New use of SCH772984 in treating SPSB1 high expression tumor

Abstract

The application discloses a new application of SCH772984 in treating SPSB1 high-expression tumors. It is found for the first time that SCH772984 can be used as an SPSB1 inhibitor and has a therapeutic effect on SPSB1 high-expression diseases such as tumors and infectious diseases, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically involving the novel application of SCH772984 in the treatment of SPSB1-overexpressing tumors. Background Technology

[0002] SPSB1 (SPSB1 protein, encoded by the SPSB1 gene) is an E3 ubiquitin ligase that plays a crucial role in various cellular processes and biological responses. It primarily regulates the degradation of target proteins through ubiquitination, playing a vital role in immune responses, inflammatory responses, tumorigenesis and development, and cell cycle regulation. Studies have shown that SPSB1 has potential biological significance in various diseases, particularly in immune system-related diseases and tumors. Therefore, specific inhibition of SPSB1 has become a potential therapeutic strategy.

[0003] Currently, researchers mainly study the functional inhibition of SPSB1 through gene knockout, RNA interference (siRNA), and other methods. However, these methods still have problems such as insufficient specificity, drug resistance, and poor long-term efficacy. Summary of the Invention

[0004] In view of this, the present invention provides a new application of SCH772984 in the treatment of SPSB1-overexpressing tumors.

[0005] The present invention achieves the above objectives using the following technical solution: The first aspect of the present invention provides the use of SCH772984 or a pharmaceutically acceptable salt thereof in the preparation of a therapeutic agent for diseases with high SPSB1 expression, wherein the diseases with high SPSB1 expression include tumors with high SPSB1 expression and infectious diseases with high SPSB1 expression.

[0006] In this invention, the chemical name of SCH772984 is (R)-1-(2-oxo-2-[4-(4-pyrimidin-2-ylphenyl)piperazin-1-yl]ethyl)-N-(3-(pyridin-4-yl)-1H-indazol-5-yl)pyrrolidine-3-carboxamide, the CAS number is 942183-80-4, and the molecular formula is C 33 H 33 N9O2 has a molecular weight of 587.67.

[0007] In this invention, the pharmaceutically acceptable salt refers to a salt of an active compound (such as SCH772984) and is prepared by reacting the active compound with a suitable organic or inorganic acid or acid derivative. Pharmaceutically acceptable salts include, but are not limited to, hydrochlorides, sulfates, phosphates, citrates, hydrobromides, acetates, benzoates, benzenesulfonates, tartrates, carbonates, citrates, gluconates, lactates, malates, methanesulfonates, stearates, valerates, nitrates, sodium salts, calcium salts, potassium salts, zinc salts, and meglumine salts.

[0008] In this invention, SPSB1 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed SPSB1, as well as any form of SPSB1 derived from cell-processed sources. The term encompasses naturally occurring variants of SPSB1 (e.g., splice variants or allelic variants). The term encompasses, for example, the SPSB1 gene, human SPSB1, and SPSB1 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in this invention, SPSB1 is a human gene with gene ID 80176.

[0009] In some embodiments, the inhibition of SPSB1 activity includes reducing the expression level of SPSB1 mRNA and / or reducing the expression level of SPSB1 protein.

[0010] In some embodiments, the SPSB1-overexpressing tumors include lung cancer, colorectal cancer, triple-negative breast cancer, liver cancer, cervical cancer, prostate cancer, pancreatic cancer, melanoma, glioma, lymphoma, gastric cancer, and ovarian cancer.

[0011] In some embodiments, the SPSB1-overexpressing tumors are selected from lung cancer and colorectal cancer.

[0012] In some embodiments, the SPSB1-overexpressing infectious diseases include infectious diseases caused by bacteria, fungi, or viruses.

[0013] In some embodiments, the bacterial infectious diseases include Listeria monocytogenes infection, Staphylococcus aureus infection, Escherichia coli infection, and Mycobacterium tuberculosis infection.

[0014] In some embodiments, the infectious diseases caused by fungi include Candida albicans infection and Aspergillus infection.

[0015] In some embodiments, the infectious diseases caused by viruses include influenza virus infection, hepatitis B virus infection, and novel coronavirus infection.

[0016] In some embodiments, the SPSB1-overexpressing disease treatment drug further includes a pharmaceutically acceptable carrier.

[0017] The medicaments or compositions thereof of the present invention may further comprise pharmaceutically acceptable carriers or excipients, wherein "pharmaceutically acceptable carriers or excipients" refers to non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulation materials, or formulation aids of any type. The carrier shall be biologically acceptable, meaning it is compatible with other components in the formulation and harmless to the patient, and will not elicit an adverse reaction (e.g., an immune response) when administered to the host.

[0018] In some embodiments, the pharmaceutically acceptable carrier is selected from one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0019] In some embodiments, the dosage form of the SPSB1-overexpressing disease treatment drug includes at least one of tablets, capsules, granules, suspensions, solution injections, lyophilized powder injections, liposome injections, and polymeric complexes.

[0020] A second aspect of the present invention provides a product for inhibiting SPSB1 activity, the product comprising SCH772984 or a pharmaceutically acceptable salt thereof.

[0021] In some embodiments, the product is a pharmaceutical product, a pharmaceutical composition, a reagent kit, a system, or a device.

[0022] In this invention, the term "pharmaceutical composition" refers to a composition containing at least one bioactive compound. The pharmaceutical compositions of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted drug delivery device. In some embodiments, oral administration is preferred. The pharmaceutical compositions of this invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, excipient, or excipient. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can be administered to the receptor via any route, provided the target tissue can be reached.

[0023] In some embodiments, the inhibition of SPSB1 activity includes reducing the expression level of SPSB1 mRNA and / or reducing the expression level of SPSB1 protein.

[0024] A third aspect of this invention provides a computer-aided drug screening method based on the SCH772984 and MID2-SPSB1 complex, the method comprising the following steps: Obtain structural data of the SCH772984 and MID2-SPSB1 complex.

[0025] The binding sites of the SCH772984 and MID2-SPSB1 complex were determined based on their structural data.

[0026] Candidate drugs targeting the binding site were obtained through computer-aided screening.

[0027] Once the binding sites are identified, computer-aided drug design methods can be used to dock compounds from a small molecule library with the target protein. During docking, the computer simulates the interaction between the compound and the binding site and evaluates their binding affinity. Based on the docking results, small molecule compounds with strong binding affinity to the target protein can be screened as potential drug candidates. The determination of binding sites in virtual drug screening is crucial to the success of drug design. Therefore, multiple factors need to be considered when determining binding sites, such as the structural characteristics of the target protein and the binding modes of known ligands. Simultaneously, the binding sites need to be appropriately processed and optimized during virtual screening to improve the accuracy and efficiency of the screening.

[0028] In some embodiments, the computer-aided screening process involves: obtaining the molecular structure of the SCH772984 and MID2-SPSB1 complex and inputting the molecular structure into a pharmacophore module library for matching; clustering all interaction sites based on the interaction patterns with SCH772984 and MID2-SPSB1 to obtain a pharmacophore model; and inputting the pharmacophore model into a molecular compound database for high-throughput screening to obtain candidate drugs.

[0029] In some embodiments, the computer-aided screening process is as follows: First, obtain the molecular structure of SCH772984 and / or the molecular structure of a small molecule inhibitor of MID2 / SPSB1. Then, based on the molecular structure, screen to obtain a library of small molecules with similar structures. Next, perform molecular docking of the library of small molecules with similar structures to the binding site to obtain the score of the docking molecules. Finally, sort them to obtain candidate drugs.

[0030] In some embodiments, the method further includes performing inhibitory activity tests on candidate drugs, calculating the inhibition rate after mixing the small molecule compound with MID2 / SPSB1 protein solution, and screening to obtain small molecule compounds with inhibitory effects.

[0031] It should be noted that the present invention does not impose any particular limitation on the specific type of small molecule compound. Any small molecule compound that may be used in the computer-aided drug screening method provided by the present invention and may produce the corresponding effect falls within the protection scope of the present invention.

[0032] In some embodiments, the small molecule compound is sourced from either newly synthesized compounds or existing databases. Existing databases include, but are not limited to, general natural product databases (COCONUT, Super Natural II, NPASS), plant natural product databases (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), traditional Chinese medicine natural product databases (CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), microbial natural product databases (StreptomeDB, NP Altas, ProCarDB, PAMDB, Lichen Database), marine natural product databases (MNPD, SWMD), natural product databases from different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB), food natural product databases (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet database), toxic natural product databases (Exposome-Explorer, T3DB, SnakeNeurotoxin Database, TPPT), and natural product industry catalogs (Greenpharma). Databases for deduplication using MS data include: AnalytiCon Discovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol, etc.; databases for deduplication using MS data include: MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS; and databases for deduplication using NMR data include: NMRShiftDB, NAPROC-13, etc.

[0033] In some embodiments, the candidate drug is a therapeutic drug for diseases with high SPSB1 expression.

[0034] In some embodiments, the SPSB1-overexpressing diseases include SPSB1-overexpressing tumors and SPSB1-overexpressing infectious diseases.

[0035] In some embodiments, the SPSB1-overexpressing tumors include lung cancer, colorectal cancer, triple-negative breast cancer, liver cancer, cervical cancer, prostate cancer, pancreatic cancer, melanoma, glioma, lymphoma, gastric cancer, and ovarian cancer.

[0036] In some embodiments, the SPSB1-overexpressing tumors are selected from lung cancer and colorectal cancer.

[0037] In some embodiments, the SPSB1-overexpressing infectious diseases include infectious diseases caused by bacteria, fungi, or viruses.

[0038] In some embodiments, the bacterial infectious diseases include Listeria monocytogenes infection, Staphylococcus aureus infection, Escherichia coli infection, and Mycobacterium tuberculosis infection.

[0039] In some embodiments, the infectious diseases caused by fungi include Candida albicans infection and Aspergillus infection.

[0040] In some embodiments, the infectious diseases caused by viruses include influenza virus infection, hepatitis B virus infection, and novel coronavirus infection.

[0041] In this invention, the term "treatment" refers to the process of intervening in or altering a specific health condition, including eliminating the cause, symptomatic treatment, or supportive treatment, and does not necessarily mean curing or completely eliminating a disease, symptom, or the symptoms of a disease or symptom. Treatment may refer to a reduction or improvement in the severity of symptoms / effects of an identified disease by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0042] The pharmaceutical compositions of the present invention, or recombinant human SCH772984, or its pharmaceutically acceptable salts, are administered to mammals or tumor cells of said mammals. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes, or Homo sapiens. The recipient may be a patient suffering from a tumor, or an ex vivo tumor cell from a patient suffering from a tumor.

[0043] Advantages and beneficial effects of this invention: This invention is the first to discover that SCH772984 can act as an SPSB1 inhibitor, specifically inhibiting SPSB1 activity and reducing non-specific interference with other related genes or signaling pathways. Compared with traditional methods such as RNA interference, SCH772984, as a small molecule compound, has stronger pharmacokinetic characteristics and can maintain its activity and stability in vivo for a longer period. This application broadens its indications, discovering its therapeutic effects on diseases with high SPSB1 expression, such as tumors and infectious diseases, and has broad clinical application prospects. Attached Figure Description

[0044] Figure 1 The figure shows the results of screening small molecule compounds with SPSB1-regulating activity. In the figure, a represents large-scale drug screening in A549 / Vector and A549 / SPSB1 cells, b represents the cell activity results after treating the two cell types with 10 μM small molecule compounds with known activity, and c represents the cell activity results after treating the two cell types with small molecule compounds selected in one round of screening at 10 μM and 5 μM.

[0045] Figure 2 The figure shows the results of the second round of validation of the screening results. In the figure, a and e represent the cell viability results after treating two types of cells with the five compounds selected in the second round of screening at 1, 5, and 10 μM, respectively. f represents the expression levels of iNOS and SPSB1 proteins detected by Western blotting.

[0046] Figure 3 The figure shows the results of the investigation into the mechanism of action of SCH772984, where a is the chemical structural formula of SCH772984, and b is the sensitivity test of A549 and MDA-MB-231 cell lines to SCH772984, ASN007, DEL-22397, Ravoxertinib, FR180204, and Ulixertinib.

[0047] Figure 4 The figure shows the interaction between SPSB1 and SCH772984. In figure a, the expression levels of SPSB1 and iNOS proteins were detected by Western Blot after treatment with different concentrations of SCH772984 for 8 hours. In figure b, the affinity of SPSB1 to SCH772984 for direct binding was detected by SPR.

[0048] Figure 5 The figure shows the specific inhibitory effect of SCH772984 on SPSB1 and its in vivo antitumor effect. In the figure, a is the tumor volume curve of SPSB1 overexpression, and b is the tumor weight statistics at doses of 0, 12.5, and 25 mg / kg.

[0049] Figure 6 The graphs show the function of SPSB1 in an in vivo acute infection model and the therapeutic effect of SCH772984. In the graphs, a is the survival rate of mice with systemic SPSB1 knockout and WT mice under the same infection conditions, b is the survival rate of mice with SPSB1 knockout in myeloid cells and WT mice under the same infection conditions, and c is the survival rate of mice after SCH772984 inhibits SPSB1 in vivo. Detailed Implementation

[0050] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0051] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0052] Example 1: Screening of SPSB1-specific inhibitors I. Experimental Materials 1. Cell lines: Wild-type A549 (human lung adenocarcinoma cell line) and wild-type HCT116 (human colorectal cancer cell line) cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, high-glucose; Gibco, USA) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin solution (Penicillin–Streptomycin, Gibco, Cat#15140-122) added to the medium. Cells were cultured at 37°C, 5% CO2, and saturated humidity.

[0053] 2. Construction of stable cell lines: A549 / Vector (empty vector control) and A549 / SPSB1 (SPSB1 stable overexpression line) were obtained via lentiviral infection and screened for resistance with puromycin. The vectors used included: pCDHO-PURO-CMV-3×FLAG (empty vector) and pCHO-PURO-CMV-SPSB1-3×FLAG (SPSB1 overexpression vector). The lentiviral packaging system included plasmid Δ8.9 (gag / pol), PLP2 (rev), and VSVG (env). All vectors and packaging plasmids were provided by Professor Binghui Li's research group at the Beijing Institute of Liver Diseases.

[0054] 3. Small molecule drug library: The small molecule compound library used in the experiment was purchased from Shanghai Tauto Biotech Co., Ltd. The drug library contains more than 20,000 small molecule compounds with known biological activities, product code L4010.

[0055] 4. Cell viability assay reagents: Cell viability is evaluated by detecting intracellular ATP levels. The ATP assay kit used was purchased from Promega Corporation (USA), product code G924C.

[0056] 5. Instruments and equipment: The microplate multi-functional enzyme-linked immunosorbent assay (ELISA) analyzer used for cell viability detection was purchased from Berthold Technologies GmbH & Co. KG (Germany).

[0057] 6. Antibodies: iNOS antibody (Proteintech, Cat#22226-1-AP); β-Actin antibody (Proteintech, Cat#66009-1-Ig); SPSB1 antibody (Abbexa Ltd., UK, Cat#abx127125).

[0058] II. Experimental Methods 1. Preliminary screening experiment of small molecule compounds: This invention establishes a high-throughput small molecule compound screening system based on cell activity detection. By comparing the difference in activity between A549 / Vector and A549 / SPSB1 cells after drug treatment, small molecule compounds that can regulate SPSB1 function are screened. The specific steps are as follows: (1) 384-well plates are used as experimental carriers, and 1,000 A549 / Vector or A549 / SPSB1 cells are seeded in each well; (2) The cells are cultured at 37°C and 5% CO2 for 8 h to allow them to adhere fully; (3) The small molecules to be tested from the compound library are added to each well to make the final concentration 10 μM; (4) After culturing for another 48 h, cell activity is measured using a cell activity detection reagent based on ATP levels; (5) A total of 20,980 small molecule compounds are screened, and preliminary candidate compounds are obtained based on the activity differences between the two cell lines.

[0059] 2. Secondary screening experiment of small molecule compounds: In order to further narrow down the candidate range, the candidate compounds obtained in the initial screening were screened again. The specific contents are as follows: (1) 188 candidate compounds obtained in the initial screening were selected; (2) The screening process was the same as that in the initial screening; (3) The final concentration of the compound was set as two gradients of 10 μM and 5 μM; (4) The secondary screening candidate compounds were obtained by screening according to the activity difference between A549 / Vector and A549 / SPSB1 at different concentrations.

[0060] 3. Three-stage screening experiment of small molecule compounds: In order to further confirm the SPSB1 dependence of the compounds, a third round of screening was carried out on the candidate compounds obtained from the secondary screening. The specific steps are as follows: (1) Select 5 candidate compounds obtained from the secondary screening; (2) The screening method is the same as that of the primary screening; (3) Set the final concentration of the compounds to 10 μM, 5 μM and 1 μM; (4) Use A549 / Vector and A549 / SPSB1 cell lines, and add HCT116 cell line with high endogenous SPSB1 expression as a positive control; (5) Based on the activity changes of different cell lines and different concentration conditions, the small molecules with SPSB1-dependent effects were finally determined.

[0061] 4. Western Blot Validation Experiment To further verify the effect of candidate small molecules on SPSB1-related pathways, protein levels of candidate drugs obtained from the secondary screening were detected. The specific procedures are as follows: (1) HCT116 cells were seeded into 12-well plates, with 1×10 cells seeded per well. 4 (2) Add candidate compounds to each well to a final concentration of 10 μM and incubate for 2 h; (3) Collect cells after incubation and extract total protein using RIPA lysis buffer; (4) Detect the protein expression levels of iNOS, SPSB1 and β-actin using Western Blot; (5) Further screen small molecule compounds with SPSB1 regulatory activity based on the above protein expression changes.

[0062] III. Experimental Results To identify small molecule compounds with more selective inhibitory effects on SPSB1-overexpressing cells, we first conducted large-scale drug screening in A549 / Vector and A549 / SPSB1 cells. Figure 1 (a) in the middle.

[0063] In the first round of screening, two cell lines were treated with a compound library containing 20,980 small molecule compounds (10 μM) with known activities, and cell viability was measured. Based on the screening criteria of "A549 / Vector cell viability greater than 70%, A549 / SPSB1 cell viability less than 30%", a total of 188 candidate compounds were obtained. Figure 1 (b) in the middle.

[0064] The 188 compounds were then subjected to a second round of screening at 10 μM and 5 μM. Based on the criteria of "A549 / Vector survival rate greater than 60% and A549 / SPSB1 survival rate less than 40% at 5 μM," five compounds were ultimately selected: Hit#2-51, Hit#2-62, Hit#2-89, Hit#2-98, and Hit#2-120. Figure 1 (c in the text)

[0065] Furthermore, we evaluated the cell viability of these five compounds at 1, 5, and 10 μM in A549 / Vector, A549 / SPSB1, and HCT116 / WT cells that naturally highly express SPSB1. The results showed ( Figure 2 Hit#2-51 and Hit#2-62 significantly inhibited the cell activity of A549 / SPSB1 and HCT116 / WT at all three concentrations, while having almost no effect on A549 / Vector, demonstrating outstanding SPSB1 selective inhibition. The other three compounds also showed inhibitory effects, but their selectivity was significantly weaker than that of Hit#2-51 and Hit#2-62.

[0066] To further verify whether these compounds could inhibit SPSB1 function, we performed Western blot analysis on cells treated with these five compounds. Figure 2 (i) Given that SPSB1 promotes the ubiquitination and degradation of iNOS, if a compound inhibits SPSB1 function, the iNOS protein level should correspondingly increase. Western Blot results showed that only Hit#2-51 significantly downregulated SPSB1 protein expression and simultaneously and significantly upregulated iNOS expression levels, while no similar effects were observed with the other compounds. In summary, Hit#2-51 was identified as a candidate small molecule for SPSB1 inhibition with optimal activity and selectivity, and can serve as a core compound for subsequent mechanism studies and drug development.

[0067] Example 2: Investigation into the inhibitory mechanism of SCH772984 on SPSB1 I. Experimental Materials SCH772984 (product number T6066) was purchased from Shanghai Taoshu Biotechnology Co., Ltd. Ravoxertinib (HY-15947), Ulixertinib (HY-15816), ASN007 (HY-136579), DEL-22397 (HY-18932), and FR180204 (HY-12275) were all purchased from MedChemExpress (MCE, USA / China). The overexpression plasmid pcDNA-3Flag-SPSB1 was provided by Professor Li Binghui's research group at the Beijing Institute of Liver Diseases.

[0068] II. Experimental Methods A dose gradient treatment was performed in a 96-well plate to detect cell viability. The specific steps are as follows: (1) The cells to be tested (including A549 / Vector and A549 / SPSB1) were seeded in a 96-well plate, with 3,000 cells seeded in each well; (2) The cells were cultured at 37°C and 5% CO2 for 12 h to allow the cells to adhere fully; (3) A concentration gradient of candidate compounds was prepared, with the highest concentration being 200 μM, and a two-fold serial dilution was performed (200 μM → 100 μM → 50 μM → …); (4) The corresponding concentration of the compound was added to each well and cultured for another 24 h; (5) Cell viability was detected in each well using a cell viability assay reagent (either ATP-based or CCK8-based methods are acceptable, but ATP-based methods are used in this example); (6) A dose-response curve was plotted based on the changes in cell viability at different concentrations, and the IC50 was calculated. 50 Differences in activity can be used to further confirm the effectiveness and selectivity of candidate small molecules.

[0069] III. Experimental Results Based on the preliminary screening results, we confirmed that the chemical structure of Hit#2-51 is the known small molecule inhibitor SCH772984 ( Figure 3 (a) Previous studies have shown that SCH772984 can inhibit the catalytic activity of ERK1 / 2 by competing for ATP binding sites and blocking the phosphorylation process of ERK1 / 2, thereby inhibiting ERK pathway activity. To clarify whether the inhibitory effect of SCH772984 on SPSB1 depends on the ERK pathway, we further selected five ERK pathway inhibitors with different mechanisms of action (DEL-22397, Ulixertinib, Ravoxertinib, FR180204, and ASN007) as controls for comparative experiments.

[0070] Drug sensitivity was assessed after overexpressing SPSB1 in A549 and MDA-MB-231 cells, respectively. The results are as follows: Figure 3As shown in b, only SCH772984 showed a significantly enhanced inhibitory effect under SPSB1 overexpression conditions, while no significant difference in sensitivity was observed between the other five ERK inhibitors under SPSB1 overexpression and the control group.

[0071] The above results indicate that the inhibitory effect of SCH772984 on SPSB1 is not due to its classic ERK pathway inhibitory function, but is more likely to exert its effect through atypical mechanisms such as directly binding to SPSB1 or regulating its stability.

[0072] Example 3: Investigation into the binding mechanism of SCH772984 and SPSB1 I. Experimental Methods To verify the effect of SCH772984 on SPSB1 protein levels and to evaluate its direct binding ability to SPSB1, this invention conducted Western blotting experiments and surface plasmon resonance (SPR) binding experiments on cell treatment.

[0073] In the Western Blot experiment, HCT116 cells were seeded and cultured to an appropriate density, and then treated with different concentrations of SCH772984 (0, 1, 5, 10 μM) for 8 hours. After treatment, the cells were collected and total protein was extracted. The protein expression levels of SPSB1, iNOS and internal control GAPDH were detected by Western Blot.

[0074] In the SPR experiment, recombinant SPSB1 protein with a His tag was used as a ligand to immobilize on the surface of the Ni2+-NTA sensor chip: after the chip was activated by nickel charging, His-SPSB1 was injected to immobilize it, and the reference channel of the unimmobilized protein was used to subtract nonspecific signals; SCH772984 was prepared with running buffer to a series of gradient concentrations (0.39, 0.78, 1.56, 3.12, 6.25, 12.5 μM), and injected into the flow cell in ascending order. The response curves of the binding and dissociation phases were recorded, and the binding parameters were calculated by fitting the sensing curves using a 1:1 Langmuir binding model to obtain the equilibrium dissociation constant Kd.

[0075] II. Experimental Results Western blotting results showed that SCH772984 treatment reduced SPSB1 protein levels in HCT116 cells, accompanied by an increase in iNOS protein levels. Figure 4 (a) The SPR results show that SCH772984 and His-SPSB1 have a detectable direct binding, and the equilibrium dissociation constant Kd obtained by fitting is approximately 1.10 μM ( Figure 4(b) indicates that SCH772984 can specifically interact with SPSB1 and has micromolar affinity. The above results together support that SCH772984 can affect downstream substrate-related pathways by binding to SPSB1 and regulating its protein level. Example 4: SCH772984 on tumors with high SPSB1 expression Intratumoral inhibition I. Experimental Materials Female BALB / c nude mice (4-5 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0076] II. Experimental Methods Mice were housed in accordance with the principles and procedures outlined in the *Guideline for the Care and Use of Laboratory Animals* issued by the National Institutes of Health (NIH), and in a specific pathogen-free (SPF) barrier facility. The housing conditions for mice in this study were as follows: temperature upper limit 23°C, lower limit 19°C; humidity upper limit 70%, lower limit 40%; and a 12-hour light / 12-hour dark cycle. The maximum tumor burden permitted by the ethics committee should not exceed 10% of the host mouse's body weight; in this study, the tumor volume did not exceed this limit. Each mouse was subcutaneously inoculated with 4 × 10⁴ mmol / L of [unspecified substance]. 6 231 SCH772984 cells were dissolved in 10% dimethyl sulfoxide, 40% PEG300, 5% Tween-80, and 45% physiological saline and administered intraperitoneally daily at doses of 12.5 mg / kg or 25 mg / kg starting from day 20. Tumor volume was measured every five days, and final weight and volume were measured by dissection at day 30. Five mice were used in each group. Tumor volume was calculated using the following formula: Volume (mm²) 3 = Length (mm) × (Width (mm)) 2 ×1 / 2.

[0077] III. Experimental Results To further verify the specific inhibitory effect of SCH772984 on SPSB1 and its in vivo antitumor effect, we inoculated MDA-MB-231 / Vector and MDA-MB-231 / SPSB1 cells into female nude mice for tumorigenesis experiments. The results showed that, compared with the control group, tumors overexpressing SPSB1 exhibited significantly faster tumor growth, and tumor volume increased rapidly after inoculation. Figure 5 (a) in the middle.

[0078] Once the tumor reached a certain size (day 20), SCH772984 treatment was initiated. Results showed that in the SPSB1 overexpression group, SCH772984 significantly inhibited tumor growth in a dose-dependent manner, especially at a dose of 25 mg / kg, where the tumor almost completely regressed. Figure 5(b) In contrast, in the Vector control group, SCH772984 had no significant effect on tumor growth.

[0079] The above results clearly demonstrate that SCH772984 has a highly specific inhibitory effect on SPSB1 in vivo and can effectively reverse the SPSB1-mediated tumor-promoting effect, further proving that targeted degradation of SPSB1 has significant anti-tumor potential.

[0080] Example 5: In vivo inhibitory effect of SCH772984 on SPSB1 infectious disease I. Experimental Materials C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All laboratory animals used were healthy mice and housed in a barrier environment that complies with laboratory animal management regulations.

[0081] Listeria monocytogenes ( Listeria monocytogenes The strain was purchased from the China Microbial Strains Resource Platform (BioBW), platform number Bio-52964. It is preserved in glycerol / culture form and is primarily intended for research and quality control studies.

[0082] II. Experimental Methods To evaluate the functional role of Spsb1 in host resistance to Listeria monocytogenes infection, in vivo infection experiments were conducted using systemic Spsb1 knockout mice and myeloid cell-specific Spsb1 knockout mice.

[0083] Spsb1 whole-body knockout mice were obtained by targeting and deleting key coding exons of the Spsb1 gene in a C57BL / 6J background using CRISPR / Cas9 technology. Stable genetic lines were established after PCR typing and sequencing verification. Spsb1 myeloid-specific knockout mice were obtained by crossing mice carrying the Spsb1 flox allele with LysM-Cre transgenic mice, thus achieving specific knockout of Spsb1 in myeloid cells. Wild-type mice served as controls.

[0084] All mice of the above different genotypes were administered 4 × 10⁴ mg / L via tail vein injection. 5 CFU of Listeria monocytogenes was used to continuously monitor the survival status of mice after infection and to plot survival curves. In the drug intervention experiment, wild-type mice were simultaneously treated with SCH772984 or an equal volume of solvent control after infection, with SCH772984 administered at a dose of 20 mg / kg. The survival status of the mice was then continuously monitored and the time of death was recorded to compare the differences in tolerance to Listeria monocytogenes infection in mice under different genotypes and drug treatment conditions.

[0085] III. Experimental Results To further evaluate the function of SPSB1 in an in vivo acute infection model and the therapeutic effect of SCH772984, we compared the survival of mice under different gene backgrounds or drug treatments. The results showed that, under the same infection conditions, the survival of Spsb1 knockout mice (Spsb1 knockout mice) was significantly lower than that of mice with different gene backgrounds or drug treatments. / The survival rate of mice was significantly higher than that of wild-type mice (WT). Figure 6 (a) suggests that SPSB1 deficiency can significantly improve survival outcomes in acute infection states. Furthermore, in a mouse model of Spsb1 specific knockout in myeloid cells (Spsb1... fl / fl In LysM-Cre, a phenotype with significantly improved survival was also observed. Figure 6 (b) indicates that SPSB1 expression in myeloid cells plays a key role in infection-related lethality.

[0086] Based on this, we evaluated the in vivo effects of the drug in inhibiting SPSB1. Compared with the DMSO control group, mice treated with SCH772984 (20 mg / kg) showed significantly prolonged survival time and higher survival rate. Figure 6 (c) The protective effect is highly consistent with the phenotype of genetically knocked-out SPSB1. Based on the aforementioned in vitro and in vivo experimental results, we hypothesize that during acute infection, SPSB1 knockout or drug inhibition can relieve its negative regulation of iNOS, thereby significantly upregulating iNOS expression levels, enhancing the bactericidal and antibacterial capabilities of M1 macrophages, and thus more effectively controlling the infection process and significantly prolonging host survival. These results further suggest that SCH772984 exerts its anti-infective protective effect by inhibiting SPSB1, and has the potential application prospect as a potential antibacterial therapeutic drug.

[0087] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The application of SCH772984 or its pharmaceutically acceptable salt in the preparation of therapeutic drugs for diseases with high SPSB1 expression, characterized in that, The diseases with high SPSB1 expression include tumors with high SPSB1 expression and infectious diseases with high SPSB1 expression.

2. The application according to claim 1, characterized in that, The SCH772984 or its pharmaceutically acceptable salts achieve the treatment of diseases with high SPSB1 expression by inhibiting SPSB1 activity; Preferably, the inhibition of SPSB1 activity includes reducing the expression level of SPSB1 mRNA and / or reducing the expression level of SPSB1 protein.

3. The application according to claim 1, characterized in that, The tumors that highly express SPSB1 include lung cancer, colorectal cancer, triple-negative breast cancer, liver cancer, cervical cancer, prostate cancer, pancreatic cancer, melanoma, glioma, lymphoma, gastric cancer, and ovarian cancer. Preferably, the SPSB1-overexpressing tumors are selected from lung cancer and colorectal cancer.

4. The application according to claim 1, characterized in that, The infectious diseases in which SPSB1 is highly expressed include infectious diseases caused by bacteria, fungi, or viruses; Preferably, the infectious diseases caused by bacteria include Listeria monocytogenes infection, Staphylococcus aureus infection, Escherichia coli infection, and Mycobacterium tuberculosis infection; Preferably, the infectious diseases caused by fungi include Candida albicans infection and Aspergillus infection; Preferably, the infectious diseases caused by viruses include influenza virus infection, hepatitis B virus infection, and novel coronavirus infection.

5. The application according to claim 1, characterized in that, The SPSB1-overexpressing disease treatment also includes pharmaceutically acceptable carriers.

6. The application according to claim 1, characterized in that, The dosage forms of the SPSB1-overexpressing disease treatment drugs include at least one of the following: tablets, capsules, granules, suspensions, solution injections, lyophilized powder injections, liposome injections, and polymeric complexes.

7. A product for inhibiting SPSB1 activity, characterized in that, The product contains SCH772984 or its pharmaceutically acceptable salts; Preferably, the product is a drug, a drug composition, a reagent kit, a system, or a device; Preferably, the inhibition of SPSB1 activity includes reducing the expression level of SPSB1 mRNA and / or reducing the expression level of SPSB1 protein.

8. A computer-aided drug screening method based on the SCH772984 and MID2-SPSB1 complex, characterized in that, The steps of the method include: Obtain structural data of the SCH772984 and MID2-SPSB1 complex; The binding sites of the SCH772984 and MID2-SPSB1 complex were determined based on their structural data. Candidate drugs targeting the binding site were obtained through computer-aided screening.

9. The method according to claim 8, characterized in that, The computer-aided screening process is as follows: obtain the molecular structure of the SCH772984 and MID2-SPSB1 complex and input the molecular structure into the pharmacophore module library for matching; and cluster all the interaction sites based on the interaction mode with SCH772984 and MID2-SPSB1 to obtain the pharmacophore model. The pharmacophore model is input into a molecular compound database for high-throughput screening to obtain candidate drugs; Preferably, the computer-aided screening process is as follows: first, obtain the molecular structure of SCH772984 and / or the molecular structure of the small molecule inhibitor that inhibits MID2 / SPSB1; then, based on the molecular structure, screen to obtain a small molecule library with similar structures; then, perform molecular docking of the small molecule library with similar structures to the binding site to obtain the score of the docking molecules; finally, sort them to obtain candidate drugs. Preferably, the method further includes conducting inhibitory activity tests on candidate drugs, calculating the inhibition rate after mixing the small molecule compound with MID2 / SPSB1 protein solution, and screening to obtain small molecule compounds with inhibitory effects.

10. The method according to claim 8, characterized in that, The candidate drug is a treatment for diseases with high SPSB1 expression. Preferably, the diseases with high SPSB1 expression include tumors with high SPSB1 expression and infectious diseases with high SPSB1 expression; Preferably, the SPSB1-overexpressing tumors include lung cancer, colorectal cancer, triple-negative breast cancer, liver cancer, cervical cancer, prostate cancer, pancreatic cancer, melanoma, glioma, lymphoma, gastric cancer, and ovarian cancer. Preferably, the SPSB1-overexpressing tumors are selected from lung cancer and colorectal cancer; Preferably, the infectious diseases in which SPSB1 is highly expressed include infectious diseases caused by bacteria, fungi, or viruses; Preferably, the infectious diseases caused by bacteria include Listeria monocytogenes infection, Staphylococcus aureus infection, Escherichia coli infection, and Mycobacterium tuberculosis infection; Preferably, the infectious diseases caused by fungi include Candida albicans infection and Aspergillus infection; Preferably, the infectious diseases caused by viruses include influenza virus infection, hepatitis B virus infection, and novel coronavirus infection.

Images