Computer-aided drug screening method based on FBXO2 and PKM2

Abstract

The invention discloses a computer-aided drug screening method, system or device based on FBXO2 and PKM2. The invention provides a brand-new method, system or equipment for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2, provides a tool for new drug development and clinical application for treatment of oral squamous cell carcinoma, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of computer-aided drug screening, and particularly relates to a computer-aided drug screening method based on FBXO2 and PKM2. BACKGROUND

[0002] Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the head and neck, with high invasiveness and metastasis, and its five-year survival rate has long hovered between 50%-60%. Traditional treatment methods include surgery, radiotherapy and chemotherapy, but there are significant limitations: surgery can destroy facial structure and function, leading to a decline in the patient's quality of life; radiotherapy can easily cause complications such as oral mucosa damage and salivary gland dysfunction; chemotherapy drugs such as cisplatin and 5-fluorouracil can inhibit tumor growth, but are accompanied by serious side effects such as bone marrow suppression and gastrointestinal reactions, and are prone to drug resistance. In addition, the molecular heterogeneity of OSCC makes it difficult to achieve precise treatment with a single therapy, and recurrence and metastasis remain the main challenge in clinical treatment. Therefore, developing new targeted drugs to overcome the shortcomings of traditional therapies has become a research hotspot in the field of OSCC treatment.

[0003] Computer-aided drug design (CADD) provides an efficient and low-cost solution for drug development by integrating computational chemistry, molecular biology and artificial intelligence technology. However, there are still some shortcomings in the computer screening method for OSCC: existing methods focus on a single target, making it difficult to cope with the molecular heterogeneity of OSCC. The occurrence and development of OSCC involve the cross-regulation of multiple signaling pathways, and existing methods lack evaluation of the synergistic effect of multiple targets, making it difficult to find candidate drugs with combined efficacy. SUMMARY

[0004] Therefore, the present application provides a computer-aided drug screening method for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2.

[0005] The present application achieves the above-mentioned purposes by adopting the following technical solutions: The first aspect of the present application provides a computer-aided drug screening method for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2, the steps of the method comprising: obtaining FBXO2 protein and PKM2 protein data; determining the binding site of the two according to the FBXO2 protein and PKM2 protein data; obtaining the candidate drug targeting the binding site of the two by computer-aided screening.

[0006] Further, the process of the computer-aided screening is: performing molecular docking of small molecules in a small molecule library with the binding site and scoring to obtain scores of the docked small molecules; sorting the scores, and selecting top n small molecules to obtain candidate compounds, n being a natural number greater than or equal to 10. In some embodiments, the process of the computer-aided screening is: obtaining the molecular structures of FBXO2 protein and PKM2 protein, and inputting the molecular structures into a pharmacophore module library for matching, clustering all interaction sites based on the interaction modes with FBXO2 and PKM2 to obtain a pharmacophore model; and inputting the pharmacophore model into a molecular compound database for high-throughput screening to obtain candidate drugs.

[0007] In some embodiments, the process of the computer-aided screening is: first obtaining the molecular structure of a small molecule inhibitor of FBXO2 and / or the molecular structure of a small molecule inhibitor of PKM2; then screening a small molecule library with similar structures based on the molecular structure of the small molecule inhibitor, performing molecular docking of the small molecule library with similar structures with the binding site to obtain scores of the docked molecules, and finally sorting to obtain candidate drugs.

[0008] In some embodiments, the process of the computer-aided screening is: screening protein analogs / antibodies / RNA drugs through the binding site to obtain candidate drugs.

[0009] Further, the candidate drugs of the small molecule type are tested for inhibitory activity, the inhibition rates of small molecule compounds mixed with FBXO2 protein solution and PKM2 protein solution are calculated, and small molecule compounds with inhibitory effects are screened.

[0010] In some embodiments, the candidate drugs of protein analogs / antibodies / RNA drugs are tested for inhibitory activity, an oral squamous cell carcinoma cell model or an oral squamous cell carcinoma animal model is obtained, the oral squamous cell carcinoma cell model or the oral squamous cell carcinoma animal model is treated with a small molecule compound, and small molecule compounds that inhibit the proliferation, migration, and invasion of oral squamous cell carcinoma cells are screened.

[0011] Further, the steps of the method of the computer-aided drug screening include: obtaining the binding site of the FBXO2 protein and PKM2 protein complex; screening small molecule compounds with similar structures in a molecular database based on the spatial structure of the binding site of the FBXO2 protein and PKM2 protein complex; performing molecular docking of the screened small molecule compounds with FBXO2 or PKM2 protein to calculate the affinity / binding energy of the target receptor to obtain scores, and sorting the scores to obtain candidate drugs.

[0012] In some embodiments, the binding site comprises at least one of Glu226 of FBXO2 and Thr41 of PKM2.

[0013] In some embodiments, the binding site is Glu226 of FBXO2 and Thr41 of PKM2.

[0014] The second aspect of the present application provides a computer-aided screening drug system for screening a drug for treating oral squamous cell carcinoma based on FBXO2 and PKM2, comprising: An acquisition unit: acquiring FBXO2 protein and PKM2 protein data.

[0015] A site unit: determining a binding site of the two based on the FBXO2 protein and PKM2 protein data.

[0016] A screening unit: obtaining a candidate drug targeting the binding site of the two by computer-aided screening.

[0017] The third aspect of the present application provides a computer-aided screening drug device for screening a drug for treating oral squamous cell carcinoma based on FBXO2 and PKM2, comprising: A memory and a processor, the memory being used to store program instructions. The processor is used to call the program instructions, and when the program instructions are executed, the method for computer-aided screening of a drug for treating oral squamous cell carcinoma based on FBXO2 and PKM2 according to the first aspect of the present application is realized.

[0018] The fourth aspect of the present application provides a computer-readable storage medium having a computer program thereon, comprising: The computer program is executed by the processor to realize the method for computer-aided screening of a drug for treating oral squamous cell carcinoma based on FBXO2 and PKM2 according to the first aspect of the present application.

[0019] The fifth aspect of the present application provides a substance for inhibiting the binding of FBXO2 and PKM2 for use in the preparation of a drug for screening a drug for treating oral squamous cell carcinoma.

[0020] In some embodiments, the substance for inhibiting the binding of FBXO2 and PKM2 comprises one or more of the following: a protein analogue / antibody / RNA and / or a small molecule drug for inhibiting the binding of FBXO2 and PKM2.

[0021] In some embodiments, the substance for inhibiting the binding of FBXO2 and PKM2 comprises shRNA as shown in SEQ ID NO: 1, 2, and 4.

[0022] The sixth aspect of the present application provides a pharmaceutical composition comprising a protein analogue / antibody / RNA and / or a small molecule drug that inhibits the binding of FBX02 and PKM2, and / or an excipient. The excipient is a diluent or a binder or a disintegrant or a lubricant.

[0023] In some embodiments, the excipient comprises one or more of the following: starch, powdered sugar, mannitol, starch paste, cellulose derivatives, magnesium stearate, microfine silica, talc, gelatin, water, ethanol, polyethylene glycols, magnesium lauryl sulfate, hydrogenated vegetable oil.

[0024] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995), and these substances are used as needed to help the stability of the formulation or to help improve the bioavailability of the active or active substances. In some embodiments, when the pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition described in the present application is administered to a human. The dose and administration method of the pharmaceutical composition are not particularly limited, and a skilled physician can easily determine the prescription and the dose of the prescription effective for the desired treatment and / or prevention, and the administration method can be, for example, injection or other treatment.

[0025] In some embodiments, the pharmaceutical composition has any one of the dosage forms selected from the group consisting of a solution, a suspension, an emulsion, a tablet, a pill, a powder, a granule, a capsule, a syrup, a sterile aqueous solution, a non-aqueous solution, a lyophilized preparation, a suppository. In addition, it can be administered once or multiple times. At this time, the biological preparation is administered in the form of a liquid preparation, a powder, an aerosol, a capsule, or a suppository. The administration route can include, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, intrarectal, etc. When administered orally, it can be formulated to protect the active ingredients in the biological preparation from degradation in the stomach. In addition, the active ingredients can be administered by any device capable of transferring to the target tissue. In specific embodiments, the biological preparation provided by the present application can be formulated into various dosage forms as needed, and the dose beneficial to the patient can be administered by a clinician according to the species, age, weight, and general disease condition of the subject, administration method, etc. The administration method can be, for example, injection or any other suitable administration method known to those skilled in the art.

[0026] The application has the advantages and beneficial effects that the application provides a computer-aided drug screening method for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2, and screens protein analogs / antibodies / RNA and / or small molecule drugs that inhibit the combination of Glu226 of FBXO2 and Thr41 of PKM2, that is, candidate drugs for treating oral squamous cell carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A computer-aided drug screening method for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2 provided by the embodiment of the application is shown in the flowchart.

[0028] Figure 2 A computer-aided drug screening system for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2 provided by the embodiment of the application is shown in the schematic diagram.

[0029] Figure 3 A computer-aided drug screening device for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2 provided by the embodiment of the application is shown in the schematic diagram.

[0030] Figure 4 A diagram of FBXO2-targeted substrates and corresponding biological processes is shown, wherein A is the result of CoIP, GST pull-down combined with mass spectrometry to identify FBXO2-targeted substrates, B is the result of GO enrichment analysis, and C is the result of KEGG enrichment analysis.

[0031] Figure 5 A diagram of the effect of FBXO2 overexpression / knockdown on extracellular acidification rate (ECAR) is shown, wherein A-B are the results of detecting ECAR (A) and the corresponding glycolysis statistical diagram (B) after FBXO2 overexpression in the CAL27 cell line, C-D are the results of detecting ECAR (C) and the corresponding glycolysis statistical diagram (D) after FBXO2 knockdown in the SCC9 cell line.

[0032] Figure 6 A diagram of the effect of FBXO2 overexpression / knockdown on glucose uptake and glycolysis products is shown, wherein A-C are the results of detecting glucose uptake (A), ATP (B), and lactic acid level (C) after FBXO2 overexpression in the CAL27 cell line, D-F are the results of detecting glucose uptake (D), intracellular ATP (E), and lactic acid production (F) level after FBXO2 knockdown in the SCC9 cell line.

[0033] Figure 7Figure 2-DG on FBXO2 glycolysis regulation results. A-B are the results of FBXO2 overexpression on ECAR (A) and corresponding glycolysis value (B) in CAL27 cell line before and after 2-DG treatment.

[0034] Figure 8 Figure 3 FBXO2 on CAL27 cell proliferation, migration and invasion after glycolysis inhibition by 2-DG results. A is the CCK8 detection of FBXO2 overexpression on CAL27 cell proliferation before and after glycolysis inhibition, B-C are the staining results (B) and statistical chart (C) of EdU detection of FBXO2 overexpression on CAL27 cell proliferation before and after glycolysis inhibition, D-E are cell images (D) and statistical chart (E) of scratch test detection of FBXO2 overexpression on CAL27 cell migration before and after glycolysis inhibition, F-G are staining results (F) and statistical chart (G) of Transwell experiment detection of FBXO2 overexpression on CAL27 cell migration and invasion before and after glycolysis inhibition.

[0035] Figure 9 Figure 4 FBXO2 on OSCC tumor growth and glycolysis in vivo results. A-B are the differences in OSCC tumor bioluminescence live imaging (A), volume and weight (B) in FBXO2 control and overexpression groups in mice in vivo, C-D are the differences in OSCC tumor bioluminescence live imaging (C), volume and weight (D) in FBXO2 control and knockdown groups in mice in vivo, E is the effect of FBXO2 overexpression and knockdown on the average content of lactic acid in OSCC tumor, F-G are the results of immunohistochemical detection of FBXO2 overexpression (F) and knockdown (G) on the expression level of FBXO2, PKM2, Ki67 and N-cadherin in OSCC tumor.

[0036] Figure 10 Figure 5 FBXO2 and glycolysis complex binding results. A is the CoIP detection of FBXO2, ENO1, LDHA combined with PKM2 in CAL27 and SCC9 cell lines, B is the CoIP detection of PKM2, ENO1, LDHA combined with FBXO2 in CAL27 and SCC9 cell lines, C is the detection of FBXO2, PKM2, ENO1 co-localization in CAL27 and SCC9 cell lines by immunofluorescence.

[0037] Figure 11Figure for FBXO2 up-regulate the protein level and assembly efficiency of glycolytic complex. A-B, the results of Western blot (WB) detection of the protein expression of PKM2, ENOl, LDHA in CAL27 cell line overexpressing FBXO2 and SCC9 cell line knocking down FBXO2 (A) and the gray value statistics (B), C, the results of CoIP detection of the binding efficiency of PKM2, ENOl, LDHA, i.e. the assembly efficiency of glycolytic complex in CAL27 cell line overexpressing FBXO2 and SCC9 cell line knocking down FBXO2.

[0038] Figure 12 Figure for FBXO2-PKM2 direct binding verification. A, the results of GST pull-down verification, B, the results of SPR verification.

[0039] Figure 13 Figure for the effect of FBXO2 on the ubiquitination level of PKM2 and its related ubiquitination degradation pathway detection results. A, the results of IP detection of the effect of FBXO2 overexpression / knocking down on the ubiquitination level of PKM2, B, WB detection of the protein level of PKM2 after FBXO2 knocking down and then MG132 / CQ treatment to clarify the degradation pathway of PKM2 inhibited by FBXO2.

[0040] Figure 14 Figure for molecular docking analysis of FBXO2-PKM2 binding site.

[0041] Figure 15 Figure for FBXO2 mutant binding PKM2 and regulating glycolytic complex, PKM2 ubiquitination. A, the results of CoIP detection of the difference in binding ability of FBXO2 wild type and mutant to PKM2, B, the results of CoIP detection of the difference in the content and assembly efficiency of glycolytic complex regulated by FBXO2 wild type and mutant, C, the results of IP detection of the difference in the ubiquitination level of PKM2 regulated by FBXO2 wild type and mutant.

[0042] Figure 16 Figure for the effect of FBXO2 mutant on the regulation of glucose uptake and glycolytic product of CAL27 cells. A, glucose uptake level, B, intracellular ATP level, C, lactic acid production level.

[0043] Figure 17 Figure for FBXO2 mutant regulating ECAR of CAL27 cells. A-B, the results of the effect on ECAR (A) and the corresponding glycolysis value (B).

[0044] Figure 18Figure for supplementing the result of verifying that the regulation of FBXO2 on glucose uptake and glycolysis products depends on PKM2 by PKM2 wild type and mutant. A is the result of glucose uptake, B is the level of ATP, and C is the level of lactic acid.

[0045] Figure 19 Figure for supplementing the result of verifying that the regulation of FBXO2 on ECAR depends on PKM2 by PKM2 wild type and mutant. A-B are the result graphs of the influence on ECAR (A) and the corresponding glycolysis value (B). DETAILED DESCRIPTION

[0046] The reagents, raw materials and experimental consumables used in the present application are easily obtained by those skilled in the art, and can be obtained from commercial channels if not otherwise specified. The experimental methods not specified in the present application are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present application and should not limit the scope of the present application in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present application and should not limit the present application as described in detail in the claims.

[0047] In order for those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in conjunction with the drawings in the embodiments of the present application. In some of the descriptions in the specification and claims and the above-mentioned drawings, a plurality of operations appearing in a specific order are included, but it should be clearly understood that these operations can be executed in the order appearing in this text or in parallel. The serial numbers of the operations, such as S101, S102, S103, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes can include more or fewer operations, and the operations can be executed in sequence or in parallel.

[0048] The present application will be further described below in conjunction with specific embodiments. The following specific embodiments are only used to explain the present application and should not be understood as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art.

[0049] Figure 1 The computer-aided drug screening method provided by the embodiment of the present application provides a computer-aided drug screening method for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2. Specifically, the computer-aided drug screening includes the following steps: S101: obtaining FBXO2 protein and PKM2 protein data.

[0050] In some embodiments, the FBXO2 is also known as F-box protein 2, FBG1, FBX2, Fbs1, OCP1, NFB42. Its corresponding Gene ID in NCBI is 26232, and its corresponding detailed information (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0051] In some embodiments, the PKM2 is also known as Pyruvate kinase isozyme type M2, muscle pyruvate kinase isozyme 2. Its corresponding Gene ID in NCBI is 5315, and its corresponding detailed information (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0052] In some embodiments, the treatment refers to any action aimed at improving the health status of the patient, such as treatment, prevention, prevention and delay of disease. In certain embodiments, such a term refers to improving or eradicating a disease or symptoms associated with the disease. In other embodiments, the term refers to minimizing the spread or worsening of the disease caused by the administration of one or more therapeutic agents to a subject suffering from such a disease.

[0053] In the present application, it is first proved that the promotion of FBXO2 to the malignant phenotype (proliferation, migration, invasion) of OSCC cells depends on the driving effect on glycolysis, which is achieved by directly binding to the core component PKM2 to inhibit its ubiquitination degradation, forming a stable FBXO2-PKM2 platform to promote the assembly of the glycolysis complex to promote the progression of OSCC. That is, the present application first discovers the interaction between FBXO2 and PKM2, and confirms it through scientific experiments. Based on this, the present application provides an effective computer-aided drug screening method for screening therapeutic drugs based on FBXO2 and PKM2 for OSCC treatment.

[0054] S102: determining the binding site of the two according to the FBXO2 protein and PKM2 protein data.

[0055] In some embodiments, the binding site of the FBXO2 protein and the PKM2 protein in the FBXO2 and PKM2 protein complex can be determined by adopting the method of protein-protein docking or any other method known in the prior art which can be used to determine the interaction site between proteins and proteins.

[0056] In some embodiments, the protein-protein docking refers to a computational simulation process of docking the structures of proteins together by certain algorithms and procedures. This process can be used to study the interaction between proteins and possible biological functions.

[0057] In some embodiments, the binding site of the FBXO2 protein and the PKM2 protein is the spatial structure of the key region of the interaction between the FBXO2 protein and the PKM2 protein.

[0058] In some embodiments, the binding site of the FBXO2 protein and the PKM2 protein comprises at least one of Glu226 of FBXO2 and Arg383, His379, Thr41 of PKM2.

[0059] In some embodiments, the binding site is Glu226 of FBXO2 and Thr41 of PKM2.

[0060] S103: obtaining a candidate drug targeting the binding site of the two through computer-aided screening.

[0061] In some embodiments, the step of computer-aided screening comprises: molecular docking of small molecules in a small molecule library with the binding site and scoring to obtain scores of the docked small molecules; sorting the scores to select the top n small molecules to obtain candidate compounds, n being a natural number greater than or equal to 10.

[0062] In some embodiments, the step of computer-aided screening comprises: obtaining the molecular structure of the FBXO2 protein and the PKM2 protein and inputting the molecular structure into a pharmacophore module library for matching, clustering all interaction sites based on the interaction mode with FBXO2 and PKM2 to obtain a pharmacophore model; inputting the pharmacophore model into a molecular compound database for high-throughput screening to obtain a candidate drug.

[0063] In some embodiments, the step of computer-aided screening comprises: first obtaining the molecular structure of the FBXO2 small molecule inhibitor and / or the molecular structure of the PKM2 small molecule inhibitor; then screening a small molecule library with similar structures based on the molecular structure of the small molecule inhibitor, and then performing molecular docking of the small molecule library with similar structures with the binding site to obtain scores of the docked molecules, and finally sorting to obtain a candidate drug.

[0064] In some embodiments, the step of computer-aided screening further comprises: performing inhibition activity experiment test on the candidate drug of small molecules, calculating the inhibition rate after mixing the small molecule compounds with FBXO2 protein solution and PKM2 protein solution respectively, and screening small molecule compounds with inhibitory effect.

[0065] In some embodiments, the step of computer-aided screening further comprises: performing an inhibitory activity experiment test on the candidate drug of the protein analogue / antibody / RNA drug, obtaining an oral squamous cell carcinoma cell model or an oral squamous cell carcinoma animal model, treating the oral squamous cell carcinoma cell model or the oral squamous cell carcinoma animal model with the small molecule compound, and screening the small molecule compound that inhibits the proliferation, migration and invasion of the oral squamous cell carcinoma cells.

[0066] In some embodiments, the step of computer-aided screening comprises: obtaining the binding site of the FBXO2 protein and the PKM2 protein complex; screening small molecule compounds with similar structures in a molecular database based on the spatial structure of the binding site of the FBXO2 protein and the PKM2 protein complex; performing molecular docking calculation on the screened small molecule compounds and the FBXO2 or PKM2 protein to obtain the score of the affinity / binding energy of the targeted receptor, and sorting the candidate drugs according to the score.

[0067] It should be noted that the specific type of the small molecule compound is not particularly limited in the present application, and any small molecule compound that can be used in the computer-aided drug screening method provided by the present application and can produce the corresponding effect falls within the protection scope of the present application.

[0068] In some embodiments, the source of the small molecule compounds is selected from the group consisting of: newly synthesized or existing databases; wherein the 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), Chinese medicine natural product databases (TCM@Taiwan, 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 of 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, Snake Neurotoxin Database, TPPT), natural product industry catalogs (Greenpharma, AnalytiCon Discovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol), databases for MS data dereplication (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), databases for NMR data dereplication (NMRShiftDB, NAPROC-13), and the like.

[0069] In some embodiments, the mode of screening drugs using computer-aided drug screening technology can include any one or more of the following modes according to the different types of small molecule compounds (inorganic small molecule compounds, protein analogs, nucleic acid analogs, polypeptides, antibodies, siRNA, shRNA, dsRNA, microRNA, or antisense nucleic acids, etc.): protein-small molecule docking, protein-protein docking, protein-nucleic acid docking.

[0070] In one specific embodiment, the present application demonstrates that FBX02 is closely related to the glycolysis process of OSCC cells.

[0071] Specifically, to identify the targeting substrate set of FBXO2 protein systematically and with high confidence, the strategy of CoIP combined with GST pull-down technology and high-precision mass spectrometry analysis was adopted. The physiological binding existing in the intracellular environment reflected by CoIP and the direct binding in the in vitro environment reflected by GST pull-down were included, and the intersection was taken to reduce the false positive rate of a single method and ensure the reliability of the screening results. After CAL27 cells were cultured to the appropriate density, the cells were collected, and the pre-cooled RIPA buffer containing protease inhibitors and phosphatase inhibitors was used to lyse the cells. After centrifugation, the supernatant was collected as the total protein sample. First, CoIP was performed using FBXO2 antibody (Proteintech, 14590-1-AP), and the steps were as follows: after the Protein A / G agarose beads were balanced, they were incubated with the antibody at room temperature overnight to complete the fixation. Then, the total protein lysate was added to specifically capture FBXO2 and its interacting protein complexes. After washing to remove non-specifically bound proteins, the complexes on the beads were subjected to protein reduction, alkylation blocking, and trypsin digestion. The obtained peptides were desalted, concentrated, and then resuspended in formic acid solution. After centrifugation, the supernatant was collected for mass spectrometry analysis. Subsequently, in vitro GST pull-down was performed using GST-FBXO2 fusion protein (Proteintech, Ag6122), and the steps were as follows: after the glutathione agarose beads were balanced, they were incubated with GST-FBXO2 fusion protein at 4°C to fix them, and the total protein sample was added for incubation to capture interacting proteins. The subsequent washing, digestion, and peptide preparation steps were consistent with the CoIP process described above. Through liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis, the raw file of the mass spectrometry raw data was obtained, which was analyzed by software MaxQuant (1.6.2.10) to match the data and obtain the identification results. CoIP mass spectrometry identified 156 interacting proteins, and GST mass spectrometry identified 335 interacting proteins. The intersection obtained 58 FBXO2 candidate substrates, which were ranked according to the Score value, and the top 3 proteins were all glycolytic enzymes (A in Figure 4 ). We performed GO and KEGG enrichment analysis on the FBXO2 candidate substrates, and the top 1 entry was glycolysis, suggesting that FBXO2 is closely related to the glycolysis process of OSCC cells (B-C in Figure 4 ).

[0072] In one embodiment, the present application demonstrates that FBXO2 can significantly increase the level of glycolysis in OSCC cells. Specifically, two human OSCC cell lines, CAL27 and SCC9 (both purchased from the China Academy of Sciences Typical Culture Collection Cell Bank), were routinely cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Based on the background FBXO2 expression level of CAL27 and SCC9, FBXO2 was overexpressed in CAL27 and knocked down in SCC9 for subsequent experiments. First, the complete coding sequence of human FBXO2 (NCBI Reference Sequence: NM_012168.6) was cloned into a plasmid vector and packaged into a slow virus particle with infectious activity, and the corresponding control was a slow virus particle with the empty vector. CAL27 cells were infected and selected by puromycin to obtain CAL27 cells stably overexpressing FBXO2 and controls. Specific short hairpin RNA (shRNA) sequences targeting human FBXO2 mRNA were designed and synthesized (shFBXO2#1: 5'-CAGCAGUUCUACUUCCUGATT-3' (SEQ ID NO: 1), shFBXO2#2: 5'-GCAAAGCACAGGUCAUUGATT-3' (SEQ ID NO: 2), shNC: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 3)), and SCC9 cells were also infected with shRNA slow virus to obtain SCC9 cells stably knocking down FBXO2 and controls. Real-time glycolytic rate, i.e., ECAR value, was determined using a Seahorse XF96 energy metabolism analyzer. FBXO2 overexpression / knocking down and the corresponding control cells were seeded in a Seahorse special culture plate at an appropriate density, and the Seashore XF DMEM medium without buffer was replaced before detection, and incubated at 37°C in a CO2-free incubator for 1 hour. Through the Seahorse XF glycolysis stress test kit, glucose, oligomycin (ATP synthase inhibitor), and 2-deoxy-D-glucose (2-DG, hexokinase competitive inhibitor) were injected in turn, and the changes in ECAR were monitored in real time by the instrument. This parameter directly reflects the glycolytic flux of the cells, and the results are shown in Figure 5 As can be seen, overexpression or knockdown of FBXO2 in both CAL27 and SCC9 OSCC cell lines can significantly increase or decrease the glycolytic flux of the cells. In addition, the levels of glucose uptake and glycolysis products were also detected by a glucose uptake detection kit (Bi Yun Tian, S0554), an ATP content detection kit (Nanjing Jiancheng, A095-1), and a lactic acid test kit (Nanjing Jiancheng, A019-2) according to the instructions, and the results are shown in Figure 6As shown. It can be seen that overexpression or knockdown of FBXO2 can significantly increase or inhibit the glucose uptake, intracellular ATP and lactic acid production levels of OSCC cells.

[0073] In the present application, both CAL27 cell line and SCC9 cell line are human tongue squamous cell carcinoma cell lines, which have important application value in oral squamous cell carcinoma (OSCC) research.

[0074] In the present application, the extracellular acidification rate (ECAR) is an index for measuring the rate of acid substances (mainly lactic acid and carbon dioxide) released to the extracellular environment during the metabolic process of cells, which is usually detected by real-time cell metabolism analysis system (such as Seahorse XF technology). It reflects the activity of cell glycolysis (anaerobic metabolism) and partial aerobic metabolism, and is an important parameter for evaluating the energy metabolism characteristics of cells.

[0075] In one specific embodiment, the present application proves that the promotion of FBXO2 to the malignant phenotype (proliferation, migration and invasion) of OSCC cells depends on the driving effect on glycolysis.

[0076] Specifically, the groups are designed as follows: oeNC (+DMSO), oeFBXO2 (+DMSO), oeNC+2-DG (10 mM), oeFBXO2+2-DG (10 mM), in order to detect the difference in the influence of FBXO2 overexpression on the malignant phenotype (proliferation, migration and invasion) of OSCC cells before and after 2-DG (glycolysis inhibitor) treatment. First, the ECAR values of each group of cells were detected by Seahorse XF96 energy metabolism analyzer, and the results verified that 2-DG treatment could completely eliminate the promotion of FBXO2 to glycolysis Figure 7 ). Subsequently, a series of cell function experiments were performed to detect whether the promotion of FBXO2 to the proliferation, migration and invasion of CAL27 cells was affected after 2-DG inhibited glycolysis. Cell proliferation was detected by CCK-8 and EdU experiments. The CCK8 detection method is as follows: the cells were seeded at 6×10 3Cell Counting Kit-8 (CK04, Dojindo) was used to detect cell proliferation. Briefly, cells were seeded in 96-well plates at a density of 1,000 cells per well. After 0, 24, 48, and 72 h of culture, CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 2 h. The absorbance at 450 nm (OD450 value) was then measured using a microplate reader. EdU detection was performed using an EdU detection kit (C0071S, Biyun Tian). Cells were incubated with 10 mM EdU solution at 37 °C for 2 h. After fixation with 4% paraformaldehyde for 20 min, the cells were permeabilized with permeabilization solution (P0097, Biyun Tian) for 15 min and incubated with Click reaction solution at room temperature for 30 min. The nuclei were stained with DAPI (C1005, Biyun Tian). Cell proliferation rate was determined by calculating the ratio of EdU-positive cells (red) to DAPI-positive cells (blue). Cell migration and invasion were detected by scratch and Transwell assays. For the scratch assay, cells were seeded in 6-well plates, and when the confluence reached 90%, a scratch was made with a pipette tip. The cells were then cultured in serum-free DMEM for another 24 h. The scratch closure was photographed, and the cell migration rate was calculated. For the Transwell assay, serum-free DMEM medium resuspended cells were added to the upper chamber of Transwell chambers (3422, Corning) coated with Matrigel matrix gel (356234, BD), and DMEM medium containing 10% FBS was added to the lower chamber. After 24 to 48 h of culture, the invasive cells adhering to the lower chamber were fixed with 4% paraformaldehyde, stained with crystal violet (G1063, Solarbio) for 15 min, and then counted. For the migration assay, no Matrigel was used. The results showed that FBXO2 overexpression significantly promoted the proliferation, migration, and invasion of CAL27 cells, but this promoting effect was significantly weakened under the condition of glycolysis inhibition caused by 2-DG Figure 8 ).

[0077] In one embodiment, the present application demonstrates that FBXO2 significantly promotes OSCC tumor growth and glycolysis in vivo through in vivo experiments.

[0078] Specifically, a total of 28 (4 groups, 7 mice per group) 6-8-week-old healthy BALB / c female nude mice were used for subcutaneous tumor-bearing experiments in nude mice. FBXO2 overexpression and knockdown stable cell lines were collected, counted, and prepared into cell suspensions, which were subcutaneously injected into the left axillary of nude mice at a volume of 100 μL of stable cell lines (5 x 10 7The model was established using cells / mL. After modeling, in vivo imaging was performed regularly to monitor tumor size, record mouse weight, and observe the general condition and diet of the mice. After the experiment, tumor tissues from each group of mice were photographed and tissue samples were preserved for subsequent experiments. It can be seen that FBXO2 overexpression significantly promoted the growth rate of OSCC tumors in mice, and the tumor volume and weight were significantly higher than those in the control group; conversely, FBXO2 knockdown significantly inhibited the growth of OSCC tumors in mice. Figure 9 (AD in the text). To detect differences in lactate content within tumor tissue, a lactate test kit (Nanjing Jiancheng, A019-3) was used. 1g of tumor tissue was weighed, 9ml of physiological saline was added, and the sample was homogenized thoroughly using a homogenizer. After centrifugation, the supernatant was collected and tested according to the kit instructions. The results showed that FBXO2 overexpression or knockdown significantly increased or decreased the mean lactate content in OSCC tumor tissue in mice. Figure 9 (E in the text). To detect the expression of FBXO2, key glycolytic enzymes, and proliferation and metastasis markers in tumor tissues, the collected tissues were fixed and embedded, and tissue sections were prepared for immunohistochemistry. After dewaxing, hydration, and antigen retrieval with sodium citrate buffer, the sections were sequentially subjected to endogenous peroxidase blocking and non-specific site blocking. After incubation overnight at 4°C with FBXO2 antibody (Proteintech, 14590-1-AP), PKM2 antibody (Proteintech, 15822-1-AP), Ki67 antibody (Proteintech, 27309-1-AP), and N-cadherin antibody (Proteintech, 22018-1-AP), the sections were incubated at room temperature with secondary antibody (PV-9000, ZSGB-BIO). After staining with DAB (ZSGB-BIO, ZLI-9017), hematoxylin was used for counterstaining, and images were acquired. It can be seen that the expression levels of FBXO2, PKM2 (a key enzyme in glycolysis), Ki67 (a proliferation marker), and N-cadherin (a transfer marker) in the FBXO2 overexpression group were significantly higher than those in the control group. Figure 9 In the F group), FBXO2 knockdown group is the opposite ( Figure 9 The presence of G in FBXO2 suggests that FBXO2 promotes the glycolysis, proliferation, and metastasis of OSCC tumors in vivo.

[0079] In one specific embodiment, the present invention verified the presence of the glycolysis complex and the direct binding of FBXO2 to the glycolysis complex using Co-IP.

[0080] Specifically, Co-IP experiments were performed in CAL27 and SCC9 cells using PKM2 antibody (Proteintech, 15822-1-AP) and FBXO2 antibody (Proteintech, 14590-1-AP), and the specific method was as previously described. Subsequently, FBXO2 and glycolytic complex proteins (PKM2, ENOl, LDHA) in the immune complex were detected by WB. The WB method was as follows: the above obtained samples were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins in the gel were transferred to a PVDF membrane activated in advance by methanol by wet transfer method. After the transfer was completed, the PVDF membrane was blocked with blocking solution, and then incubated with specific primary antibody at 4°C overnight. The primary antibodies used were as follows: FBXO2 antibody (Proteintech, 14590-1-AP), PKM2 antibody (Proteintech, 15822-1-AP), ENOl (Proteintech, 11204-1-AP), and LDHA antibody (Proteintech, 19987-1-AP). After TBST washing, the corresponding anti-rabbit (Proteintech, SA00001-2) or anti-mouse (Proteintech, SA00001-1) secondary antibody was incubated at room temperature. Finally, the target band was developed using ECL chemiluminescence reagent. It can be seen that in CAL27 and SCC9 cells, the down-drawing of PKM2 can detect the binding of ENOl, LDHA and FBXO2, and the down-drawing of FBXO2 can also detect the binding of PKM2, ENOl and LDHA, proving the existence of glycolytic complex and the binding of FBXO2 and the complex (A-B in FIG. 6). Figure 10 Subsequently, the co-localization of FBXO2 and glycolytic complex was observed by immunofluorescence staining. The steps of immunofluorescence were as follows: after the cells were fixed, permeated and blocked, they were sequentially incubated with FBXO2, PKM2 and ENOl specific primary antibodies at 4°C overnight, and then washed. The corresponding Cy5 fluorescently labeled secondary antibody (Invitrogen, A10523), Alexa Fluor 594 fluorescently labeled secondary antibody (Invitrogen, A-11005) and Alexa Fluor 488 fluorescently labeled secondary antibody (Invitrogen, A-11001) were used at room temperature in the dark, and the staining and image acquisition were completed. Antibody elution was performed between different indicators. Finally, the cell nucleus was counterstained with DAPI. The results showed that in CAL27 and SCC9 cells, FBXO2 (purple), PKM2 (red) and ENOl (green) in the cytoplasm showed obvious co-localization (C in FIG. 6). Figure 10

[0081] ​In one embodiment, the present application proves that FBXO2 up-regulates the expression of each component of the glycolysis complex, participates in the assembly of the complex and improves the assembly efficiency.

[0082] Specifically, the expression amount and assembly efficiency of the glycolysis complex in FBXO2 overexpression / knockdown and corresponding control group cells were detected by the aforementioned WB and CoIP methods. First, the protein levels of FBXO2, PKM2, ENOl and LDHA in each group were detected by WB, with β-actin (Proteintech, 66009-1-Ig) as the internal reference, and the gray values were statistically analyzed. The results showed that the protein levels of FBXO2, PKM2, ENOl and LDHA in the FBXO2 overexpression group were significantly higher than those in the control group, and the results were opposite in the FBXO2 knockdown group, indicating that FBXO2 significantly up-regulated the protein levels of each component of the glycolysis complex. Figure 11 Then, the assembly efficiency of each group of glycolysis complexes was detected by CoIP, which was reflected by the average amount of ENOl and LDHA combined with the core protein PKM2 of the complex. The results showed that more FBXO2, ENOl and LDHA were combined with PKM2 in the IP group of the FBXO2 overexpression group than in the control group, and the results were opposite in the FBXO2 knockdown group, proving that FBXO2 participated in the assembly of the glycolysis complex and enhanced the assembly efficiency of the complex. Figure 11

[0083] In one embodiment, the present application proves that FBXO2 directly targets and binds to the core component PKM2 of the glycolysis complex to play a regulatory role.

[0084] Specifically, according to the previous results of FBXO2 interacting protein spectrum identification, PKM is the Top 1 protein, and PKM2 is the dominant subtype in cancer cells. Therefore, first, the direct binding of FBXO2 and PKM2 was verified in vitro by GST pull-down+WB. The E. coli BL21 (DE3) expression system was used for recombinant protein preparation. The pGEX-4T-2-FBXO2 and pET28a(+)-PKM2 plasmids were transformed into competent cells, respectively, and positive clones were obtained by antibiotic screening. Single colonies were picked and expanded to OD 600 ​≈0.6, 0.3 mM IPTG was added to induce expression for 14 hours at 16°C. After ultrasonic disruption, the supernatant of GST-FBXO2 fusion protein was directly used for subsequent experiments, and the PKM2-His fusion protein was purified by Ni-NTA affinity chromatography column. To verify the interaction between proteins, GST-FBXO2 was incubated with glutathione agarose beads, then co-incubated with purified PKM2-His protein, and the bound protein was collected after washing and using elution buffer. During the whole experiment, empty GST tag protein was set as a negative control, and all interaction results were verified by Western blot. The results showed that significant His-PKM2 was detected in the direct binding protein of GST-FBXO2, while no signal was detected in the control GST, proving that FBXO2 directly binds to PKM2 (A in FIG. 6). Figure 12 Subsequently, FBXO2 (Proteintech, Ag6368) and PKM2 (Sino Biological, 11430-H07E) recombinant proteins were used for SPR technology to quantitatively analyze the interaction kinetics between the two. The specific steps are as follows: PKM2 protein was used as a ligand to be immobilized on the surface of the sensor chip. This process activated the chip surface by mixing 400 mM EDC with 100 mM NHS immediately, then covalently coupled by injecting 30 μg / mL of PKM2 protein solution, and finally used 1 M ethanolamine hydrochloride to block the unreacted active sites. FBXO2 protein was used as an analyte, which was diluted to six concentration gradients of 100, 50, 25, 12.5, 6.25, and 0 nM using running buffer. Using the multi-cycle kinetics method, each FBXO2 solution was injected into the sample channel at a flow rate of 20 μL / min in order from low to high concentration, with an association and dissociation time of 240 seconds and 360 seconds, respectively. By analyzing the sensorgram of the association and dissociation process, the affinity constant (KD) between the two was calculated. The measured association rate constant kon (1 / (M*s)) was 6.01E5, the dissociation rate constant koff (1 / s) was 1.90E-2, the high kon+medium koff showed dynamic and reversible binding, and the calculated equilibrium dissociation constant KD (M) was 3.16E-8, indicating high affinity between the two (B in FIG. 6). Figure 12

[0085] In one specific embodiment, the present application proves that FBXO2 reduces the ubiquitination-mediated proteasome pathway degradation of PKM2.

[0086] ​Specifically, to clarify the biological regulation of FBXO2 on PKM2, based on the cognition that FBXO2 is a ubiquitin ligase, first, the ubiquitination level of PKM2 was detected by IP experiment. In the FBXO2 overexpression / knockdown and the corresponding control group cells, after pulling down the PKM2 protein, the total ubiquitination and K48 ubiquitination levels were detected by WB, the method was the same as CoIP, and the ubiquitin antibody used was anti-total Ub antibody (Cell Signaling Technology, 3936) and specific anti-K48-Ub antibody (Cell Signaling Technology, 8081). The results showed that the ubiquitination degree of PKM2 in the FBXO2 overexpression group was significantly higher than that in the control group, and the FBXO2 knockdown group was the opposite, suggesting that FBXO2 reduced the total ubiquitination and K48 ubiquitination levels of PKM2 (A in Figure 13 Further, FBXO2 knockdown and control group cells were treated with MG132 (proteasome inhibitor) and CQ (autophagy inhibitor), and then the protein level of PKM2 was detected by WB. The results showed that the down-regulation of PKM2 by FBXO2 knockdown was eliminated by MG132, while CQ had no obvious effect (B in Figure 13 The degradation of the proteasome pathway is the classic effect of K48 ubiquitination, and this result suggests that the up-regulation of FBXO2 on PKM2 is achieved by inhibiting the proteasome pathway, which is consistent with the previous observation that FBXO2 reduces the K48 ubiquitination of PKM2. These results reveal that FBXO2 exerts a non-canonical deubiquitination-mediated up-regulation on PKM2.

[0087] In one specific embodiment, the present application screens the key binding site of FBXO2 and PKM2 by molecular docking.

[0088] Specifically, first, the full-length AlphaFold predicted structure of FBXO2 (Uniprot ID: Q9UK22) was selected as the ligand protein, and the X-ray structure 6B6U of PKM2 (Uniprot ID: P14618) was selected as the receptor protein. Global protein-protein docking was performed based on the HDOCKlite v1.1 server, and the PLIP platform and PyMOL software were used to analyze the binding interface at the atomic level. According to the molecular docking results (A in Figure 14), FBXO2 GLU-226 forms salt bridges with PKM2 ARG-383, HIS-379, and hydrogen bonds with PKM2 THR-41, which in turn forms hydrogen bonds with FBXO2 ASP-183. FBXO2-E226 and PKM2-T41 are identified as the key binding sites between the two proteins.

[0089] In one embodiment, the present application demonstrates that the ubiquitination level of PKM2 and the assembly efficiency of glycolytic complex are restored after blocking the binding of FBXO2 to PKM2.

[0090] Specifically, based on the previous molecular docking results, FBXO2 mutants FBXO2 E226A were constructed to block the binding of FBXO2 to PKM2, and then to verify that the deubiquitination of FBXO2 to PKM2 is caused by its direct binding to PKM2. First, the FBXO2 or PKM2 wild type and mutant ORF were cloned as templates, and specific primers were used for PCR amplification to obtain a Flag-tagged fusion fragment carrying homologous arms, and then a pcDNA3.1 linearized vector was prepared. After KpnI and BamHI double enzyme digestion, the target fragment and the linearized vector were directionally recombined by a seamless cloning system, and the ligation product was transformed into competent cells and plated on a resistant plate for screening. After single colony selection and expansion, the correct recombination vector was verified by colony PCR and sequencing analysis, and then used for subsequent experiments. Mock group, FBXO2 E226A group and FBXO2 WT group were set up, and empty vector, FBXO2 E226A (mutant) and FBXO2 WT (wild type) were used to transfect CAL27 cells, respectively. First, the binding of FBXO2 E226A to PKM2 was detected by CoIP, and it was found that the FBXO2 E226A protein level in the IP complex of FBXO2 WT (pulled down) was significantly lower than that in the FBXO2 E226A group, that is, the binding of FBXO2 Figure 15 to PKM2 was effectively reduced (A in FIG. 1). Subsequently, the regulation of glycolytic complex by FBXO2 E226A and FBXO2 WT was detected by CoIP. The input results showed that the protein levels of PKM2, ENO1 and LDHA in the FBXO2 E226A group were significantly less than those in the FBXO2 WT group and close to the Mock group, indicating that FBXO2 E226A cannot up-regulate the protein level of the glycolytic complex; the IP results showed that the protein levels of PKM2, ENO1 and LDHA in the FBXO2 E226AThe binding of ENO1, LDHA and PKM2 is more complex than that of FBXO2. WT The group showed a significant reduction, approaching that of the mock group, suggesting FBXO2 E226A Unable to promote the assembly of glycolytic complexes ( Figure 15 (B in the text). Finally, the ubiquitination status of PKM2 in each group was detected by IP, and the results showed that FBXO2 E226A Unable to be like FBXO2 WT Similarly, it reduces the total ubiquitination level of PKM2 and the ubiquitination level of K48. Figure 15 (C in the text). These results demonstrate that the regulatory effect of FBXO2 on PKM2 ubiquitination and glycolysis complexes depends on direct binding to PKM2.

[0091] In one specific embodiment, the present invention demonstrates that blocking the binding of FBXO2 to PKM2 restores the level of glycolysis in OSCC cells.

[0092] Specifically, let's assume the Mock group and FBXO2 as described above. E226A Group and FBXO2 WT In this group, ECAR values ​​were measured using a Seahorse XF96 energy metabolism analyzer, and the levels of cellular glucose uptake and glycolysis products were detected using biochemical reagent kits, all using the methods described above. Results showed that FBXO2... E226A The glycolysis flux reflected by the ECAR value measured in the group was significantly lower than that of FBXO2. WT Group and close to the Mock group ( Figure 16 ), and FBXO2 E226A The levels of glucose uptake, intracellular ATP, and lactate production in the group were significantly lower than those in the FBXO2 group. WT Group and approach the Mock group ( Figure 17 These results validate FBXO2. E226A The FBXO2 did not enhance the glycolysis level of CAL27 cells, meaning that the FBXO2-promoting effect on glycolysis depends on direct binding to PKM2.

[0093] In one specific embodiment, the present invention demonstrates that PKM2 is an indispensable downstream effector molecule for FBXO2 regulation of glycolysis.

[0094] Specifically, based on previous molecular docking results, a PKM2 mutant that blocks FBXO2 binding was constructed. T41A The method is the same as described above. Groups were established including a control group, an FBXO2 overexpression group, an FBXO2 overexpression + PKM2 knockdown group, and an FBXO2 overexpression + PKM2 knockdown + PKM2 reinjection group. WT Group, FBXO2 overexpression + PKM2 knockdown + PKM2 replenishment T41AThe shPKM2 sequence used to knock down PKM2 was 5'-ACGTGGATGATGGGCTTATTT-3' (SEQ ID NO:4). ECAR values ​​were measured using a Seahorse XF96 energy metabolism analyzer, and the levels of cellular glucose uptake and glycolysis products were detected using biochemical reagent kits, all according to the methods described above. Results are as follows... Figure 18 , Figure 19 The results showed that overexpression of FBXO2 but knockdown of PKM2 reduced glycolytic flux, glucose uptake, intracellular ATP, and lactate production levels as indicated by ECARs. This suggests that knockdown of PKM2 eliminated the promoting effect of FBXO2 on glycolysis. Subsequent PKM2 refilling... WT It can restore the promoting effect of FBXO2 on glycolysis, but it also replenishes PKM2. T41A This promoting effect could not be reversed. This indicates that PKM2 is a necessary downstream effector molecule for FBXO2-driven glycolysis, and that FBXO2 relies on direct binding between the two to exert its driving effect. According to in vitro and in vivo experimental results, FBXO2 significantly drives glycolysis by directly binding to PKM2, reducing its ubiquitination level, upregulating the protein levels of various components of the glycolytic multienzyme complex, and promoting its assembly, thereby promoting the malignant phenotype (proliferation, migration, and invasion) of OSCC cells. Therefore, blocking the binding of FBXO2 and PKM2 can effectively alleviate the development of OSCC, and computer-aided drug screening for OSCC treatment can be achieved based on their binding sites.

[0095] Figure 2 This diagram illustrates a computer-aided drug screening system based on FBXO2 and PKM2 for screening therapeutic agents for oral squamous cell carcinoma, according to an embodiment of the present invention. Specifically, the system includes: Acquisition Unit: Acquire data for FBXO2 and PKM2 proteins.

[0096] Site unit: The binding sites of the FBXO2 and PKM2 proteins are determined based on the data.

[0097] In some embodiments, the binding sites of FBXO2 and PKM2 proteins in the FBXO2 and PKM2 protein complex may be determined by protein-protein docking or any other prior art known to be able to determine the interaction sites between proteins.

[0098] In some embodiments, the binding sites of FBXO2 and PKM2 proteins are the spatial structures of key regions where FBXO2 and PKM2 proteins interact.

[0099] In some embodiments, the binding site of the FBXO2 protein and the PKM2 protein comprises Glu226 of FBXO2 and at least one of Arg383, His379, and Thr41 of PKM2.

[0100] In some embodiments, the binding site is Glu226 of FBXO2 and Thr41 of PKM2.

[0101] The screening unit: through computer-aided screening, a candidate drug targeting the binding site of the two is obtained.

[0102] In some embodiments, the step of computer-aided screening comprises: molecular docking of small molecules in a small molecule library to the binding site and scoring to obtain scores of the docked small molecules; sorting the scores, and selecting the top n small molecules to obtain candidate compounds, n being a natural number greater than or equal to 10.

[0103] In some embodiments, the step of computer-aided screening comprises: obtaining the molecular structure of the FBXO2 protein and the PKM2 protein and inputting the molecular structure into a pharmacophore module library for matching, clustering all interaction sites based on the interaction mode with FBXO2 and PKM2 to obtain a pharmacophore model; inputting the pharmacophore model into a molecular compound database for high-throughput screening to obtain a candidate drug.

[0104] In some embodiments, the step of computer-aided screening comprises: first obtaining the molecular structure of a small molecule inhibitor of FBXO2 and / or the molecular structure of a small molecule inhibitor of PKM2; then screening a small molecule library with similar structures based on the molecular structure of the small molecule inhibitor, and molecular docking of the small molecule library with similar structures to the binding site to obtain scores of the docked molecules, and finally sorting to obtain a candidate drug.

[0105] Figure 3 An apparatus for computer-aided screening of drugs for screening of oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2 is shown, and specifically, the computer device comprises: a memory and a processor, the memory being used to store program instructions; the processor being used to invoke the program instructions, when the program instructions are executed, realizing the method for computer-aided screening of drugs for screening of oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2 as described above.

[0106] In some embodiments, the computer device can further comprise: an input device and an output device.

[0107] In some embodiments, the memory, the processor, the input device, and the output device can be connected through a bus or other means.Figure 3 As shown is an example of a bus connection mode; wherein the memory is used for storing program instructions; the processor is used for calling the program instructions, when the program instructions are executed, for realizing the above method. In some embodiments, the memory can be understood as any saving device of the program, and the processor can be understood as the using device of the program.

[0108] The embodiment of the present application further provides a computer readable storage medium, wherein the computer readable storage medium stores a computer program, and the computer program is executed by a processor to realize the computer-aided drug screening method for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2.

[0109] In several embodiments provided in the present application, it should be understood that the disclosed system, device and method can be implemented by other manners. For example, the above-described device embodiments are only illustrative, for example, the division of the units is only a logical function division, and actual implementation can have another division manner, for example, a plurality of units or components can be combined or integrated into another system, or some features can be ignored or not executed. In addition, the coupling or direct coupling or communication connection between the units or components shown or discussed can be indirect coupling or communication connection through some interfaces, devices or units, and can be electrical, mechanical or other forms.

[0110] The units described as separate components can or can not be physically separated, and the components shown as units can or can not be physical units, that is, they can be located in one place, or can be distributed on a plurality of network units. According to actual needs, part or all of the units can be selected to achieve the purpose of the embodiment scheme.

[0111] In addition, each functional unit in each embodiment of the present application can be integrated in one processing unit, or each unit can be physically present separately, or two or more units can be integrated in one unit. The integrated unit can be realized in the form of hardware or in the form of a software functional unit. The present application does not have special restrictions on the specific implementation form.

[0112] Those skilled in the art can understand that all or part of the steps of various methods in the above embodiments can be completed by instructing the relevant hardware through a program, and the program can be stored in a computer readable storage medium, which can include Read Only Memory (ROM), Random Access Memory (RAM), a magnetic disk or an optical disc, etc. The above has introduced in detail the computer device provided by the present application. For those skilled in the art, according to the idea of the embodiments of the present application, the specific implementation manner and application scope will be changed. In conclusion, the content of the specification should not be understood as a limitation of the present application.

[0113] It should be noted that, for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A computer-aided drug screening method for screening a drug for treating oral squamous cell carcinoma based on FBX02 and PKM2, characterized by, The steps of the method include: Obtaining FBXO2 protein and PKM2 protein data; Determining the binding sites of the two according to the FBXO2 protein and PKM2 protein data; Obtaining the candidate drugs targeting the binding sites through computer-aided screening.

2. The computer-aided drug screening method according to claim 1, characterized in that, The process of the computer-aided screening is: Molecular docking of small molecules in a small molecule library with the binding sites and scoring to obtain the scores of the docked small molecules; Ranking the scores to select the top n small molecules to obtain candidate compounds, n being a natural number greater than or equal to 10; Preferably, the process of the computer-aided screening is: Obtaining the molecular structure of FBXO2 protein and PKM2 protein and inputting the molecular structure into a pharmacophore module library for matching, clustering all interaction sites based on the interaction mode with FBXO2 and PKM2 to obtain a pharmacophore model; Inputting the pharmacophore model into a molecular compound database for high-throughput screening to obtain candidate drugs; Preferably, the process of the computer-aided screening is: first obtaining the molecular structure of an FBXO2 small molecule inhibitor and / or the molecular structure of a PKM2 small molecule inhibitor; then screening a small molecule library with similar structures based on the molecular structure of the small molecule inhibitor, and then performing molecular docking of the small molecule library with similar structures with the binding sites to obtain the scores of the docked molecules, and finally ranking to obtain candidate drugs; Preferably, the process of the computer-aided screening is: screening protein analogs / antibodies / RNA drugs through the binding sites to obtain candidate drugs.

3. The method of computer-aided drug screening according to claim 2, characterized in that, Performing inhibition activity experiment test on the candidate drugs of small molecule type, calculating the inhibition rate after mixing the small molecule compounds with FBXO2 protein solution and PKM2 protein solution respectively, and screening small molecule compounds with inhibitory effect; Preferably, performing inhibition activity experiment test on the candidate drugs of protein analogs / antibodies / RNA drugs, obtaining an oral squamous cell carcinoma cell model or an oral squamous cell carcinoma animal model, treating the oral squamous cell carcinoma cell model or the oral squamous cell carcinoma animal model with small molecule compounds, and screening small molecule compounds that inhibit the proliferation, migration and invasion of oral squamous cell carcinoma cells.

4. The method of computer-aided drug screening according to claim 1, characterized in that, The steps of the computer-aided drug screening method include: Obtaining the binding sites of the FBXO2 protein and PKM2 protein complex; Screening structurally similar small molecule compounds in a molecular database based on the spatial structure of the binding sites of the FBXO2 protein and PKM2 protein complex; Performing molecular docking of the screened small molecule compounds with FBXO2 or PKM2 protein to calculate the affinity / binding energy of the target receptor to obtain scores, and ranking the scores to obtain candidate drugs; Preferably, the binding sites include at least one of Glu226 of FBXO2, Arg383, His379 and Thr41 of PKM2; Preferably, the binding sites are Glu226 of FBXO2 and Thr41 of PKM2. 5.A computer-aided screening drug system for screening a drug for treating oral squamous cell carcinoma based on FBXO2 and PKM2, characterized in that, The steps include: An obtaining unit: obtaining FBXO2 protein and PKM2 protein data; Site unit: determining the binding site of the two according to the FBXO2 protein and PKM2 protein data; Screening unit: obtaining a candidate drug targeting the binding site of the two by computer-aided screening.

6. A computer-aided drug screening device for screening a drug for oral squamous cell carcinoma based on FBXO2 and PKM2, characterized by, Comprising: a memory and a processor, the memory being used to store program instructions; the processor being used to call the program instructions, when the program instructions are executed, realizing the method for computer-aided screening of drugs for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2 according to any one of claims 1-4.

7. A computer readable storage medium having a computer program thereon, characterized in that, Comprising: the computer program is executed by the processor to realize the method for computer-aided screening of drugs for screening oral squamous cell carcinoma treatment drugs based on FBXO2 and PKM2 according to any one of claims 1-4.

8. The use of a substance inhibiting the binding of FBXO2 and PKM2 in the preparation of a drug for screening oral squamous cell carcinoma treatment drugs.

9. Use according to claim 8, characterized in that, The substance inhibiting the binding of FBXO2 and PKM2 comprises one or more of the following: protein analogues / antibodies / RNA and / or small molecule drugs inhibiting the binding of FBXO2 and PKM2; Preferably, the substance inhibiting the binding of FBXO2 and PKM2 comprises shRNA as shown in SEQ ID NO: 1, 2, 4.

10. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises protein analogues / antibodies / RNA and / or small molecule drugs inhibiting the binding of FBXO2 and PKM2, and excipients; the excipients are diluents or binders or disintegrants or lubricants; Preferably, the excipients comprise one or more of the following: starch, sugar powder, mannitol, starch paste, cellulose derivatives, magnesium stearate, micro-powder silica, talc, gelatin, water, ethanol, polyethylene glycol, magnesium lauryl sulfate, hydrogenated vegetable oil.

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