RSK2 inhibitors and uses thereof

Abstract

This invention discloses an RSK2 inhibitor and its uses, belonging to the field of pharmaceutical technology. The RSK2 inhibitor of this invention is a compound shown in formula (1). The RSK2 inhibitor is used in the preparation of drugs for treating and / or preventing cancer. The cancers include breast cancer and ovarian cancer cells. This invention has conducted in vitro biochemical and intracellular activity studies on the compound shown in formula (1), demonstrating that it has strong RSK2 inhibitory activity and good therapeutic effects on ovarian cancer, breast cancer, and other related cancers.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an RSK2 inhibitor and its uses. Background Technology

[0002] RSK2 is a serine / threonine kinase belonging to the p90 ribosomal S6 kinase (p90 RSK; RSKs) family, which contains four members (RSK1-4). RSK2 is an atypical protein kinase composed 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 phosphorylation of the RSK2 C-terminal kinase initiates a series of highly coordinated phosphorylation events, ultimately leading to complete 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 signaling. RSK2 is a downstream signaling protein of ERK1 / 2 and can phosphorylate a variety of downstream substrates, playing an important role in cell proliferation, differentiation, and survival. In recent years, overexpression and aberrant activation of RSK2 have been shown to be associated with many human diseases, including breast cancer, prostate cancer, and human head and neck squamous cell carcinoma. In triple-negative breast cancer, RSK2 has been identified as one of the most important kinases because it can phosphorylate various transcription factors, thereby promoting tumor growth and metastasis. Conversely, knocking down the RSK2 gene can lead to apoptosis, inhibit proliferation and migration, and reduce tumor formation. These studies indicate that RSK2 is a highly promising target for cancer therapy. Currently, only one RSK2 inhibitor has entered clinical trials; therefore, the discovery of more new RSK2 inhibitors is of great significance for cancer treatment and clinical drug discovery.

[0003] Currently, there are no reports on the development of novel RSK2 inhibitors with high activity and high drug-likeness.

[0004] The compounds described in this invention have not been reported for use as RSK2 inhibitors. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention provides an RSK2 inhibitor, which is a compound of the following formula (1):

[0007]

[0008] Equation (1).

[0009] Based on the same inventive concept, the present invention also provides the use of the above-mentioned RSK2 inhibitor in the preparation of medicaments for treating and / or preventing cancer.

[0010] According to one aspect of the invention, the cancer includes breast cancer cells and ovarian cancer cells.

[0011] Based on the same inventive concept, the present invention also provides an anticancer drug composition comprising an RSK2 inhibitor and a pharmaceutically acceptable carrier; wherein the RSK2 inhibitor is a compound of the following formula (1):

[0012]

[0013] Equation (1).

[0014] The beneficial effects of this invention are:

[0015] The present invention has demonstrated through computer simulation that the compound of formula (1) has a good affinity for RSK2; the present invention has demonstrated through in vivo cell experiments that the compound of formula (1) has a good inhibitory effect on the proliferation of breast cancer and ovarian cancer; the present invention has demonstrated through SPR experiment that the compound of formula (1) has a high affinity for RSK2 protein; the present invention has demonstrated through Western blot experiment that the compound of formula (1) can downregulate RSK2 expression. Attached Figure Description

[0016] Figure 1 This is the binding mode of the compound of formula (1) of this invention with the RSK2 crystal structure;

[0017] Figure 2 This invention presents the affinity strength between compounds of formula (1) at different concentrations and RSK2 protein.

[0018] Figure 3 The Western blot technique of this invention is used to detect the downregulation of RSK2 expression by compound (1); wherein, A is a polyacrylamide gel electrophoresis diagram of SKOV3 cells treated with different concentrations of compound (1) and 5 nM LJH685; B is a polyacrylamide gel electrophoresis bar diagram of SKOV3 cells treated with different concentrations of compound (1) and 5 nM LJH685. Detailed Implementation

[0019] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art; unless otherwise specified, the raw materials and reagents involved herein can be purchased commercially or obtained by known methods.

[0020] It should be noted that this application form (1) was purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0021] Example 1

[0022] The design concept of the RSK2 inhibitor of this invention

[0023] The crystal structure of RSK2 was obtained from the Protein Data Bank (RSCB PDB) website. Representative crystal complexes were selected, and key residues for protein-ligand interaction were summarized using molecular dynamics simulations and the PLIF module of MOE, thus constructing a basic pharmacophore model for RSK2 inhibitors. Molecular docking was performed using Glide software. First, standard-precision docking mode was 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 constant between the compound and the protein, and molecules with poor binding constants were filtered out. Molecular dynamics simulations were used to calculate the binding free energy of the 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. Active small molecules with good binding ability to RSK2 protein (1) were selected for bioactivity verification.

[0024]

[0025] Equation (1).

[0026] Computer simulation docking analysis results show:

[0027] In the combination mode of the active molecular formula (1) and the RSK2 crystal structure ( Figure 1 These compounds can bind to the N-terminal region of the RSK2 protein. The nitrogen on the imidazole ring acts as a hydrogen bond acceptor and interacts with the Leu150 of the RSK2 protein via hydrogen bonding, while the hydrogen on the hexa-amide ring interacts with the Asp154 via hydrogen bonding.

[0028] It can be seen that the compound of formula (1) has a good affinity for RSK2.

[0029] Example 2

[0030] Inhibitory effect of compound (1) on tumor cell proliferation

[0031] 1. Inhibitory effect of the compound on tumor cell proliferation

[0032] Detection Principle: CCK8 Method. The CCK-8 Cell Viability Assay 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 agent, WST-8 is oxidized and reduced by intracellular dehydrogenases to generate a water-soluble orange-yellow formazan dye that dissolves in tissue culture medium. The amount of formazan generated is directly proportional to the number of viable cells. The OD value is measured at 450 nm using a microplate reader, which indirectly reflects the number of viable cells. The lower the OD value, the weaker the cell activity, and the greater the drug toxicity.

[0033] Experimental Procedure: Tumor cells (breast cancer and ovarian cancer) in logarithmic growth phase were seeded at a density of 2000 cells / well in 96-well culture plates, 100 μL per well. After overnight culture, different concentrations (starting from 10 μM, with 9 five-fold dilutions) of the drug were added and incubated for 72 h, with three replicates for each concentration. A corresponding concentration of dimethyl sulfoxide (DMSO) solvent control and a cell-free zeroing well were also included. 10 µL of CCK8 reagent was added to each well, and the plates were incubated at 37°C for 3 h. The absorbance at 450 nM was measured using a microplate reader. MDA-MB-231 and MDA-MB-436 were breast cancer cells; A2780 and SKOV3 were ovarian cancer cells.

[0034] The degree of inhibition of cell proliferation by the drug is calculated using the following formula:

[0035] Inhibition rate (%) = (OD control - OD administration well) / OD control well × 100%

[0036] The experiment was repeated three times, and the 10 μM cell viability of the compound was calculated using Prism GraphPad software through a nonlinear regression curve fitted with a normalized dose response. The results are shown in Table 1. Further activity values ​​showed that the IC50 value of compound (1) in SKOV3 cells was 4.1 μM.

[0037] Table 1: Survival rate of compound (1) at 10 μM in breast cancer and ovarian cancer cells.

[0038]

[0039] 2. Surface plasmon resonance (SPR) technique for detecting the affinity of compound (1) for RSK2 protein.

[0040] Detection Principle: Surface plasmon resonance (SPR) technology is an analytical technique developed based on optical principles. The SPR biosensor uses polarized light as its light source. A gold film is deposited on the surface of the sensor chip. During the experiment, a biomolecule (target molecule) is first immobilized on the gold film surface. Then, molecules interacting with it are dissolved in a solution (or mixture) and flow through the chip surface. 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) changes accordingly. The detector detects this change, and by plotting and analyzing this change curve, the binding constant Ka, dissociation constant Kd, or affinity constant KD between molecules can be determined.

[0041] Experimental Procedure: PBST (pH=7.4, 0.1% Tween 20) was added to the RSK2 protein sample stock solution to dilute it to five concentration gradients: 10 nM, 40 nM, 160 nM, 640 nM, and 2560 nM. All samples were flowed sequentially for testing. PBST was used as the flow carrier throughout the experiment. In the interaction testing phase, the analytes were flowed at a rate of 0.5 μL / s. -1 The solution flows over the chip surface; in the surface regeneration process, Glycine-HCl (pH=2.0) solution is used as the regeneration fluid at a flow rate of 2 μL·s-1.

[0042] In the experiment, samples of compound (1) with different concentration gradients were loaded sequentially from low to high concentration, at a flow rate of 0.5 μL·s. -1 The reaction temperature was 4 ℃, the binding time was 600 s, and the dissociation time was 360 s. Glycine-HCl (pH=2.0) solution was used as the regeneration solution for regeneration.

[0043] Kinetic analysis was performed using Berthold bScreen LB 991 (V4 device) control software. The KD value of the compound sample in formula (1) was obtained by fitting the data using Berthold bScreen LB 991 analysis software. The smaller the KD value, the stronger the affinity of the sample for RSK2 protein.

[0044] Test results: by Figure 2 It can be seen that the compound shown in Formula 1 can bind to the RSK2 protein, with a KD of 156 nM.

[0045] 3. Western blot analysis of compounds downregulating RSK2 expression

[0046] Detection Principle: Western blotting utilizes polyacrylamide gel electrophoresis (PAGE). Protein samples separated by PAGE are transferred to a solid support, where proteins are adsorbed non-covalently, preserving the types and biological activities of the separated peptides. The proteins on the solid support serve as antigens, triggering an immunoreaction with corresponding antibodies, followed by a reaction with enzyme- or isotope-labeled secondary antibodies. Substrate color development allows for the detection of protein components expressing specific target genes separated by electrophoresis.

[0047] Experimental procedure: SKOV3 cells were seeded into six-well plates and incubated overnight. They were then treated with different concentrations of compounds (1 nM, 2 nM, 5 nM) and LJH685 (5 nM) for 48 h. A 1‰ (v / v) DMSO medium was used as a blank control. Electrophoresis results are shown below. Figure 3 As shown in Figure A. Cell samples were collected and lysed in ice-cold RIPA buffer for 30 min. The lysate was then removed by centrifugation at 14,000 rpm for 10 min at 4°C, and the supernatant was discarded. Protein concentration was determined using a PierceBCA protein assay kit. Cell lysates were loaded onto 10% SDS-PAGE, electrophoresed, and the separated proteins were then electrotransferred to a polyvinylidene fluoride membrane and blocked with 5% bovine serum albumin for 1 h. The membrane was hybridized with a 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 h. Bands were observed using enhanced chemiluminescence immunoassay, and then quantified using ImageJ software.

[0048] Western blot analysis was conducted to detect the effect of compound 1 on RSK2 protein expression. The results are as follows: Figure 3 Compound B, of formula (1), can downregulate the expression of RSK2 protein.

[0049] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of the compound of formula (1) as an RSK2 inhibitor in the preparation of a medicament for treating breast cancer and / or ovarian cancer, characterized in that, The compound of formula (1) is .

Images