RSK2 inhibitor and application thereof

Abstract

The invention discloses an RSK2 inhibitor and application thereof, and belongs to the technical field of medicines. The RSK2 inhibitor is a compound shown in the following formula (1). The invention also discloses application of the RSK2 inhibitor in preparation of medicines for treating and / or preventing cancers. The cancers comprise breast cancer and ovarian cancer cells. In-vitro biochemical level and intracellular activity research is carried out on the compound shown in the formula (1), and it is proved that the compound has high RSK2 inhibitory activity and has a good treatment effect on related cancers such as ovarian cancer and breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a RSK2 inhibitor and a use thereof. Background Art

[0002] RSK2 is a serine / threonine kinase that belongs to the p90 ribosomal S6 kinase (p90 RSK; RSKs) family, which contains four members (RSK1-4). RSK2 is an atypical protein kinase consisting of two kinase domains separated by a linker region consisting of multiple phosphorylation sites. The N-terminal kinase domain is responsible for RSK2 substrate phosphorylation, while the C-terminal kinase plays an autoregulatory role. Mechanistically, MAPK binds to the C-terminus of RSK2, and subsequent mitogen-activated protein kinase (MAPK)-mediated C-terminal kinase phosphorylation of RSK2 initiates a series of highly coordinated phosphorylation events, ultimately leading to the full activation of RSK2. The RSK kinase family is a convergence point in the EGFR, MAPK, and PDK-1 pathways. They are involved in the regulation of transcriptional and translational signals. RSK2 is a downstream signaling protein of ERK1 / 2. RSK2 can phosphorylate a variety of downstream substrates and plays an important role in cell proliferation, differentiation, and survival. In recent years, overexpression and abnormal activation of RSK2 have been shown to be associated with many human diseases, including breast cancer, prostate cancer, human head and neck squamous cell carcinoma, etc. In triple-negative breast cancer, RSK2 has been identified as one of the most important kinases in triple-negative breast cancer because RSK2 can phosphorylate multiple transcription factors, thereby promoting tumor growth and metastasis. At the same time, knocking down the RSK2 gene can lead to cell apoptosis, inhibit proliferation and migration, and weaken tumor formation. These studies show that RSK2 is a very potential cancer treatment target. Currently, only one RSK2 inhibitor has entered clinical research, so the discovery of more new RSK2 inhibitors is of great significance for cancer treatment and clinical drug discovery.

[0003] At present, there are no reports on the development of new RSK2 inhibitors with high activity and high drugability.

[0004] The compounds of the present invention have not been reported to be used as RSK2 inhibitors. Summary of the invention

[0005] In view of the above-mentioned shortcomings, the present invention provides an RSK2 inhibitor and its use. Based on summarizing the structure-activity relationship and key pharmacophores of RSK2 inhibitors, the present invention constructs a pharmacophore model, uses virtual screening techniques such as molecular docking and molecular dynamics simulation, screens a series of small molecules, and conducts in vitro biochemical level and intracellular activity studies on them, and obtains compounds with strong RSK2 inhibitory activity, which have good therapeutic effects on the treatment of ovarian cancer, breast cancer and other related cancers.

[0006] In order to achieve the above object, the present invention provides a RSK2 inhibitor, which is a compound represented by the following formula (1):

[0007]

[0008] Based on the same inventive concept, the present invention also provides use of the above-mentioned RSK2 inhibitor in the preparation of a drug for treating and / or preventing cancer.

[0009] According to one aspect of the present invention, the cancer includes breast cancer and ovarian cancer cells.

[0010] Based on the same inventive concept, the present invention also provides an anticancer drug composition, which comprises an RSK2 inhibitor and a pharmaceutically acceptable carrier; wherein the RSK2 inhibitor is a compound represented by the following formula (1):

[0011]

[0012] Beneficial effects of the present invention:

[0013] The present invention confirms through computer simulation that the compound of formula (1) has good affinity for RSK2; the present invention confirms through in vivo cell experiments that the compound of formula (1) of the present invention has a good inhibitory effect on the proliferation of breast cancer and ovarian cancer; through SPR experiments, it is confirmed that the compound of formula (1) has a high affinity for RSK2 protein; and through Western blot experiments, it is confirmed that the compound of formula (1) can downregulate RSK2 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the binding mode of the compound of formula (1) of the present invention and the RSK2 crystal structure;

[0015] Figure 2 The affinity strength of the compound samples of formula (1) of the present invention at different concentrations to the RSK2 protein;

[0016] Figure 3 The Western blot technique of the present invention is used to detect the down-regulation of RSK2 expression by the compound of formula (1); wherein A is a polyacrylamide gel electrophoresis diagram of SKOV3 cells treated with different concentrations of the compound of formula (1) and 5 nM LJH685; B is a bar graph of polyacrylamide gel electrophoresis of SKOV3 cells treated with different concentrations of the compound of formula (1) and 5 nM LJH685. DETAILED DESCRIPTION

[0017] To make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0018] It should be noted that the present application formula (1) was purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0019] Example 1

[0020] Design ideas of the RSK2 inhibitor of the present invention

[0021] The RSK2 crystal structure was obtained from the Protein Data Bank (RSCB PDB) website. Representative crystal complexes were selected. The key residues for protein-ligand interactions were summarized using molecular dynamics simulation and the PLIF module of MOE, thereby constructing a basic pharmacophore model of RSK2 inhibitors. Molecular docking was performed using Glide software. Standard-precision docking mode was first used for screening, and then ultra-precision docking was performed to retain compounds with high docking scores. Deep learning was used to predict the binding constants between compounds and proteins, and molecules with poor binding constants were filtered out. Molecular dynamics simulation was used to calculate the binding free energy of protein ligands, and molecules with good binding free energy were retained. ADMET property prediction was performed to screen out compounds with good drug-like properties, and active small molecules with good binding ability to RSK2 protein (1) were selected for biological activity verification:

[0022]

[0023]

[0024] The results of computer simulation docking analysis showed that:

[0025] In the binding mode between the active molecular formula (1) and the RSK2 crystal structure ( Figure 1 ), such compounds can bind to the N-terminal region of the RSK2 protein, the nitrogen on the imidazole ring acts as a hydrogen bond receptor and interacts with Leu150 of the RSK2 protein through hydrogen bonds, and the hydrogen on the hexaamide ring interacts with Asp154 through hydrogen bonds.

[0026] It can be seen that the compound of formula (1) has good affinity for RSK2.

[0027] Example 2

[0028] The inhibitory effect of the compound of formula (1) on the proliferation of tumor cells

[0029] 1. Inhibitory effect of compounds on tumor cell proliferation

[0030] Detection principle: CCK8 method. The CCK-8 cell viability detection kit contains WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt). In the presence of an electron coupling reagent, WST-8 is oxidized and reduced by intracellular dehydrogenase to generate a water-soluble orange-yellow formazan dye that can be dissolved in tissue culture medium. The amount of formazan generated is proportional to the number of living cells. Using an enzyme reader to measure the OD value at a wavelength of 450nm can indirectly reflect the number of living cells. The smaller the OD value, the weaker the cell activity, which means the greater the drug toxicity.

[0031] Experimental operation: Tumor cells (breast cancer and ovarian cancer) in the logarithmic growth phase were inoculated into 96-well culture plates at a density of 2000 breast cancer cells or ovarian cancer cells per well, with 100 μL per well. After overnight culture, different concentrations of drugs (final concentration starting from 10 μM, 5-fold dilution and 9 gradients) were added for 72 hours, and three replicate wells were set for each concentration, and dimethyl sulfoxide (DMSO) solvent controls and cell-free zero wells of corresponding concentrations were set. 10 μl of CCK8 reagent was added to each well, and it was placed in a 37°C incubator for incubation for 3 hours, and the absorbance at 450 nM was measured with an enzyme marker. Among them, MDA-MB-231 and MDA-MB-436 are breast cancer cells; A2780 and SKOV3 are ovarian cancer cells.

[0032] The degree of drug inhibition on cell proliferation was calculated according to the following formula:

[0033] Inhibition rate (%) = (OD control - OD drug well) / OD control well × 100%

[0034] The whole experiment was repeated three times, and Prism GraphPad software was used to calculate the 10 μM survival rate of the compound on cells by normalizing the dose-response fitting nonlinear regression curve. The results are shown in Table 1. Further activity value results showed that the IC50 value of the compound of formula (1) on SKOV3 cells was 4.1 μM.

[0035] Table 1: Survival rate of the compound of formula (1) at 10 μM on breast cancer and ovarian cancer cells

[0036]

[0037] 2. Surface plasmon resonance (SPR) technology to detect the affinity of the compound of formula (1) and RSK2 protein

[0038] Detection principle: Surface plasmon resonance (SPR) technology is an analytical technology developed based on optical principles. The light source of the SPR biosensor is polarized light, and the surface of the sensor chip is coated with a layer of gold film. During the experiment, a biological molecule (target molecule) is first fixed on the surface of the gold film, and then the molecules that interact with it are dissolved in a solution (or mixed solution) and flowed over the surface of the chip. During the process of binding and dissociation between the protein on the gold film chip and the molecules in the flow path, the resonance angle (i.e., the SPR angle) will change accordingly. The detector detects this change, and based on the analysis of this change curve, the binding constant Ka, dissociation constant Kd or affinity constant KD between molecules can be obtained.

[0039] Experimental procedure: PBST (pH=7.4, 0.1% Tween 20) was added to the RSK2 protein sample stock solution to dilute it into 5 concentration gradients: 10nM, 40nM, 160nM, 640nM, 2560nM. All samples were circulated and tested in turn. PBST was used as the flow carrier throughout the experiment. In the interaction test phase, the analyte was diluted at 0.5μL·s -1 Flowing over the chip surface; in the surface regeneration stage, Glycine-HCl (pH=2.0) solution is used as the regeneration solution, and the flow rate is 2μL·s-1.

[0040] The experiment was carried out by loading different concentration gradients of the compound of formula (1) in order from low to high concentrations, with a flow rate of 0.5 μL·s -1 The binding reaction temperature was 4°C, the binding time was 600s, and the dissociation time was 360s. Glycine-HCl (pH=2.0) solution was used as the regeneration solution for regeneration.

[0041] The kinetic analysis was performed using the Berthold bScreen LB 991 (V4 device) control software. The KD value of the compound sample of formula (1) was obtained by fitting according to the Berthold bScreen LB 991 analysis software. The smaller the KD value, the stronger the affinity of the sample to the RSK2 protein.

[0042] Test results: Figure 2 It can be seen that the compound represented by formula 1 can bind to the RSK2 protein with a KD of 156 nM.

[0043] 3. Western blot technology to detect compounds down-regulating RSK2 expression

[0044] Detection principle: Western Blot method uses polyacrylamide gel electrophoresis. The protein sample separated by PAGE (polyacrylamide gel electrophoresis) is transferred to a solid phase carrier, adsorbing the protein in the form of non-covalent bonds, and maintaining the type of polypeptide separated by electrophoresis and its biological activity unchanged. The protein on the solid phase carrier is used as an antigen, reacts with the corresponding antibody, and then reacts with the enzyme or isotope-labeled second antibody. The protein component expressed by the specific target gene separated by electrophoresis is detected through substrate color development.

[0045] Experimental procedure: SKOV3 cells were inoculated into six-well plates, incubated overnight, and then treated with different concentrations of compounds (1nM, 2nM, 5nM) and LJH685 (5nM) for 48h, and 1‰ (v / v) DMSO culture medium was used as blank control. The electrophoresis diagram is shown in Figure 3 As shown in A. The cell samples were collected and lysed in ice-cold RIPA buffer for 30 minutes. Then, the lysate was cleared by centrifugation at 14,000 rpm for 10 minutes at 4°C, and the supernatant was removed and the protein concentration was determined using the Pierce BCA protein assay kit. The cell lysate was loaded onto 10% SDS-PAGE, and the separated proteins were then electrophoresed and transferred to a polyvinylidene difluoride membrane and blocked with 5% bovine serum albumin for 1 hour. The membrane was hybridized with the secondary antibody (RSK2) and incubated overnight at 4°C. The membrane was placed in a secondary antibody tube and incubated at room temperature for 1 hour, and the bands were observed using enhanced chemiluminescence, and then quantitatively detected using ImageJ software.

[0046] Western Blot experiments were performed to detect the effect of the compound of formula 1 on the expression of RSK2 protein. Figure 3 B. The compound of formula (1) can down-regulate the expression of RSK2 protein.

[0047] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A RSK2 inhibitor, characterized in that The RSK2 inhibitor is a compound represented by the following formula (1):

2. Use of the RSK2 inhibitor according to claim 1 in the preparation of a medicament for treating and / or preventing cancer.

3. The use according to claim 2, characterized in that: The cancers include breast cancer and ovarian cancer cells.

4. An anticancer drug composition, characterized in that: The anticancer drug composition comprises a RSK2 inhibitor and a pharmaceutically acceptable carrier; wherein the RSK2 inhibitor is a compound represented by the following formula (1):

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