Targeting CD147 small molecule compound and application thereof in liver cancer metastasis inhibition

Abstract

The invention relates to the technical field of cancer treatment, in particular to a CD147 targeting small molecule compound and application thereof in liver cancer metastasis inhibition. The technical problem to be solved by the invention is to inhibit liver cancer metastasis. According to the corresponding technical scheme, a compound HIT106114483, N-(2-isoxypheyl)-2-(3-pheyl-1H-1, 2, 4-triazol-5-yl) acetamide is used for being specifically combined with CD147, the interaction of CD147-NME1 is inhibited, and then the migration and invasion capacity of liver cancer cells is remarkably reduced. The effect of the small molecule compound in inhibition of liver cancer metastasis is determined for the first time, and the small molecule compound has accurate selectivity and targeting ability and no obvious cytotoxicity, provides a new strategy and potential medicine for treatment of liver cancer metastasis, and has important clinical application value and wide market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a small molecule compound targeting CD147 and application thereof in inhibiting liver cancer metastasis. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is the most common type of liver cancer, with poor prognosis, and is the fourth leading cause of cancer-related death worldwide. Although surgical resection and transplantation can be used for the treatment of early-stage HCC, most patients are diagnosed at an advanced stage. Currently, the drugs commonly used for patients with advanced liver cancer include small molecule kinase inhibitors (sorafenib) targeting multiple targets such as anti-VEGF, multi-target kinase inhibitors (lenvatinib), and monoclonal anti-PD-1 antibodies (nivolumab), etc. However, due to drug resistance and tumor recurrence, the overall survival rate of HCC is still low. Therefore, there is an urgent need to discover potential therapeutic drugs with anti-tumor effect and low toxicity for HCC patients.

[0003] CD147, also known as Basigin or EMMPRIN, is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD147 plays an important role in the occurrence and development of tumors. The design of biological technology drugs targeting CD147 and the clinical transformation application of major diseases such as inflammation and cancer are widely used. The national class I biological product "iodine [ 131 I] Mertixibat Injection (Licaitin)" was registered for marketing in 2007 (Guojiazhengzi S20060064), becoming the first tumor monoclonal antibody drug listed in China. Its component is iodine [ 131 I] labeled CD147 antibody F(ab')2. The results of a series of clinical trials show that Licaitin can significantly improve the survival rate of liver cancer patients, reduce the recurrence rate, and prolong the recurrence time. In addition to antibodies, small molecule compounds targeting CD147 have also been studied. AC-73 is a small molecule compound targeting CD147, which binds to Glu64 and Glu73, two amino acid residues in the N-terminal IgC2 domain of CD147, prevents CD147 dimerization, and inhibits the CD147-ERK1 / 2-STAT3-MMP-2 signaling pathway, thereby inhibiting liver cancer metastasis. Although small molecule compounds such as AC-73 have been discovered through virtual screening and other methods, the activity and selectivity of these compounds still need to be further improved. SUMMARY

[0004] The present inventors have found that an increase in the cytoplasmic distribution of CD147 in tumor cells or an increase in the interaction between CD147 and NME1 in the cytoplasm promotes tumor metastasis, and inhibiting the interaction between CD147 and NME1 in the cytoplasm of tumor cells can inhibit tumor metastasis.

[0005] Based on the above research results, the present application claims the following technical solutions:

[0006] In one aspect, the present application provides the following technical solutions of any one of the following applications:

[0007] The application of a drug for inhibiting the distribution of CD147 in the cytoplasm or a drug for inhibiting the interaction between CD147 and NME1 in the cytoplasm in the preparation of a drug for preventing and / or treating a disease;

[0008] The application of a drug for inhibiting the distribution of CD147 in the cytoplasm or a drug for inhibiting the interaction between CD147 and NME1 in the cytoplasm in the preparation of a drug for inhibiting the interaction between CD147 and NME1 to form a complex;

[0009] The application of a drug for inhibiting the distribution of CD147 in the cytoplasm or a drug for inhibiting the interaction between CD147 and NME1 in the cytoplasm in the preparation of a drug for inhibiting the activation of TGF-β signaling pathway or inhibiting the TGF-β-Par polarity protein complex pathway.

[0010] In any of the above applications, the disease is a disease directly or indirectly caused by the cytoplasmic distribution (or increased distribution) of CD147; or, the disease is a disease directly or indirectly caused by the interaction between CD147 distributed in the cytoplasm and NME1 to form a complex (or increased complex); or, the disease is a disease directly or indirectly inhibited by the activation of TGF-β signaling pathway or TGF-β-Par polarity protein complex pathway due to the interaction between CD147 distributed in the cytoplasm and NME1 to form a complex (or increased complex).

[0011] As a specific example, the disease is cancer, specifically metastasis of cancer;

[0012] As a specific example, the cancer is a CD147-expressing cancer, specifically metastasis of a cancer with cytoplasmic distribution of CD147, further specifically cancer metastasis promoted by increased cytoplasmic distribution of CD147 or cancer metastasis promoted by increased interaction between CD147 and NME1 in the cytoplasm;

[0013] As a specific example, the cancer is liver cancer, further specifically hepatocellular carcinoma;

[0014] As a specific example, the metastasis is lung metastasis;

[0015] The above-mentioned cytoplasm is the cytoplasm of a cancer cell.

[0016] As a specific example, the drug prevents or treats the disease by inhibiting the interaction between CD147 and NME1 in the cytoplasm.

[0017] As a specific embodiment, the drug prevents or treats the disease by inhibiting the TGF-β signal pathway and / or the TGF-β-Par polarity protein complex pathway.

[0018] As a specific embodiment, the drug prevents or treats the disease by inhibiting the interaction between CD147 and NME1 in the cytoplasm, and then inhibiting the downstream TGF-β signal pathway and / or the TGF-β-Par polarity protein complex pathway of CD147-NME1.

[0019] In any of the above technical solutions, the drug for inhibiting the distribution of CD147 in the cytoplasm can be a drug for preventing or reducing or slowing down or eliminating the distribution of CD147 in the cytoplasm. It can be a small molecule compound, an antibody, an RNA, a cell, etc.

[0020] In any of the above technical solutions, the drug for inhibiting the interaction between CD147 and NME1 can be a drug for preventing or reducing or slowing down or eliminating the interaction between CD147 and NME1 to form a complex. It can be a small molecule compound, an antibody, an RNA, a cell, etc.

[0021] As a specific embodiment, the drug specifically binds to CD147, and then specifically targets and inhibits the interaction between CD147 and NME1 in the cytoplasm.

[0022] As a specific embodiment, the drug is a triazole compound or an isomer, a pharmaceutically acceptable salt, or a hydrate or a solvate thereof, as shown in the structural formula (I) or (II),

[0023]

[0024] In general formula (I), n is 0 or 1; R1 is an unsubstituted C6 aryl ring group or a C6 aryl ring group substituted with one substituent, wherein the substituent is C1 alkoxy or C2 alkoxy or C3 straight-chain or branched alkoxy or C4 straight-chain or branched alkoxy; R2 is an unsubstituted C6 aryl ring group or a C6 aryl ring group substituted with one substituent, wherein the substituent is halogen, specifically F or Cl or Br;

[0025] In general formula (II), n is 0 or 1; R1 is an unsubstituted C6 aryl ring group or a C6 aryl ring group substituted with one substituent, wherein the substituent is C1 alkyl or C2 alkyl or C3 straight-chain or branched alkyl or C4 straight-chain or branched alkyl; R2 is a 5-membered aromatic heterocyclic group containing 1 heteroatom selected from oxygen, sulfur and nitrogen;

[0026] Further preferably, the compound is as shown below:

[0027]

[0028] In another aspect, the present application provides a model, which is any one of the following:

[0029] 1) a cell model, which is a tumor cell having CD147 distributed in the cytoplasm or increased distribution in the cytoplasm;

[0030] 2) a protein interaction model, which is a complex formed by the interaction of CD147 and NME1;

[0031] 3) a cell model, which is a tumor cell having the complex formed by the interaction of CD147 and NME1 in the cytoplasm;

[0032] As a specific embodiment, the cell model of 1) or 3) above is a recombinant cell obtained by introducing a mutant gene encoding CD147 into a tumor cell, wherein the mutant gene is expressed in the recombinant cell, and the protein encoded by the mutant gene has a leucine (L) at position 249 mutated to an alanine (A), and the distribution of CD147 in the cytoplasm is increased compared to the original tumor cell.

[0033] The introduction of the mutant gene can be achieved by using existing conventional means by those skilled in the art, which include but are not limited to: introduction of an exogenous mutant coding gene, gene editing, homologous recombination, site-directed mutagenesis, etc.

[0034] As a specific embodiment, the tumor cell is a CD147 expression positive cell, specifically a cancer cell, further specifically a hepatocarcinoma cell, and still further specifically a hepatocellular carcinoma cell.

[0035] The cell model has the following properties: compared to the tumor cell before the introduction of the CD147 mutant gene, the cell proliferation, migration and invasion ability of the cell model are significantly enhanced, but there is no significant difference in cell apoptosis.

[0036] In another aspect, the present application provides technical solutions for the use of the model in any one of the following:

[0037] 1) the use of the model as a model of a disease directly or indirectly caused by the distribution of CD147 in the cytoplasm; or as a model of a disease directly or indirectly caused by the formation of a complex by the interaction of CD147 and NME1 in the cytoplasm;

[0038] 2) the use of the model in screening drugs for preventing or treating diseases;

[0039] 3) the use of the model in screening drugs for inhibiting the interaction of CD147 and NME1;

[0040] As a specific example, the disease is a disease directly or indirectly caused by the cytoplasmic distribution of CD147 or an increase in the cytoplasmic distribution.

[0041] As a specific example, the disease is a disease directly or indirectly caused by the formation of a complex of CD147 distributed in the cytoplasm and NME1 or an increase in the complex.

[0042] As a specific example, the disease is a tumor.

[0043] As a specific example, the disease is metastasis of a tumor.

[0044] As a specific example, the disease is a tumor positive for CD147 expression.

[0045] As a specific example, the disease is metastasis of a tumor in which CD147 is present in the cytoplasm.

[0046] As a specific example, the cytoplasm is the cytoplasm of a tumor cell.

[0047] As a specific example, the tumor is a cancer, specifically a liver cancer, further specifically a hepatocellular carcinoma.

[0048] In another aspect, the present application provides a technical solution for the use of any of the following:

[0049] 1) Use of the distribution of CD147 in the cytoplasm of tumor cells as a detection target in the preparation of a product for evaluating whether a treatment method is effective, or use of a reagent and / or instrument for detecting the distribution of CD147 in the cytoplasm of tumor cells in the preparation of a product for evaluating whether a treatment method is effective, wherein a tumor patient is administered a certain treatment method, and the cytoplasmic distribution of CD147 in the tumor cells of the patient is detected after the administration of the treatment method, and if the cytoplasmic distribution of CD147 increases compared to before the administration of the treatment method, then the treatment method is not effective in controlling the progression of the tumor for the patient;

[0050] 2) Use of the interaction of CD147 with NME1 in the cytoplasm of tumor cells as a detection target in the preparation of a product for evaluating whether a treatment method is effective, or use of a reagent and / or instrument for detecting the interaction of CD147 with NME1 in the cytoplasm of tumor cells in the preparation of a product for evaluating whether a treatment method is effective; wherein a patient is administered a certain treatment method, and the interaction of CD147 with NME1 in the tumor cells of the patient is detected after the administration of the treatment method, and if the interaction is stronger compared to before the administration of the treatment method, then the treatment method is not effective in controlling the progression of the tumor for the patient;

[0051] As a specific example, preferably, the tumor is a tumor positive for CD147 expression, specifically a cancer, more specifically a liver cancer, further specifically a hepatocellular carcinoma.

[0052] In the application of the above diagnosis or evaluation, the "reagent and / or instrument for detecting the distribution of CD147 in the cytoplasm" can be any reagent or instrument used in the prior art for detecting the distribution of proteins known to those skilled in the art, and the reagent related to CD147 (monoclonal antibody Hab18 specific to CD147). Any prior art for detecting the distribution of proteins includes but is not limited to: Western blot, immunofluorescence, cell membrane / cytoplasm protein separation, fluorescent protein labeling, immunohistochemistry, biotin labeling, antibody chip. The reagent for detecting whether CD147 is distributed in the cytoplasm includes but is not limited to, monoclonal antibody Hab18 specific to CD147, Minute TM Cell membrane protein separation kit (SM-005, Invitrogen), immunohistochemistry universal two-step detection kit (PV9000, Zhongshan Jinqiao); fluorescent secondary antibody Alexa Fluor TM 555 labeled donkey anti-mouse IgG (Invitrogen, A31570), Alexa Fluor TM 555 labeled donkey anti-rabbit IgG (Invitrogen, A31572), Alexa Fluor TM 488 labeled donkey anti-rabbit IgG (Invitrogen, A21206) and Alexa Fluor TM 488 labeled donkey anti-mouse IgG (Invitrogen, A21202).

[0053] In the application of the above diagnosis or evaluation, the "reagent and / or instrument for detecting the distribution of CD147 in the cytoplasm" can be any reagent or instrument used in the prior art for detecting the distribution of proteins known to those skilled in the art, and the reagent related to CD147 (monoclonal antibody Hab18 specific to CD147). Any prior art for detecting the distribution of proteins includes but is not limited to: Western blot, immunofluorescence, cell membrane / cytoplasm protein separation, fluorescent protein labeling, immunohistochemistry, biotin labeling, antibody chip. The reagent for detecting whether CD147 is distributed in the cytoplasm includes but is not limited to, monoclonal antibody Hab18 specific to CD147, Minute

[0054] The prior art for detecting the interaction between proteins includes but is not limited to: liquid chromatography-tandem mass spectrometry, co-immunoprecipitation, cryo-EM, microscale thermophoresis, surface plasmon resonance technology, protein chip. Key reagents used therein include, for example, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Red-NHS amino coupling kit (BR-1000-50, Cytiva); co-immunoprecipitation kit (26149, Thermo Scientific); horseradish peroxidase labeled goat anti-mouse IgG (Invitrogen, 31466) and goat anti-rabbit IgG (Invitrogen, 32260); heavy metal staining / negative staining: 2% uranyl acetate or phosphotungstic acid.

[0055] In another aspect, the present application provides a drug screening method.

[0056] The drug screening method provided by the present application comprises the following steps: taking the cytoplasmic distribution of CD147 in tumor cells as a target, and searching for a drug that can inhibit the cytoplasmic distribution of CD147 in tumor cells, i.e., a target drug.

[0057] Or, taking the interaction between CD147 and NME1 in the cytoplasm of tumor cells as a target, and searching for a drug that can inhibit the interaction between CD147 and NME1 in the cytoplasm of tumor cells, i.e., a target drug.

[0058] As a specific embodiment, the drug screening method provided by the present application comprises the following steps: screening a compound that can bind to a small molecule binding pocket on the CD147 protein from a compound database as a candidate target drug; wherein the small molecule binding pocket on the CD147 protein is composed of residues ASP136, TRP137, ALA138, TRP139, LEU150, MET151, ASN152, VAL160, SER161, SER162 and SER167.

[0059] As a specific embodiment, the drug screening method comprises the following steps: applying a candidate drug to the model, and if the distribution of CD147 in the cytoplasm of the model is inhibited, or the interaction between CD147 and NME1 in the model is inhibited, then the drug is a target drug.

[0060] As a specific embodiment, the application to the model is contact with the model or entering the inside of the model.

[0061] As a specific embodiment, the drug screening method comprises the following steps:

[0062] Constructing a three-dimensional model of the CD147 protein by using software;

[0063] Identifying a small molecule binding pocket on the CD147 protein in the region or near the region where CD147 interacts with NME1 by using software;

[0064] Screening a compound that can bind to the small molecule binding pocket from a compound database as a candidate target drug.

[0065] As a further specific embodiment, the method can further comprise the step of further screening the candidate target drug according to affinity and / or drug properties.

[0066] As a further specific embodiment, the method can further comprise the step of experimentally verifying the screened compound.

[0067] As a further specific embodiment, the drug properties include, but are not limited to: drug-drug interaction risk (DDI risk), Ames mutagenicity, plasma protein binding (PPB), hepatotoxicity, aqueous solubility, lipid-water partition coefficient, blood brain barrier level penetration, intestinal absorption.

[0068] In the drug screening method, the drug is for treating or preventing a disease caused directly or indirectly by cytoplasmic distribution or increased distribution of CD147; or a disease caused directly or indirectly by CD147 in cytoplasm interacting with NME1 to form a complex or increased complex.

[0069] The disease is a tumor.

[0070] As a specific embodiment, the disease is metastasis and / or proliferation of a tumor.

[0071] As a specific embodiment, the disease is metastasis and / or proliferation of a tumor.

[0072] As a specific embodiment, the disease is a tumor positive for CD147 expression.

[0073] As a specific embodiment, the disease is metastasis and / or proliferation of a tumor with cytoplasmic distribution of CD147.

[0074] As a specific embodiment, the tumor is a cancer, specifically a liver cancer, further specifically a hepatocellular carcinoma.

[0075] Definitions:

[0076] Cytoplasm: also known as cytoplasm or cytoplasm, refers to all substances inside the cell membrane and outside the nucleus, including cytoplasmic matrix, endomembrane system, cytoskeleton and inclusions. Unless otherwise specified, cytoplasm refers to the cytoplasm of tumor cells. In some cases, it refers to the cytoplasm of tumor cells positive for CD147 expression; in some cases, it refers to the cytoplasm of tumor cells with cytoplasmic distribution of CD147; in some cases, it refers to the cytoplasm of cancer cells; in some cases, it refers to the cytoplasm of liver cancer cells; in some cases, it refers to the cytoplasm of hepatocellular carcinoma cells.

[0077] CD147: CD147, also known as Basigin or EMMPRIN, is a type I transmembrane glycoprotein, which belongs to the immunoglobulin superfamily. CD147 exists in multiple isoforms, and isoform 2 is particularly referred to herein, which contains two immunoglobulin (Ig) domains in its extracellular segment.

[0078] NME1 : non-metastatic cells 1, also known as nucleoside diphosphate kinase A, NDPK-A.

[0079] Drug: drug, as used herein without any special indication, refers to any type of drug, including but not limited to: small molecule compounds, antibodies, RNA, cells, etc. In some aspects refers to small molecule compounds.

[0080] Inhibition: inhibition, as used herein without any special indication, refers to any spatial and / or temporal level of inhibition, including but not limited to: prevention or deterrence, reduction, attenuation, slowing, elimination.

[0081] Metastasis of a cancer: refers to migration and / or invasion of cancer cells, without any special indication.

[0082] The triazoles of the present application can be used in actual use in the form of a pharmaceutical composition, which can include a triazole or an isomer or hydrate or solvate thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or excipient.

[0083] Pharmaceutically acceptable carrier, adjuvant or excipient: refers to a non-toxic carrier, adjuvant or excipient that does not destroy the pharmacological activity of the compound with which it is formulated.

[0084] Isomer: refers to a stereoisomer or a tautomer of a compound.

[0085] Pharmaceutically acceptable salt: refers to a salt that retains the biological effectiveness and properties of the compound.

[0086] Prevention: disease refers to the prevention of the occurrence and / or overall development of a disease.

[0087] Treatment: refers to therapeutic intervention that ameliorates signs or symptoms of a disease or pathological condition after its onset. In the case of cancer, treatment can reduce the number of cancer cells, reduce the proliferation of cancer cells, reduce tumor size, slow to some extent the infiltration of cancer cells into peripheral organs, prevent the infiltration of cancer cells into peripheral organs, slow to some extent tumor metastasis, stop tumor metastasis, inhibit to some extent tumor growth, and / or relieve to some extent one or more symptoms associated with the cancer.

[0088] Patient: refers to an animal, preferably a mammal, most preferably a human.

[0089] The application screens a small molecule compound with liver cancer treatment potential acting on CD147, the compound specifically binds to CD147, and then specifically targets and inhibits the interaction between CD147 and NME1 in the cytoplasm, thereby inhibiting the migration and invasion of tumor cells. The compound has precise selectivity and targeting ability, and no obvious cytotoxicity, providing a new strategy and potential drug for the treatment of liver cancer metastasis, and has important clinical application value and broad market prospect. The application provides a new target and new drug for the treatment of liver cancer, which has important significance for the treatment of liver cancer and broad application prospect. The model and drug screening method of the application also have important significance for the development of liver cancer drugs. BRIEF DESCRIPTION OF DRAWINGS

[0090] Figure 1 is a cell overexpressing CD147 wild type or mutant.

[0091] Figure 2 is the effect of the interaction between cytoplasmic CD147 and NME1 on HCC metastasis.

[0092] Figure 3 is the 3D structure of CD147 and the drug screening process.

[0093] Figure 4 is the interaction of the compound with CD147-NME1.

[0094] Figure 5 is that the small molecule compound can specifically inhibit CD147 / NME1 and inhibit liver cancer cell metastasis.

[0095] Figure 6 is that the small molecule compound can inhibit liver cancer metastasis. DETAILED DESCRIPTION

[0096] The following are some specific embodiments of the application, wherein the technical means or reagents used are conventional methods and reagents known in the art unless otherwise specified.

[0097] 1 Cytoplasmic CD147 promotes liver cancer progression

[0098] 1.1 Experimental method

[0099] Western Blot: Total cell lysate was electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (ISEQ00010, Millipore). After blocking with 5% skim milk, the membrane was incubated with primary antibody at 4°C overnight. Secondary antibodies include horseradish peroxidase-labeled goat anti-mouse IgG (Invitrogen, 31466) and goat anti-rabbit IgG (Invitrogen, 32260), and reacted at room temperature for 1 h. Reaction bands were visualized using ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA, USA). TM Touch imaging system (Bio-Rad, Hercules, CA, USA).

[0100] Immunofluorescence: Cells in culture dishes were washed twice with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde for 10 min. After three PBS washes, cells were permeabilized with 0.2% Triton X-100, blocked with 1% goat serum albumin (in PBS for 30 min), and then incubated with the corresponding primary antibody overnight at 4°C. After three independent washes, cells were incubated with secondary antibody for 1 h at room temperature and then stained with 4', 6-diamidino-2-phenylindole (DAPI) (C1006, BiShen) for 15 min at room temperature. The secondary antibodies used included Alexa Fluor 555-labeled donkey anti-mouse IgG (Invitrogen, A31570), Alexa Fluor 488-labeled donkey anti-rabbit IgG (Invitrogen, A21206), and Alexa Fluor 488-labeled donkey anti-mouse IgG (Invitrogen, A21202). Images were taken using a Nikon AlR-A1 confocal laser microscope system (Nikon, Tokyo, Japan) or a Leica Stellaris 5 (Leica, Wetzlar, Germany). TM TM TM TM TM Images were taken using a Nikon AlR-A1 confocal laser microscope system (Nikon, Tokyo, Japan) or a Leica Stellaris 5 (Leica, Wetzlar, Germany).

[0101] Cell membrane / plasma protein separation: The separation of cell membranes and cytoplasm was performed using the Minute TM Cell membrane protein isolation kit (SM-005) from Invitrogen. The proteins were quantified in the cytoplasm and membrane fractions by BCA protein assay kit (Invitrogen, 23227), and the expression of each fraction was analyzed by Western Blot.

[0102] Cell migration and invasion assays: Cell migration or invasion assays were performed using Transwell TM chambers (pore size 8 μm, Millipore) with or without Matrigel (354234, Corning). In the experiments, 1 x 10 5 HepG2 and HCCLM3 cells were seeded into the upper chamber containing serum-free medium, and the lower chamber was supplemented with medium containing 10% fetal bovine serum. After incubation for 24 h (HCCLM3) or 48 h (HepG2), the Transwell TM ​​​​Chambers were stained with 0.2% crystal violet in 95% ethanol for 30 min, and then washed with pure water at room temperature. Five random fields were photographed from each chamber, and the cells on the lower surface were counted.

[0103] 1.2 Experimental results

[0104] In hepatoma cell lines, the basal-lateral membrane localization signal of mutant CD147, in which the leucine (L) at the 249th amino acid was replaced by alanine (A) (L249A) (Figure 1A). The plasmid pEGFP-CD147 Figure 1A was transfected into hepatoma cells. WT and pEGFP-CD147 L249A (the plasmid encoding the mutant L249A of the complete mature peptide of human CD147 (also known as Basigin, EMMPRIN), as described in detail in Figure 1A ). It was found that, compared with the membrane localization of EGFP-CD147 WT , EGFP-CD147 L249A mainly exhibited cytoplasmic distribution Figure 1B . Western blot analysis showed that, in experiments for isolating cell membrane / plasma proteins, the cytoplasmic accumulation of EGFP-CD147 L249A was significantly increased Figure 1C . In EGFP-CD147 WT cells, CD147 was mainly located in the cell membrane, and its cytoplasmic distribution was very small; in EGFP-CD147 L249A cells, compared with EGFP-CD147 WT , the cytoplasmic distribution of CD147 was significantly increased, and the cytoplasmic distribution of CD147 was significantly more than the membrane distribution of CD147. Therefore, the mutation of the basal-lateral membrane localization signal of CD147 resulted in cytoplasmic mislocalization, thereby serving as a cell model for cytoplasmic distribution of CD147 in this study.

[0105] In order to evaluate the significance of cytoplasmic CD147 in tumor progression, the biological functions of CD147-WT and CD147-L249A in vitro were compared. Compared with cells overexpressing EGFP-CD147 WT , the migration and invasion abilities of cells overexpressing EGFP-CD147 L249A were significantly enhanced Figure 1D , and cytoplasmic CD147 promoted the progression of hepatoma.

[0106] 2 Cytoplasmic CD147 promotes the progression of hepatoma through NME1

[0107] 2.1 Experimental methods

[0108] Liquid chromatography-tandem mass spectrometry (LC-MS / MS): Shotgun LC-MS analysis was performed in this experiment. Protein solution or protein bands were digested into peptide mixture by protease, and then separated by high performance liquid chromatography, and then introduced into high resolution mass spectrometer for analysis. After the peptide was ionized in the mass spectrometer, it carried a certain amount of charge, and the mass-to-charge ratio (m / z) of each peptide was obtained by detector analysis. At the same time, the mass spectrometer further bombarded the peptide ion to obtain the secondary mass spectrum signal. The mass spectrometry data was analyzed using search software and the corresponding proteome database.

[0109] Co-Immunoprecipitation (Co-IP) and Western Blot analysis: To detect the interaction of CD147 and NME1 in HepG2 and HCCLM3 cells, Co-Immunoprecipitation kit (26149, Thermo Scientific) was used. In Western Blot analysis, the co-precipitates were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (ISEQ00010, Millipore). The membrane was blocked with 5% skim milk and incubated with primary antibody at 4°C overnight, and secondary antibody was reacted at room temperature for 1 h. The reaction band was transferred to ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA) for visualization. TM Touch imaging system (Bio-Rad, Hercules, CA, USA) for visualization.

[0110] 2.2 Experimental results

[0111] Membrane and cytoplasmic proteins were isolated from HCCLM3-CD147 WT and HCCLM3-CD147 L249A cells, and the differential proteins interacting with membrane and cytoplasmic CD147 were identified by liquid chromatography tandem mass spectrometry (LC-MS / MS) to screen the key proteins interacting with cytoplasmic CD147. Co-IP experiment showed that CD147-L249A enhanced the binding between CD147 and NME1 compared with CD147-WT Figure 2A ). It was confirmed by membrane / plasma separation that NME1 interacted with CD147 in the cytoplasm Figure 2B ). The role of the interaction between cytoplasmic CD147 and NME1 in HCC metastasis was verified. The migration and invasion induced by CD147-L249A were inhibited after NME1 knockdown compared with the control group Figure 2C ). The changes in CD147-L249A activated TGF-β pathway and altered Par polarity complex (Par3, Par6B and aPKC) expression were also inhibited after NME1 knockdown Figure 2D). These results suggest that cytoplasmic CD147 promotes cancer progression and activates downstream signaling, which requires cooperation with NME1.

[0112] 3 Lead compounds for CD147 / NME1 binding identified by virtual screening

[0113] 3.1 Experimental methods

[0114] Model building: In this study, we tried to explore whether the NME1 binding site of CD147 has a small molecule binding pocket and aimed to identify a series of compounds that can bind to this pocket with high affinity and appropriate drug properties. Based on the AlphaFold model, we optimized and protonated the three-dimensional structure of human CD147 isoform 2 using MOE software. The optimized structure was further used to detect potential small molecule binding pockets, especially in the NME1 binding region. MOE-SiteFinder (Volkamer, Griewel et al. 2010) was used to detect small molecular binding pockets around the NME1 binding region. There is a pocket near the NME1 binding region, with a ligand binding propensity score (PLB score) of 2.25, indicating that it is a suitable area for small molecule binding. The characteristics of this pocket, such as size, hydrophobic atoms, ligand binding propensity score (PLB), and included residues, were calculated. This pocket contains 10 hydrophobic atoms, which can accommodate up to 15 atoms, indicating that this pocket is suitable for accommodating low molecular weight compounds. The formation of this pocket is composed of residues ASP136, TRP137, ALA138, TRP139, LEU150, MET151, ASN152, VAL160, SER161, SER162, and SER167.

[0115] Preparation of CD147 receptor: The three-dimensional model of human CD147 isoform-2 was protonated and structurally optimized. MOE-SiteFinder (Volkamer, Griewel et al. 2010) was used to detect small molecular binding pockets around the NME1 binding region. The pocket served as the docking region for virtual screening. The Apopdb2receptor tool (version 3.2.0.2) of Openeye (software 2015) was used to process and generate receptor files for OEDock virtual screening. The docking region was defined by a docking box with a length, width, and height of x x with an inner contour volume of

[0116] ​Preparation of chemical databases: Two chemical databases (D001 and T001) were used to screen potential inhibitors of human CD147. A total of 1,661,725 compounds were stored in these databases. To ensure that all possible conformations of each compound could be docked with the receptor, the omega2 plugin (version 3.0.1.2) in the openeye software (version 2015) was applied to generate multiple conformations of each compound, with an average of 42 conformations for each compound.

[0117] Virtual screening: Ubuntu kylin 15.10 operating system and program OEDock (version 3.2.0.2) were used for virtual screening on a workstation (CPU: 40; memory: 64; SSD: 512G), with the running parameters set as “-save_component_scores=true, -hitlist_size=30,000, -docked_molecule_file=sdf, docking_resolution=high, and the rest of the parameters set as default parameters” (software 2015). Finally, 1,657,052 compounds were successfully docked to CD147, and the top 100,000 compounds with high binding affinity to CD147 were obtained.

[0118] Calculation of drug properties: Physicochemical properties such as molecular weight, hydrogen bond donor / acceptor, water solubility (logS), lipid-water partition coefficient (logP), and surface accessible area (TPSA) were calculated by the “Calculate Molecular Properties Protocol” in Discovery Studio 2019. Compound properties such as human intestinal absorption level (HIA), drug interaction risk (2D6 pKi), blood-brain barrier level (BBB level), plasma protein binding level, liver toxicity prediction, and Ames prediction were calculated and analyzed by Discovery Studio 2019 and MOE software (2020).

[0119] 3.2 Experimental results

[0120] First, the 3D structure of human CD147 was protonated and optimized, and pocket 1 (Asp136 Trp137 Ala138 Trp139 Leu150 Met151 Asn152 Val160 Ser161 Ser162 Ser167) was selected by structure-based virtual screening using the MOE-Site Finder plugin. Figure 3A). Subsequently, two compound libraries (D001 and T001) were subjected to multi-conformation generation processing, and the docking scores of all compounds were calculated and sorted by OEDock of OpenEye, and high-affinity compounds were screened by Chemgauss4 scoring function. A total of 1657052 compounds were successfully docked, and 100000 compounds with high binding affinity to CD147 were screened. Finally, considering the properties of the selected molecules, including water solubility (logS), octanol / water partition coefficient (logP), human intestinal absorption (HIA), and CYP2D6 drug interaction, analysis was performed using Stardrop software (version 6.5.0), and finally 136 compounds were determined for further SPR screening Figure 3B ).

[0121] 4. Identification of CD147-NME1 interaction inhibitors

[0122] 4.1 Experimental methods:

[0123] Small molecule compound stimulation: Plasmids pEGFP-CD147 and pmCherry-NME1 were introduced into hepatoma cell lines HCCLM3 and HepG2, and after 24 h, different concentrations of small molecule compounds (Compound 1, Compound 2 and Compound 3) were added, and after 24 h of stimulation, Co-IP and Western Blot analysis or cell migration and invasion experiments were performed.

[0124] Protein purification: Synthetic plasmids pET21a-CD147 ECD (Extracellular Domain, ECD) and pET21a-NME1 were transformed into the expression host bacteria Origami B (DE3) E. coli strain and inoculated into LB (Luria-Bertani) solid medium containing ampicillin and incubated at 37°C overnight. Subsequently, monoclonal strains were selected and cultured in 10 mL LB liquid medium at 37°C and 220 rpm overnight, then amplified to 1 L LB medium and continued to be cultured at 220 rpm and 37°C for 6 hours until the OD 600 reached about 0.6. Isopropyl β-D-thiogalactoside (1 mol / L, 500 μL) was added to 1 L LB medium and induced to low-temperature oscillation at 16°C and 180 rpm for 16 to 24 h. The bacteria were collected and resuspended in Ni-NTA buffer after oscillation. The supernatant was obtained by centrifugation at 4°C and 12000 rpm for 15 min, and filtered using a 0.45 μm filter, and finally the protein was purified using fast protein liquid chromatography (AKTA Basic and HisTrap TM HP) and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0125] Microscale thermophoresis (MST) technique: After the protein was labeled with Red-NHS according to the instructions (MO-L011, NanoTemper), it was stored in PBST buffer at a concentration of 10 μM. After incubation at room temperature for 30 min, the labeled protein was centrifuged at 15000 x g for 10 min at 4°C. Subsequently, the ligand was diluted by 16-fold in series, and mixed with the same volume of labeled protein. The sample was then loaded into a premium capillary and analyzed using a Monolith NT.115 system (NanoTemper).

[0126] Surface plasmon resonance assay (SPR): The interaction between CD147 and NME1 was analyzed by SPR technology using a Biacore T200 SPR system (Cytiva, Wilmington). His-CD147 and His-NME1 proteins were purified in our laboratory using an AKTA purification system. NME1 was immobilized on the chip using an amino coupling kit (BR-1000-50, Cytiva). The affinity constant analysis used Biacore T200 Evaluation Software 3.2 (Cytiva).

[0127] CCK-8 (Cell Counting Kit-8) experiment: HepG2 and HCCLM3 cells were seeded into 96-well plates at a density of 2 x 10 3

[0128] 4.2 Experimental results

[0129] Virtual screening initially identified 136 (1-136) high-affinity compounds with relatively satisfactory drug properties. Through SPR analysis, 9 compounds showed higher CD147 affinity compared with the control. In addition, MST experiments confirmed that among the 9 compounds, only 3 compounds directly interacted with CD147, which were named Compound 1 (C-1), K​D = 2.175 x 10 -7 M; Compound 2 (C-2), K D = 4.7451 x 10 -5 M; Compound 3 (C-3), K D = 9.2104 x 10 -5 ( Figure 4A ). To detect the inhibitory effect of these compounds on CD147-NME1 interaction, Co-IP Figure 4B ) and SPR experiments Figure 4C ) showed that CD147-NME1 interaction was significantly reduced after C-1 compared with C-2 and C-3. In addition, C-1 could significantly inhibit the migration and invasion ability of HCC cells induced by cytosolic CD147 Figure 4D ), and the proliferation of HCC cells was not affected after 24 h treatment with C-1, C-2 and C-3 Figure 4E ), which indicated that the inhibition of HCC cell migration and invasion by C-1 was not due to cytotoxicity. The above experiments proved that C-1, C-2 and C-3 are selective inhibitors targeting CD147-NME1 interaction and show potential as anti-cancer drugs.

[0130] 5C-1 can specifically inhibit CD147 / NME1, inhibit liver cancer cell metastasis

[0131] 5.1 Experimental methods are the same as above.

[0132] 5.2 Experimental results

[0133] C-1 is N-(2-methoxyphenyl)-2-(5-phenyl-2H-1,2,4-triazol-3-yl)acetamide, HIT106114483, CAS1358320-16-7, Figure 5A . To explore whether C-1 can inhibit the progression of HCC induced by CD147-NME1, the effect of C-1 on tumor metastasis was detected. First, C-1 can significantly reduce the interaction of CD147-NME1 complex, and its inhibitory effect is stronger with the increase of dose Figure 5B ). Transwell results showed that C-1 significantly inhibited the migration and invasion ability of tumor cells induced by cytosolic CD147, and this effect was dose-dependent Figure 5C ). To confirm that C-1 is a selective inhibitor of CD147-NME1, the response of C-1 in two cells: HCCLM3-CD147 WTand HCCLM3-CD147 L249A C-1 significantly reduced the migration and invasion of HCCLM3-CD147 L249A cells, but not in HCCLM3-CD147 WT cells, indicating that C-1 selectively targets CD147-NME1 Figure 5D ) pathway downstream of CD147-NME1 Figure 5E ).

[0134] 6 C-1 Inhibits Liver Cancer Metastasis

[0135] 6.1 Experimental Methods

[0136] Hematoxylin and Eosin (H&E) : Tissue paraffin sections were placed in an oven at 80°C for 2h, then the sections were stained according to the following steps: xylene twice, 5min each time. Absolute ethanol twice, 5min each time. 95% ethanol twice, 2min each time. 80% ethanol 2min. 75% ethanol 2min. Tap water rinse 5min. Hematoxylin solution staining 5min, running water 5min to wash off the hematoxylin solution. 1% hydrochloric acid ethanol 5s, tap water rinse 5min. Eosin staining 5min, tap water rinse 30s, 95% ethanol 10s. Absolute ethanol twice, 2min each time, xylene twice, 2min each time, neutral resin mounting, air-dried and stored in a well-ventilated place.

[0137] Immunohistochemistry (IHC) : After paraffin-embedded sections (3pm) fixed with formalin were deparaffinized with xylene and alcohol, antigen retrieval was performed using a pH 6.0 citrate buffer (product number ZLI-9065, Beijing Zhongshanjinqiao Biotechnology Co., Ltd.). The sections were first incubated with 0.3% hydrogen peroxide solution and normal goat serum blocking solution, then incubated with the primary antibody at 4°C overnight in a wet box. After incubation, immunoperoxidase staining was completed using a streptavidin-peroxidase detection kit (product number SP9000, Beijing Zhongshanjinqiao Biotechnology Co., Ltd.), and color development detection of the target protein CD147 was performed using 3,3'-diaminobenzidine (product number ZLI-9018, Beijing Zhongshanjinqiao Biotechnology Co., Ltd.).

[0138] 6.2 Experimental Results

[0139] Inhibition of liver cancer lung metastasis by small molecule inhibitors. HCCLM3-CD147 L249A (3x106 )Cell injection into the tail vein of 8-week-old male NOD-Scid mice to establish a liver cancer lung metastasis mouse model. Twenty days after injection, the mice were randomly divided into four groups: control group, low-dose group (15 mg / kg), medium-dose group (30 mg / kg) and high-dose group (50 mg / kg). The small molecule inhibitor was injected intraperitoneally once a day at the specified dose, and the body weight was recorded every two days. After 20 days of treatment, all mice were sacrificed, and their tissues (heart, liver, spleen, lung and kidney) were analyzed by H&E method. The results showed that the proportion of CD147 positive lesions in the lung tissue of the control group was 5 / 6; while C-1 treatment showed dose-dependent inhibition of tumor metastasis: 3 / 6 in the 15 mg / kg group, 1 / 6 in the 30 mg / kg group, and 0 / 6 in the 50 mg / kg group ( Figure 6A ). In addition, C-1 inhibited tumor metastasis with low toxicity, which was verified by body weight measurement ( Figure 6B ) and HE staining of important organs ( Figure 6C and 6D ). The above data show that C-1, as a selective inhibitor targeting CD147-NME1 interaction, can effectively inhibit liver cancer metastasis.

[0140] The above examples and the like are detailed explanations of the ideas of the present application in conjunction with examples, but do not limit the protection scope of the present application. Any improvement made by anyone under the inspiration of the multiple first discoveries of the present application, as long as it is based on the multiple discoveries of the present application, should belong to the protection scope of the present application.

Claims

1. Any of the following applications: The use of drugs that inhibit the distribution of CD147 in the cytoplasm or drugs that inhibit the interaction between CD147 and NME1 in the cytoplasm in the preparation of drugs for the prevention and / or treatment of diseases; Application of drugs that inhibit the distribution of CD147 in the cytoplasm or drugs that inhibit the interaction between CD147 and NME1 in the cytoplasm in the preparation of drugs that inhibit the formation of complexes by the interaction between CD147 and NME1; The application of drugs that inhibit the distribution of CD147 in the cytoplasm or drugs that inhibit the interaction between CD147 and NME1 in the cytoplasm in the preparation of drugs that inhibit the activation of the TGF-β signaling pathway or the TGF-β-Par polar protein complex pathway.

2. The application according to claim 1, characterized in that: The disease in question is cancer, specifically metastasis of cancer; Alternatively, the cancer is a CD147-positive cancer, specifically a metastatic cancer in which CD147 is distributed in the cytoplasm, and more specifically, a cancer metastasis promoted by increased CD147 cytoplasmic distribution or a cancer metastasis promoted by increased interaction between CD147 and NME1 in the cytoplasm. Alternatively, the cancer may be liver cancer, or more specifically, hepatocellular carcinoma; Alternatively, the metastasis may be a lung metastasis; Preferably, the cytoplasm is the cytoplasm of cancer cells.

3. The application according to claim 1 or 2, characterized in that: The drug achieves disease prevention or treatment by inhibiting the interaction between CD147 and NME1 in the cytoplasm; Alternatively, the drug may prevent or treat the disease by inhibiting the TGF-β signaling pathway and / or the TGF-β-Par polar protein complex pathway; Alternatively, the drug may prevent or treat the disease by inhibiting the interaction between CD147 and NME1 in the cytoplasm, thereby inhibiting the downstream TGF-β signaling pathway and / or the TGF-β-Par polar protein complex pathway of CD147-NME1.

4. The application according to any one of claims 1-3, characterized in that: The drug is a triazole compound or its isomer, a pharmaceutically acceptable salt, or a hydrate or solvate, as shown in formula (I) or (II). In general formula (I), n is 0 or 1; R1 is an unsubstituted C6 aryl ring or a C6 aryl ring substituted with one substituent, wherein the substituent is a C1 alkoxy or a C2 alkoxy or a C3 straight-chain or branched-chain alkoxy or a C4 straight-chain or branched-chain alkoxy; R2 is an unsubstituted C6 aryl ring or a C6 aryl ring substituted with one substituent, wherein the substituent is a halogen, specifically F, Cl or Br; In general formula (II), n is 0 or 1; R1 is a C6 aryl ring group that is unsubstituted or substituted with one substituent, wherein the substituent is a C1 alkyl or C2 alkyl or C3 straight-chain or branched alkyl or C4 straight-chain or branched alkyl; R2 is a 5-membered aromatic heterocyclic group that contains one heteroatom selected from oxygen, sulfur and nitrogen. More preferably, the compound is as follows:

5. A model, as described in any of the following: 1) A cell model consisting of tumor cells with increased CD147 distribution in the cytoplasm; 2) A protein interaction model, which is a complex formed by the interaction of CD147 and NME1; 3) A cell model consisting of tumor cells containing a complex formed by the interaction of CD147 and NME1 in their cytoplasm; Preferably, the cell model described in 1) or 3) above is a recombinant cell obtained by introducing a mutant gene encoding the CD147 gene into tumor cells. The mutant gene is expressed in the recombinant cell, and the protein encoded by the mutant gene is a protein in which leucine (L) at position 249 is mutated to alanine (A). Compared with the initial tumor cells, CD147 is more distributed in the cytoplasm. More preferably, the tumor cells are CD147-positive tumor cells, specifically cancer cells, more specifically liver cancer cells, and even more specifically hepatocellular carcinoma cells.

6. The application of the model of claim 5 in any of the following: 1) The model is used as a model of diseases caused directly or indirectly by the distribution of CD147 in the cytoplasm; or as a model of diseases caused directly or indirectly by the interaction of CD147 and NME1 in the cytoplasm to form a complex. 2) The application of the model in screening drugs for the prevention or treatment of diseases; 3) Application of the model in screening drugs that inhibit the interaction between CD147 and NME1; The disease is caused directly or indirectly by the cytoplasmic distribution of CD147; or, the disease is caused directly or indirectly by the interaction of CD147 distributed in the cytoplasm with NME1 to form a complex. Preferably, the disease is a tumor, specifically a metastatic tumor, more specifically a CD147-positive tumor, and even more specifically a metastatic tumor in which CD147 is distributed in the cytoplasm. Preferably, the cytoplasm is the cytoplasm of tumor cells; More preferably, the tumor is cancer, specifically liver cancer, and more specifically hepatocellular carcinoma.

7. Any of the following applications: 1) The application of CD147 distribution in tumor cell cytoplasm as a detection target in the preparation of products for evaluating the effectiveness of treatment methods, or the application of reagents and / or instruments for detecting CD147 distribution in tumor cell cytoplasm in the preparation of products for evaluating the effectiveness of treatment methods, wherein, After a cancer patient is given a certain treatment, the cytoplasmic distribution of CD147 in the patient's tumor cells is detected. If the cytoplasmic distribution of CD147 increases compared with before the treatment is given, then the treatment is not effective in controlling the progression of the tumor for the patient. 2) The application of the interaction between CD147 and NME1 in the cytoplasm of tumor cells as a detection target in the preparation of products for evaluating the effectiveness of treatment methods; the application of reagents and / or instruments for detecting the interaction between CD147 and NME1 in the cytoplasm of tumor cells in the preparation of products for evaluating the effectiveness of treatment methods; wherein, after a patient is given a certain treatment method, the interaction between CD147 and NME1 in the patient's tumor cells is detected. If the interaction is stronger compared with before the treatment method was given, then the treatment method has not effectively controlled the progression of the tumor for the patient; Preferably, the tumor is a CD147-positive tumor, specifically cancer, more specifically liver cancer, and further specifically hepatocellular carcinoma.

8. A drug screening method, comprising the following steps: using the cytoplasmic distribution of CD147 in tumor cells as a target, searching for drugs that inhibit the cytoplasmic distribution of CD147 in tumor cells, which are the target drugs; Alternatively, the target drug can be found by targeting the interaction between CD147 and NME1 in the cytoplasm of tumor cells.

9. The drug screening method according to claim 8, characterized in that: The drug screening method includes the following steps: screening compounds from a compound database that can bind to the small molecule binding pocket on the CD147 protein as candidate target drugs; wherein the small molecule binding pocket on the CD147 protein is composed of residues ASP136, TRP137, ALA138, TRP139, LEU150, MET151, ASN152, VAL160, SER161, SER162 and SER167; And / or, the drug screening method includes the following steps: applying the candidate drug to the model described in claim 5, and if the distribution of CD147 in the cytoplasm of the model is inhibited, or the interaction between CD147 and NME1 in the model is inhibited, then the drug is the target drug.

10. The drug screening method according to claim 8, characterized in that: The method includes the following steps: A three-dimensional model of the CD147 protein was constructed using software; Using software, small molecule binding pockets on the CD147 protein can be identified in or near the region where CD147 interacts with NME1. Compounds that can bind to the small molecule binding pocket are screened from the compound database as candidate target drugs; Preferably, the method may further include a step of further screening candidate target drugs based on affinity and / or drug properties; More preferably, the method may further include a step of experimentally verifying the screened compounds.

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