein through large-scale virtual docking and CCK8 experiments, and verifies them through SPR experiments, further screens out the compound DB07268 with the highest affinity for RACGAP1, and finally verifies its effect through cell experiments and molecular experiments.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] A use of a compound DB07268 in the preparation of a RACGAP1 inhibitor, wherein the compound DB07268 is a compound as shown in Formula I:

[0009]

[0010] Furthermore, the concentration range of the compound DB07268 in the RACGAP1 inhibitor is 1.563 μM to 50 μM.

[0011] The present invention also provides a use of a compound DB07268 in the preparation of a drug for preventing and / or treating tumors, wherein the compound DB07268 is a compound as shown in Formula I:

[0012]

[0013] Furthermore, the drug is a drug that inhibits the expression of RACGAP1 in tumor cells.

[0014] Furthermore, the tumor cells are growth hormone-type pituitary tumor GH3 cells.

[0015] The present invention also provides a use of a compound DB07268 in the preparation of a drug for inhibiting tumor cell growth, wherein the compound DB07268 is a compound as shown in Formula I:

[0016]

[0017] Furthermore, the tumor cells are growth hormone-producing pituitary tumor GH3 cells.

[0018] The present invention also provides use of a RACGAP1 inhibitor in preparing a medicament for preventing and / or treating RACGAP1-related diseases.

[0019] Furthermore, the RACGAP1 inhibitor contains a compound as shown in Formula I:

[0020]

[0021] Furthermore, the RACGAP1-related diseases include tumors, diabetic nephropathy, acute kidney injury, congenital heart disease, congenital dyserythropoietic anemia and viral infectious diseases.

[0022] In addition, the present invention also provides a method for screening RACGAP1 inhibitors, the specific steps of which are as follows:

[0023] S1. Molecular docking was performed using high-throughput virtual screening and standard precision mode to screen out candidate compound A of RACGAP1 protein;

[0024] S2. Using CCK8 cell proliferation assay, detect the effect of candidate compound A obtained in step S1 on GH3 cell proliferation, and further screen out candidate compound B;

[0025] S3. Use surface plasmon resonance technology to establish a RACGAP1 protein-candidate compound B interaction determination model to screen out the compound with the highest affinity to the RACGAP1 protein;

[0026] S4. The screened compounds were evaluated and compared through Western Blot experiment and CCK8 cell proliferation experiment.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1. The present invention finds that DB07268, as a RACGAP1 inhibitor, can be used for the targeted treatment of RACGAP1-related diseases, such as various tumors, diabetic nephropathy, acute kidney injury, congenital heart disease, congenital dyserythropoietic anemia, viral infectious diseases, etc.

[0029] 2. Existing literature reports that DB07268 is a JNK inhibitor, but no studies have shown the relationship between DB07268 and RACGAP1, and there is currently no targeted inhibitor for RACGAP1. The present invention screened and verified the RACGAP1 inhibitor DB07268 for the first time. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of the structural formula of DB07268;

[0031] Figure 2 This is a superposition of the human RACGAP1 protein structure. The CDC42 receptor is included in the 5C2J and 5C2K structures;

[0032] Figure 3 Prediction of possible binding sites on the surface of RACGAP1 protein, orange is the 2OVJ structure, cyan is the 5C2J structure;

[0033] Figure 4 Comparison results of the RACGAP1 amino acid sequences of human, mouse, and rat. The black boxes are the amino acids near Ser387;

[0034] Figure 5 For the CCK8 cell proliferation assay, 6 of the 74 candidate compounds showed significant proliferation inhibition effects on GH3 cells (STSP: positive control compound);

[0035] Figure 6 In the surface plasmon resonance (SPR) experiment, compound DB07268 (T3200) interacted with protein RACGAP1, and its dissociation constant K d =0.523 μM;

[0036] Figure 7 For the SPR experiment, RACGAP1 protein interacted with all candidate compounds, and the one with the highest affinity was T3200 (DB07268);

[0037] Figure 8 The diagram of the interaction between RACGAP1 protein and compound DB07268, where A: RACGAP1 is shown as a cartoon structure, the carbon atoms of amino acid residues are shown in white, the carbon atoms of compounds are shown in green, the magenta dotted line is a hydrogen bond, and the red dotted line represents a Pi-cation interaction; B: 2D binding mode of RACGAP1 protein binding to compound DB07268;

[0038] Fig. 9 For Western Blot experiments, DB07268 inhibited RACGAP1 protein expression in GH3 cells;

[0039] Fig.10 For cytotoxicity experiments, the IC50 of DB07268 in GH3 cells was 1.770 μM. DETAILED DESCRIPTION

[0040] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0041] In the following examples, the amino acid sequences are as follows:

[0042] The amino acid sequence of human RACGAP1 is shown in SEQ ID NO.1:

[0043]

[0044] The amino acid sequence of mouse RACGAP1 is shown in SEQ ID NO.2:

[0045]

[0046]

[0047] The amino acid sequence of rat RACGAP1 is shown in SEQ ID NO.3:

[0048]

[0049] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0050] Example 1

[0051] This embodiment provides a method for screening candidate compounds of RACGAP1 protein by using large-scale virtual docking, and the specific steps are as follows:

[0052] (1) Structural analysis of RACGAP1 protein

[0053] 1) Obtaining the structural model of RACGAP1 protein

[0054] The RACGAP1 protein was searched on the Uniprot website (https: / / www.uniprot.org / ). The amino acid sequence of human RACGAP1 (Uniprot ID: Q9H0H5) is 632 amino acids long and has a molecular weight of approximately 71.03KD. In human RACGAP1, 106-285 is the region that interacts with SLC26A8, 286-335 is the zinc finger domain, 349-539 is the Rho-GAP domain, and the rest is a disordered structure. The sequence information is shown in SEQ ID NO.1.

[0055] The crystal structure of human RACGAP1 is currently available, but only the Rho-GAP domain has been resolved. The PDB ID and structure are shown in Tables 1 and Figure 2 Mouse and rat RACGAP1 only have structures predicted by AlphaFold.

[0056] Table 1 RACGAP1 crystal structure information

[0057]

[0058] 2) Analysis of small molecule ligand binding sites

[0059] Studies have reported that Ser387 is a phosphorylation modification site in the amino acid sequence of RACGAP1 with crystal structure. Therefore, 2VOJ with the lowest resolution was selected for subsequent operations. Structurally, Ser387 is located in the binding region of RACGAP1 protein and CDC42 ( Figure 3 ), therefore, the docking region of virtual screening can take Ser387 as the center to generate the grid file required for docking.

[0060] 3) RACGAP1 homology comparison

[0061] The amino acid sequences of RACGAP1 from humans, mice, and rats were compared. Figure 4 As shown, except for ILE500 in rat, the amino acids near Ser387 are completely conserved, so it is no longer necessary to screen RACGAP1 proteins from mouse and rat.

[0062] (2) Virtual screening

[0063] First, the drug similarity screening of L4010, D7800, and D8800 compound databases was performed according to the Lipinski five-fold rule. Then, molecular docking was performed in Glide using high-throughput virtual screening (HTVS) and standard precision (SP) modes to improve the efficiency and accuracy of virtual screening. The screening results were based on a docking score of -5 kcal / mol as the threshold (the lower the score, the better), and 97 molecules (ΔG < -5 kcal / mol) were screened, of which 74 compounds available for purchase were selected for further study.

[0064] Table 2 74 compounds

[0065]

[0066]

[0067] Example 2

[0068] In the first round of virtual docking screening in Example 1, 74 candidate compounds were obtained. In order to further narrow the scope of the target compounds, this example conducted a second round of CCK8 cell proliferation experiments to detect the effects of candidate compounds on GH3 cell proliferation and further screen RACGAP1 inhibitors. The specific steps are as follows:

[0069] S1. Preparation of compound working solution

[0070] The compound stock solution was diluted 100-fold with complete culture medium (Ham's F-12K medium + 15% horse serum + 2.5% fetal bovine serum) to obtain a 100 μM compound working solution. The final cell administration concentration was 50 μM, and the DMSO content of all wells was uniformly controlled at 0.5%.

[0071] S2. Cell inoculation and drug treatment

[0072] S2-1. Collect cells in the logarithmic growth phase and plate 6×10 3 Cells were seeded into 384-well plates with 25 μL complete medium and incubated at 37°C with 5% CO 2 Incubate overnight in an incubator.

[0073] S2-2. Observe under a microscope. After the cells adhere to the wall, add 25 μL of the corresponding compound working solution to each well and continue incubation for 72 hours.

[0074] S2-3. After the incubation, 5 μL of CCK8 solution was added to each well and the cells were incubated in the incubator for 2 hours. The OD value at a wavelength of 450 nm was then measured on a microplate reader.

[0075] S3. Data Processing

[0076] The calculation formula of the inhibition rate of the compound on GH3 cell proliferation is as follows:

[0077] Inhibition rate = (1-(OD S -OD BLK ) / (OD NC –OD BLK ))×100%.

[0078] Among them, OD S : Absorbance value of sample well (complete medium containing cells + CCK8 + test compound),

[0079] OD NC : Absorbance value of negative well (complete medium containing cells + CCK8),

[0080] OD BLK : Absorbance value of blank well (complete medium without cells + CCK8).

[0081] The results are as follows Figure 5As shown, there are 4 compounds with an inhibition rate of more than 90% on GH3 cell proliferation - T3200 (DB07268), T1591 (Ancitabine hydrochloride), T7004 (Tubercidin), T6908 (NSC228155), and there are 2 compounds with an inhibition rate of about 80% - T2177 (Kaempferol) and T19833 (5-Fluorooroticacid). Therefore, in this example, 6 candidate compounds were screened out through the CCK8 cell proliferation experiment.

[0082] Example 3

[0083] In order to further explore whether the 6 candidate compounds obtained in Example 2 have direct interaction with RACGAP1, this example uses surface plasmon resonance (SPR) technology to establish a RACGAP1 protein-compound interaction determination model to screen out candidate compounds with the highest affinity to the RACGAP1 protein.

[0084] SPR experiments were performed on a GE Healthcare BIAcore T200 instrument using Cytiva's CM5 chip according to the manufacturer's protocol. The specific steps are as follows:

[0085] Recombinant RACGAP1 protein was immobilized on a CM5 chip. Then, a series of analytes (DB07268, Kaempferol, Tubercidin, NSC228155, Ancitabine hydrochloride, and 5-Fluoroorotic acid) were prepared using RACGAP1 antibody (ab134972 from Abcam) as a positive control and injected at a flow rate of 30 μL / min for 150 seconds. After each injection, the chip was regenerated for 5 minutes by injecting 10 mM glycine-HCl (pH 2.0) at a flow rate of 10 μL / min. 60 seconds of binding data were collected, followed by 120 seconds of dissociation data. All operations were performed in 5% DMSO-PBS. Finally, GE Healthcare's BIAcore T200Evaluation software (v.2.0) was used for kinetic analysis using a 1:1 Langmuir binding model.

[0086] The results are as follows Figure 6 and Figure 7 As shown in Figure 2, all six candidate compounds interacted with RACGAP1, and the dissociation constant K dThe compound with the lowest value and the strongest affinity is T3200 (DB07268), therefore, compound DB07268 has a higher affinity with RACGAP1.

[0087] The interaction pattern between RACGAP1 protein and compound DB07268 constructed by molecular docking is shown in the figure below. Figure 8 As shown, A: RACGAP1 is shown as a cartoon structure, the carbon atoms of amino acid residues are shown in white, the carbon atoms of compounds are shown in green, the magenta dotted lines are hydrogen bonds, and the red dotted lines represent Pi-cation interactions. B: 2D binding mode of RACGAP1 protein binding to compound DB07268.

[0088] Example 4

[0089] To further study the ability of DB07268 to target and inhibit RACGAP1 protein in cells, this example used different concentrations of DB07268 to treat pituitary tumor GH3 cells, and detected the expression level of RACGAP1 protein in each group of cells by Western Blot experiment after 72 hours. The specific steps are as follows:

[0090] S1. Prepare 2-fold diluted DB07268 working solutions of 50 μM, 25 μM, 12.5 μM, etc. and DMSO control;

[0091] S1-1, take 6 μL of 50 mM DB07268 stock solution, dilute it to 50 μM with 6 mL of complete culture medium, take 3 mL and add it to another tube of 3 mL of complete culture medium, then the final concentration of DB07268 is 25 μM, and so on;

[0092] S1-2. Take 6 μL of DMSO and dilute it with 6 mL of complete culture medium and add it to GH3 cells to serve as the DMSO control.

[0093] S2. Dosing treatment

[0094] S2-1, GH3 cells were plated at 1.5×10 5 Cells were seeded in 12-well culture plates with 1 mL of complete culture medium and incubated at 37°C with 5% CO 2 Culture in an incubator;

[0095] S2-2. After the cells adhered on the second day, the original culture medium was discarded and 1 mL of pre-prepared DB07268 working solution of various concentrations (starting from 50 μM, 1 / 2 dilution) was added to each well;

[0096] S2-3, 72 hours after treatment, proteins were extracted for Western blot analysis;

[0097] S3. Western blot detection

[0098] S3-1, extract total cell protein using RIPA lysis buffer;

[0099] S3-2, electrophoresis the protein sample on SDS-polyacrylamide gel, and transfer the protein to polyvinylidene fluoride (PVDF) membrane by electrophoresis;

[0100] S3-3, the PVDF membrane was blocked with 5% skim milk and incubated with the following primary antibodies overnight at 4°C: anti-RACGAP1 (66056-1-Ig, Proteintech) and anti-GAPDH (60004-1-Ig, Proteintech);

[0101] S3-4, incubate the PVDF membrane with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour;

[0102] S3-5. Protein bands were detected using an enhanced chemiluminescence assay kit (Millipore).

[0103] The results are as follows Fig. 9 As shown, when DB07268 was in the concentration range of 1.563 μM to 50 μM, the expression level of RACGAP1 in GH3 cells was significantly reduced. Therefore, compound DB07268 can inhibit the expression of RACGAP1 protein in cells.

[0104] Example 5

[0105] According to the research results of Example 4, compound DB07268 directly interacts with RACGAP1 protein and inhibits the expression of RACGAP1 protein. This example uses CCK8 cell proliferation experiment to verify the effect of DB07268 on tumor cell proliferation and growth.

[0106] This example provides a method for verifying the effect of compound DB07268 using a cytotoxicity experiment, and the specific steps are as follows:

[0107] S1. Methods for preparing drugs of different concentrations

[0108] S1-1. According to mass = concentration × volume × molecular weight, weigh a certain mass of DB07268, dilute it with DMSO to a 50 mM storage solution, and store it in a -20°C refrigerator for later use;

[0109] S1-2, prepare 2-fold diluted DB07268 working solutions of 50 μM, 25 μM, 12.5 μM, etc. and DMSO control;

[0110] S1-2-1. Take 6 μL of 50 mM DB07268 stock solution, dilute it to 50 μM with 6 mL of complete culture medium, take 3 mL and add it to another tube of 3 mL of complete culture medium. The final concentration of DB07268 is 25 μM, and so on.

[0111] S1-2-2. Take 6 μL of DMSO and dilute it with 6 mL of complete culture medium and add it to GH3 cells to serve as the DMSO control.

[0112] S2. Dosing treatment

[0113] S2-1, GH3 cells were plated at 4×10 3 Cells were seeded in 96-well culture plates with 100 μL complete culture medium and incubated at 37°C with 5% CO 2 Culture in an incubator;

[0114] S2-2. After the cells adhered on the second day, the original culture medium was discarded and 100 μL of pre-prepared DB07268 working solution of various concentrations (starting from 50 μM, 1 / 2 dilution) was added to each well;

[0115] S2-3, cell viability was detected 72 hours after treatment;

[0116] S3, CCK8 detection

[0117] S3-1. Add 10 μL of CCK8 solution (Japan Tongren) to each well to be tested and incubate in a light-proof 37°C incubator for 2 hours;

[0118] S3-2. After the incubation, the OD value was measured using a Multiskan Go microplate reader (Thermo Scientific);

[0119] S3-3. The half maximal inhibitory concentration (IC) of the drug was fitted by GraphPad Prism 9.5. 50 ).

[0120] The results of CCK8 cell proliferation assay are as follows Fig.10 As shown, after DB07268 intervention in GH3 cells for 72 hours, its IC 50 The concentration of DB07268 in GH3 cells was 1.770 μM, which showed a significant proliferation inhibitory effect on GH3 cells. Therefore, compound DB07268 can effectively inhibit the growth of GH3 cells.

[0121] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Use of a compound DB07268 in the preparation of a RACGAP1 inhibitor, characterized in that: The compound DB07268 is a compound as shown in Formula I:

2. Use of a compound DB07268 according to claim 1 in the preparation of a RACGAP1 inhibitor, characterized in that: The concentration range of the compound DB07268 in the RACGAP1 inhibitor is 1.563 μM to 50 μM.

3. Use of a compound DB07268 in the preparation of a drug for preventing and / or treating tumors, characterized in that: The compound DB07268 is a compound as shown in Formula I:

4. Use of a compound DB07268 according to claim 3 in the preparation of a drug for preventing and / or treating tumors, characterized in that: The drug is a drug that inhibits the expression of RACGAP1 in tumor cells.

5. Use of a compound DB07268 according to claim 4 in preparing a drug for preventing and / or treating tumors, characterized in that: The tumor cells are growth hormone-type pituitary tumor GH3 cells.

6. Use of a compound DB07268 in the preparation of a drug for inhibiting tumor cell growth, characterized in that: The compound DB07268 is a compound as shown in Formula I:

7. Use of a compound DB07268 according to claim 6 in the preparation of a drug for inhibiting tumor cell growth, characterized in that: The tumor cells are growth hormone-type pituitary tumor GH3 cells.

8. Use of a RACGAP1 inhibitor in the preparation of a drug for preventing and / or treating RACGAP1-related diseases.

9. Use of a RACGAP1 inhibitor according to claim 8 in the preparation of a drug for preventing and / or treating RACGAP1-related diseases, characterized in that: The RACGAP1 inhibitor contains a compound as shown in Formula I:

10. Use of a RACGAP1 inhibitor according to claim 8 in the preparation of a drug for preventing and / or treating RACGAP1-related diseases, characterized in that: The RACGAP1-related diseases include tumors, diabetic nephropathy, acute kidney injury, congenital heart disease, congenital dyserythropoietic anemia and viral infectious diseases.

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