A screening method for fgfr inhibitors

Abstract

The application discloses a screening method of FGFR inhibitor, which comprises the following steps: 1) applying FGFR inhibitor to be detected to cell strains with different FGFR1OP2 protein expression amounts respectively, and testing the efficacy of the FGFR inhibitor to be detected; and 2) judging the correlation between the efficacy of different cell strains and the FGFR1OP2 protein expression amounts of different cell strains. The application also discloses a screening method of cell model suitable for specific FGFR inhibitor, which comprises the following steps: 1) detecting the FGFR1OP2 protein expression amount in the cell model to be detected; and 2) judging the FGFR1OP2 protein expression amount. The method has the advantages of high sensitivity and accurate indication, and is suitable for popularization and application of drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for screening FGFR inhibitors. Background Technology

[0002] The FGFR1OP2 protein is primarily expressed in the cytoplasm, and its function has been poorly understood to date. Current reports mainly suggest that the FGFR1OP2-FGFR1 fusion may be involved in the development of myeloproliferative syndrome (EMS) (Grand et al., 2004) and that it can induce myeloproliferative leukemia (Qin et al., 2015). FGFR1OP2 may be involved in the FGFR1 signaling pathway.

[0003] FGFR1 is a transmembrane protein. The FGFR signaling pathway receives signals through its extracellular domain, autophosphorylation activates the TK domain, and tyrosine kinases influence downstream important proteins, including PTPN11, SOS, and FRS2, participating in signaling pathways such as JAK / STAT, PI3k / ATK / mTOR, and RAS / RAF / MAPK (Brewer, et al., 2016). The JM region is an important regulatory region of the FGFR1 protein; some other TKIs or GPCRs bind to the JM region through their adopters, participating in the regulation of FGFR1 autoactivation. Inhibitors targeting FGFRs have the potential to treat related diseases, and more and more research institutions and pharmaceutical companies are using them as promising therapeutic targets. FGFR inhibitors have become one of the hot topics in targeted drug research. However, the overall response rate of FGFR inhibitors is low, and there are no obvious and useful biomarkers to indicate their efficacy and screen suitable patients. Currently, the most effective biomarker is FGFR2 fusion, but these fusions occur in a low proportion of tumors, making them unsuitable for widespread drug promotion and application.

[0004] Therefore, there is an urgent need for a new biomarker to indicate the efficacy of FGFR or to screen suitable patients. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a method for screening FGFR inhibitors.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] One of the technical solutions of this invention is: providing a method for screening FGFR inhibitors, which includes the following steps:

[0008] (1) The FGFR inhibitor to be tested was administered to cell lines with different FGFR1OP2 protein expression levels, and the efficacy of the FGFR inhibitor to be tested was tested.

[0009] (2) Determine the correlation between the efficacy of the FGFR inhibitor to be tested on different cell lines and the expression level of FGFR1OP2 protein in different cell lines.

[0010] In step (1), the different cell lines are preferably the same type of cell line to facilitate parallel comparison. For example, the KATOIII cell line, which has the same cell state but different FGFR1OP2 protein expression levels.

[0011] In step (2): if the efficacy of the drug is positively correlated with the expression level of FGFR1OP2 protein, then the FGFR inhibitor to be tested is an effective FGFR inhibitor.

[0012] The pharmacodynamics (i.e., the efficacy of the inhibitor) described in this invention are conventional in the art, typically manifested as IC50, EC50, ED50, pIC50, Ki, Kd, ​​and Score. Specific explanations are as follows.

[0013] EC50 value: The concentration at which a drug achieves 50% of its maximum clinical efficacy (either inhibitory or stimulatory). This is a pharmaceutical term.

[0014] ED50 value: The effective dose (not concentration) of a drug at which 50% of individuals exhibit a specific therapeutic effect.

[0015] IC50 value: The concentration of the inhibitor at which a 50% inhibitory effect is achieved.

[0016] pIC50 value: the negative logarithm of 10 of the IC50 value.

[0017] Ki value: The concentration of the inhibitor when a 50% inhibitory effect is detected (obtained using Michaelis-Menten kinetics).

[0018] Kd value: The equilibrium constant when a complex of two or more biomolecules is separated into components; for example, the pIC50 value when an inhibitor or substrate is separated from an enzyme.

[0019] Score: This refers to the score given for docking, which evaluates the binding affinity between small molecules and proteins or between proteins. S is generally a negative value. The larger the absolute value, the stronger the binding affinity and the higher the activity of the small molecule or protein.

[0020] The present invention preferably uses IC50 to characterize the efficacy of the FGFR inhibitor to be tested.

[0021] Preferably, the cell line is derived from a cell line from a patient with a solid tumor or a patient with leukemia.

[0022] This invention focuses on the effect of FGFR1OP2 protein expression level on the efficacy of inhibitors in cell lines. Therefore, the cell lines referred to are those derived from cell lines from which other variables have been excluded. Examples include KATOIII, KG1, NCIH716, SKNO1, KASUMI6, SUPB15, OPM2, BDCM, NCIH2009, HGC27, MFE296, NCIH1703, REH, G401, G292CLONEA141B1, AN3CA, A375, HCT116, NCIH460, MFE280, DLD1, NCIH1975, LP1, A498, SNU423, NUGC3, HCC1954, BGC823, CAPAN1, CAMA1, SNU449, and MKN7.

[0023] Regarding the result judgment of the screening method described above, in a specific embodiment of the present invention:

[0024] When the expression level of FGFR1OP2 protein is lower in the first cell line compared to the second cell line, but the IC50 value is higher, it indicates that the FGFR inhibitor to be tested is effective.

[0025] The second technical solution of the present invention is: providing a method for screening cell models suitable for specific FGFR inhibitors, which includes the following steps:

[0026] (1) Detect the expression level of FGFR1OP2 protein in the cell model to be tested;

[0027] (2) Determine the level (value) of the expression level of the FGFR1OP2 protein.

[0028] In a specific embodiment of the present invention, based on the numerical value of the protein expression level in step (2), a cell model in which the FGFR1OP2 protein expression level is higher than the general level is selected for a specific FGFR inhibitor.

[0029] Preferably, the cell model is a cell line derived from a patient with a solid tumor or a patient with leukemia.

[0030] In this invention, the detection method can be conventional in the art, such as qPCR detection. When using qPCR for detection, the nucleotide sequence of the forward primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2; or the nucleotide sequence of the forward primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 4.

[0031] The third technical solution of the present invention is: to provide a reagent for measuring the expression level of FGFR1OP2 in the screening of FGFR inhibitors or the screening of cell models suitable for specific FGFR inhibitors.

[0032] Methods for determining FGFR1OP2 expression levels can be conventional in the art, and may include, for example, microarrays (protein chips and microfluidic chips, etc.), digital single-molecule immunoassay arrays, ELISA, radioimmunoassay, immunoturbidimetry, immunohistochemistry, Western blotting, and other known methods that may be based on antibodies.

[0033] In the above technical solutions, "cell line" includes cell samples from clinical sources; "cell model" includes cells from all sources such as cell lines, cell strains, and clinical samples. In this invention, the cell model mainly refers to a drug screening cell model.

[0034] The positive and progressive effects of this invention are as follows:

[0035] This invention uses FGFR1OP2 expression level as a biomarker to indicate drug efficacy or screen suitable patients. It has the advantages of accurate indication, high sensitivity and good overall response rate, and is suitable for drug promotion and application. Attached Figure Description

[0036] Figure 1 The relationship between the efficacy of FGFR inhibitors on different cell lines and their FGFR1OP2 expression levels.

[0037] Figure 2 The results show the expression levels of FGFR1OP2 in different samples in the PDX model.

[0038] Figure 3 The relationship between the efficacy of FGFR inhibitors on different cell samples and the expression level of FGFR1OP2 in a PDX model.

[0039] Figure 4 This is a molecular simulation of the binding of FGFR1OP2 to the JM region of FGFR1 in cells.

[0040] Figure 5 Molecular simulation of how FGFR inhibitors suppress the JM region of FGFR1 via the FGFR1OP2 protein.

[0041] Figure 6 It is a combination mode of MAX-40279-01-FGFR1-FGFR1OP2.

[0042] Figure 7 It interacts with MAX-40279-01-FGFR1-FGFR1OP2.

[0043] Figure 8 It is the AZD4547-FGFR1-FGFR1OP2 binding mode.

[0044] Figure 9 It is an interaction between AZD4547-FGFR1-FGFR1OP2.

[0045] Figure 10 The binding mode is Pemigatinib -FGFR1-FGFR1OP2.

[0046] Figure 11 It is a Pemigatinib-FGFR1-FGFR1OP2 interaction.

[0047] Figure 12 The binding mode is Erdafitinib -FGFR1-FGFR1OP2.

[0048] Figure 13 It interacts with Erdafitinib-FGFR1-FGFR1OP2.

[0049] Figure 14 The binding mode is Infigratinib -FGFR1-FGFR1OP2.

[0050] Figure 15 It interacts with Infigratinib-FGFR1-FGFR1OP2. Detailed Implementation

[0051] The present invention is further illustrated below by way of examples, but these examples do not limit the invention to the scope of the embodiments described. Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions, or as selected in the product instructions. All reagents and raw materials used in this invention, unless otherwise specified, are commercially available.

[0052] Experimental methods

[0053] 1. Preparation of experimental samples

[0054] Name: MAX-40279-01, its chemical composition is N-[7-(4-fluoro-2-methoxyphenyl)-6-methylthieno[3,2-d]pyrimidin-2-yl]-1-(piperidin-4-yl)-1H-pyrazole-4-amine fumarate. Its chemical structural formula is:

[0055]

[0056] The molecular weight is 496.56.

[0057] Physical properties: Powder. Storage conditions: 2-8ºC.

[0058] Carrier solution: 1% PVP K90 - 0.1% Tween 80 aqueous solution

[0059] Preparation method: Weigh 1000 mg of PVP K90 into 1000 mL of pure water, and add 0.1 mL of Tween 80. Place the carrier solution on a magnetic stirrer and stir until clear. Stir at 300 rpm for 30 minutes. Add MAX-40279-01 to a 1% PVP K90-0.1% Tween 80 aqueous solution and store at 2-8ºC. Stir again at 300 rpm for 30 minutes before use.

[0060] 2. Detection method for FGFR1OP2:

[0061] RNA-seq sequencing; threshold TPM=9.

[0062] Probe-based qPCR detection. Dye-based qPCR detection, with GAPDH as a control, showed that FGFR1OP2 expression was 0.016 times higher than that of GAPDH. Primer sequences are shown in Table 1 below.

[0063] Table 1

[0064]

[0065] 2.1 Experimental Samples

[0066] The cDNA sample was diluted 10 times and used as a template for instrumental detection.

[0067] 2.2 Materials and Instruments for Real-Time Quantitative PCR

[0068] Quantitative PCR reagent: 2X SG Fast qPCR Master Mix (B639271, BBI).

[0069] Quantitative PCR instrument: LightCycler 480 II Real-Time PCR Instrument (Roche, Rotkreuz, Switzerland).

[0070] 2.3 PCR Reaction Procedure: Preparation of Reaction Mixture

[0071] Table 2

[0072]

[0073] 1) PCR cycling conditions

[0074] Table 3

[0075]

[0076] 2) Instrument Operation

[0077] After completing the above steps, place the 96 / 384-well plate with the sample added into a LightCycler 480 II (Roche) for reaction.

[0078] 3. Cell line experiments:

[0079] The inhibitory activity of compound MAX-40279-01 on cell proliferation was determined in various cell lines using the CellTiter-Glo assay (which is based on detecting the ATP content in living cells during cell proliferation and has advantages such as high sensitivity and convenient testing).

[0080] a. Prepare complete culture medium (generally basal culture medium with fetal bovine serum and antibiotics; some special culture media may require the addition of appropriate additives), and mix thoroughly.

[0081] b. Resuscitate the cells, passage them for about two generations, and select cell lines with good growth.

[0082] c. Remove the cell culture flask from the incubator (Thermo 3111) and check the cell name, culture medium type, and cell number marked on the flask.

[0083] d. Adherent cells: Aspirate the culture medium, wash once with trypsin, discard the waste liquid, and add 3 ml of fresh trypsin to the culture flask for digestion. When the cells are loose and about to detach from the flask wall, add 8 ml of complete culture medium to stop the trypsin digestion and mix gently. Use a pipette to transfer the cell suspension into a centrifuge tube and centrifuge at 800-1000 rpm for 3-5 minutes.

[0084] e. Suspension cells: Transfer the cell suspension into a centrifuge tube using a pipette and centrifuge at 800-1000 rpm for 3-5 minutes.

[0085] f. Discard the cell supernatant from the centrifuge tube.

[0086] g. Add an appropriate volume of culture medium to the centrifuge tube and gently pipette to resuspend the cells evenly.

[0087] h. Count the cells using a Vi-Cell XR cell counter (Backman).

[0088] i. Adjust the cell suspension to the appropriate concentration.

[0089] j. Add the cell suspension to a 96-well plate with a bottom-permeable cell culture medium, 100 μl / well. Label the cell name, seeding density, date, and other details, and incubate the plate overnight in a CO2 incubator.

[0090] k. Prepare a 2 mM stock solution of the compound using DMSO.

[0091] l. Starting with 2 mM as the highest concentration, the compounds were gradually diluted 3-fold with DMSO to obtain 10 concentration gradients.

[0092] m.STS was used as a positive control. The compound was gradually diluted 3-fold with DMSO at a maximum concentration of 0.4 mM to obtain 10 concentration gradients.

[0093] n. Transfer 0.5 μl from the corresponding compound plate and add it to the cell culture plate that has been cultured overnight.

[0094] o. Incubate at 37°C for 72 hours.

[0095] After 72 hours of treatment with the compound, cell morphology was observed under an inverted microscope (DMILLED). Cells in the DMSO control wells showed normal growth and no contamination was observed.

[0096] q. Place the cell culture plate at room temperature for 30 minutes to equilibrate.

[0097] r. Preparation of CellTiter Glo assay reagents: Dissolve CellTiter Glo Buffer in a water bath beforehand, add it to CellTiter Glo Substrate, mix well, and let stand.

[0098] s. Add 100 μl of CellTiter Glo assay reagent per well to the culture plate.

[0099] t. Mix on a vibratory plate for 2 minutes to induce cell lysis.

[0100] u. Place the 96-well plate at room temperature for 10 minutes to stabilize its emission signal.

[0101] v. Attach the white base film to the bottom of the culture plate and use an EnSpire (PE) assay plate.

[0102] w. Record the experimental results obtained from the analysis.

[0103] x.XLfit software: Fit model: Dose response one site /

[0104] f(x)205[fit=(A+((BA) / (1+((C / x)^D))))]

[0105] y. Inhibition rate calculation formula: Inhibition% = [1 - (T72)] sample -T72 blank ) / (T72 DMSO - T72 blank []×100%. After 72 hours of drug and cell interaction, T72 sample T72 blank and T72 DMSO These refer to the readings of the dosing well, the blank control, and the 0.5% DMSO negative control, respectively.

[0106] 4. MiniPDX Experimental Methods:

[0107] laboratory animals

[0108] BALB / c-nude mice were purchased from domestic companies with qualifications for breeding and selling laboratory animals, and were generally 5-6 weeks old. The mice were housed in an SPF-grade laboratory animal center. They underwent an acclimatization period of 3 days before the formal experiments.

[0109] Environmental conditions of laboratory animal housing

[0110] All laboratory animals are housed in an AAALAC-accredited laboratory animal center. The center has an independent ventilation system, with a housing temperature of 24.0-27.0 ℃, relative humidity of 40-60%, an air exchange rate of 10-20 times / hour, and a 12h / 12h day / night cycle. Animals are continuously provided with cobalt-60 radiation-sterilized complete pelleted feed, with unlimited access. Autoclaved drinking water is also continuously provided, with free access to the water.

[0111] Experimental observation

[0112] Throughout the experiment, the use and observation of laboratory animals were conducted in accordance with the relevant regulations for the use and management of animals by AAALAC. After inoculation with tumor cells, the laboratory mice were observed daily according to standard experimental procedures, and their behavior, food and water intake, weight, coat luster, and other abnormalities were recorded and monitored.

[0113] Example 1: Cytological Detection

[0114] The IC50 of MAX-40279-01 was low in cell lines with high FGFR1OP2 expression.

[0115] The activity of MAX-40279-01 and the expression of FGFR1OP2 (RNA sequencing data, TPM) in 33 cell types (purchased from ATCC) were tested. The results are shown in the table below. An IC50 value greater than 0.1 μM indicated ineffectiveness of the drug, and vice versa. The results showed that cells with high FGFR1OP2 expression had low IC50 values, indicating that the drug was effective against the cells. (See Table 4.) Figure 1 .

[0116] [Table 4]

[0117]

[0118] Example 2 mini-PDX model

[0119] 1. Immerse the patient's surgical sample in a culture flask containing RPMI-1640 culture medium (serum-free) and immediately send it to the laboratory.

[0120] 2. Using sterilized ophthalmic scissors, remove visible non-tumor tissue and necrotic tumor tissue. Moisten the tumor tissue with a small amount of RPMI-1640 culture medium (serum-free) and quickly mince it. Place these minced tumor tissues in a 50ml centrifuge tube, add 10-15ml of collagenase solution (Sigma) (collagenase 4 dissolved in D-Hanks, final concentration 1mg / ml, filtered and sterilized), and incubate in a 37℃ water bath for 1 hour. After 1 hour, remove the 50ml centrifuge tube, dilute it halfway with serum-free RPMI-1640 culture medium, repeatedly pipette, and filter the cell suspension through a sterilized 70-mesh filter. Centrifuge at 1200 rpm for 8 minutes, discard the supernatant, and take 20ml of RPMI-1640 culture medium (serum-free) to suspend the precipitated cells (containing lymphocytes, fibroblasts, mesenchymal cells, and necrotic tumor cells), and gently mix. Repeat 2-3 times to obtain the tumor tissue cell suspension.

[0121] 3. Resuspend cells, adjust cell concentration, and fill into MiniPDX experimental devices. These dedicated MiniPDX devices have a unique pore size that allows small molecule drugs below 500KD, large molecule antibody drugs, and various growth factors to freely enter and exit, while tumor cells remain inside the device. Inoculate the MiniPDX devices subcutaneously into mice and begin drug administration on the day of inoculation. The drug administration method for the treatment group was: 12mpk, PO, BID*7 (PO: oral; BID: twice daily), while the control group received no drug.

[0122] 4. In this test, the drug was administered for 7 consecutive days. After the test, the MiniPDX device was removed, and the cell viability after different drug administration regimens was detected by testing the ATPase activity in tumor tissue cells using the ATPase Kinetic ELIPAAssay Kit (Cat#BK051, cytoskeleton.Inc.).

[0123] 5. The proliferation inhibition rate (TCGI) (%) was calculated as follows: [1 - (T7 - T0) / (C7 - C0)] × 100% (T represents the treatment group, C represents the control group, T7 and C7 represent the viability value after 7 days, C0 and T0 represent the viability value before treatment, and C0 = T0). Six MiniPDX experimental devices were used in each of the treatment and control groups. Two-way ANOVA was used for p-value analysis. The effectiveness of various dosing regimens was evaluated based on changes in the proliferation inhibition rate.

[0124] 6. The weight of the medication group and the control group was measured daily. If no significant weight changes or differences were observed in either the control group or the medication group, the data were considered valid.

[0125] 7. Cells from two additional untreated MiniPDX experimental devices were used for RNA detection. FGFR1OP2 expression was detected using the following steps:

[0126] (1) The total RNA concentration was identified using Nanodrop 8000 and the total RNA integrity was identified using Agilent 2100.

[0127] (2) The Illumina RNA library was constructed using the Illumina RNA library construction kit and sequenced on HiSeq X Ten. At the same time, the expression of FGFR1OP2 was detected by quantitative PCR.

[0128] Samples from 31 cancer patients (10 with brain cancer, 8 with pancreatic cancer, 3 with cholangiocarcinoma, 1 with cholangiocarcinoma, 1 with colon cancer, 1 with duodenal cancer, 2 with gastric cancer, 3 with liver cancer, 1 with germ cell cancer, and 2 with rhabdoid tumor) were analyzed using MiniPDX for pharmacodynamics. MiniPDX drug sensitivity testing was performed using the method described in step 2. Gene expression was also obtained using the method described in step 7. The results are as follows: Figure 2 , 3 As shown, MAX-40279-01 showed good inhibitory effects on patient-derived tumor cells with high FGFR1OP2 expression.

[0129] Example 3: Detection of FGFR1OP2 in clinical samples

[0130] 1. FGFR1OP2 testing in leukemia patients

[0131] 1. Collect 2.5 mL of whole blood from the patient using a PAXgene blood RNA tube. Gently invert the tube 8-10 times to ensure thorough mixing of the preservative and blood. Incubate at room temperature for 1-4 hours, then at -20°C for 24 hours. For long-term storage, keep at -70°C. Alternatively, store at -20°C until transported on dry ice.

[0132] 2. Refer to the instruction manual to use the PAX Blood RNA Kit for RNA extraction.

[0133] 3. The total RNA concentration was determined using Nanodrop 8000, and the total RNA integrity was determined using Agilent 2100.

[0134] 4. The RNA library was constructed using the Illumina RNA library construction kit and sequenced on HiSeq X Ten.

[0135] 5. Use the RNA from step 3 to detect FGFR1OP2 expression via quantitative PCR.

[0136] 2. FGFR1OP2 detection in patients with solid tumors

[0137] 1. You will need about 5 white paraffin slides (3-5 micrometers thick), with at least 2. The tissue area should ideally be about 5 mm x 5 mm, and the slides should be attached to an anti-detachment glass slide to prevent detachment.

[0138] 2. The selection principle for paraffin sections is to select diseased tissue, diseased margins, or tissue blocks at the junction of diseased and normal tissue as much as possible, and to avoid tissues with blood clots, calcification, or a lot of fat; the proportion of tumor cells should be greater than 20%, and the proportion of necrotic cells should be less than 5%.

[0139] 3. To prevent the paraffin slides from breaking or rubbing against each other, all paraffin slides must be placed in a slide box.

[0140] 4. RNA was extracted from the white sections of the paraffin using the FFPE RNA Extraction Kit (thermo).

[0141] 5. The total RNA concentration was determined using Nanodrop 8000, and the total RNA integrity was determined using Agilent 2100.

[0142] 6. The RNA library was constructed using the Illumina RNA library construction kit and sequenced on a Hiseq X Ten.

[0143] 7. Use the RNA from step 4 to detect FGFR1OP2 expression by quantitative PCR.

[0144] Example 4 Clinical Trial

[0145] Preliminary clinical trials show that patients with high FGFR1OP2 expression levels benefit from the efficacy of MAX-40279-01.

[0146] Table 5

[0147]

[0148] Example 5 Molecular Simulation

[0149] Since no resolvable crystal structure of FGFR1OP2 is currently available, we performed sequence alignment and template retrieval in the MOE2019.0101 crystal structure protein database based on its sequence. We built 10 models using the Homologymodel module and optimized the models using the Amber10:EHT force field, selecting the best structure from each for superposition analysis. The crystal structure of FGFR1 was obtained from the PDB database.

[0150] First, the MOE site finder function was used to search for potential active pockets in the three crystal structures. Then, FGFR1OP2 was used to dock with each of the three active pockets (JM region and two TK regions). Docking was performed using the Protein-Protein docking method in the MOE Dock module. The prepared FGFR1OP2 and FGFR1 were used as receptors, the entire FGFR1 as a site, FGFR1OP2 as a ligand, and the entire structure as a ligand site. The GBVI / WSA dG score was fitted using hydrophobicpatch potential. Structural optimization was performed in the rigid docking of the Rigid Body. Pre-Placement output 10,000 conformations, Placement output 1,000 conformations, and finally, 100 Refinement conformations were input. Through Browse analysis of the 100 output conformations, combined with the score, the conformations with stable binding between FGFR1OP2 and FGFR1 were selected for interaction analysis. Protein contacts were used to search for interactions in the binding conformation, including hydrogen bonds, ionic bonds, π-π or H-π interactions, and van der Waals interactions. Then, the FGFR1OP2-FGFR1 complex was used as a site, and the small molecule as a ligand for similar docking searches and scoring, further searching for interactions in the binding conformation. Mechanistic analysis was performed using protein simulation and molecular docking. It was found that FGFR1OP2 can bind to MAX-40279-01. Both FGFR1OP2 and MAX-40279-01 bind to the juxtamembrane region (JM) of the FGFR1 protein, jointly inhibiting the FGFR1 signaling pathway and enhancing the inhibitory effect of MAX-40279-01 on FGFR1. AI analysis was also used to analyze the interactions between the small molecule and the protein.

[0151] Table 6. Score values ​​of the interaction forces between different small molecules and FGFR1-FGFR1OP2

[0152]

[0153] Figure 4 The green area represents FGFR1OP2, and the purple area represents the JM region of FGFR1.

[0154] Figure 5 MAX-40279-01 inhibits the JM region of FGFR1 by utilizing the FGFR1OP2 protein, thereby suppressing the FGFR1 signaling pathway and achieving tumor inhibition.

[0155] Figure 6 and Figure 7 The results of MAX-40279-01 binding with FGFR1-FGFR1OP2 show three interactions: strong hydrogen bonding with Leu417 and Gln426 of FGFR1, and H-π interaction with Gln70 of FGFR1OP2.

[0156] Figure 8 and Figure 9 The binding results of AZD4547 and FGFR1-FGFR1OP2 show that there is an H-π interaction between Gln71 of AZD4547 and FGFR1OP2.

[0157] Figure 10 and Figure 11 The results show that Pemigatinib does not have strong nonbonded interactions with FGFR1-FGFR1OP2, and mainly interacts with van der Waals.

[0158] Figure 12 and Figure 13 The binding results of Erdafitinib with FGFR1-FGFR1OP2 show that there are three interactions: an H-π interaction with Leu417 of FGFR1, an H-π interaction with Ser420, and an H-π interaction with Ile69 of FGFR1OP2.

[0159] Figure 14 and Figure 15 The binding results of Infigratinib to FGFR1-FGFR1OP2 show that there is an H-π interaction between Infigratinib and Ser420 of FGFR1.

[0160] Summary: Compared with MAX-40279-01, the binding affinity of Pemigatinib and Infigratinib is about 5 times weaker; that of AZD4547 and Erdafitnib is about 10 times weaker.

[0161] Example 6: Testing of four candidate compounds in four cell lines using CellTiterGlo assay

[0162] Experimental steps:

[0163] 1. Cell seeding

[0164] 1) Prepare complete culture medium: Add FBS and appropriate additives according to the supplier's information sheet. Mix gently.

[0165] 2) Check the cell names, complete culture medium, and passage number marked on the flask.

[0166] 3) Use a vacuum pump to remove and discard the culture medium.

[0167] 4) Simply rinse the cell layer with 0.25% (w / v) Trypsin-0.038% (w / v) EDTA solution to remove any trace amounts of serum containing trypsin inhibitors.

[0168] 5) Add 3.0 ml of Trypsin-EDTA solution to the flask and observe the cells under an inverted microscope until the cell layer is dispersed.

[0169] 6) Add 9.0 ml of complete growth medium and gently pipette out the cells.

[0170] 7) Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0171] 8) Use a vacuum pump to discard the supernatant.

[0172] 9) Add an appropriate volume of complete culture medium. Resuspend the cell pellet by gently pipetting.

[0173] 10) Use Vi-cell XR to calculate the cell count and adjust the cells to the appropriate density.

[0174] 11) According to the planned plate layout, add 100 μL of cell suspension to a 96-well opaque-walled transparent plate.

[0175] Table 7

[0176]

[0177] The data in the table above further demonstrate that FGFR1OP2 expression can be used to determine the effectiveness of other FGFR inhibitors. sequence list <110> Guangzhou Zaiji Pharmaceutical Technology Co., Ltd. <120> A screening method for FGFR inhibitors <130> P21017235C <150> 202010954827.1 <151> 2020-09-11 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> FGFR1OP2-F1 <400> 1 agcgagtaga agccatgaaa ca 22 <210> 2 <211> 21 <212> DNA <213> Artificial sequence <220> <223> FGFR1OP2-R1 <400> 2 cccataacta acgtggaccg t 21 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> FGFR1OP2-F2 <400> 3 gcgagtagaa gccatgaaac 20 <210> 4 <211> 23 <212> DNA <213> Artificial sequence <220> <223> FGFR1OP2-R2 <400> 4 cttgctggat tcccataact aac 23 <210> 5 <211> 21 <212> DNA <213> Artificial sequence <220> <223> ACTB-F <400> 5 cagcagatgt ggatcagcaa g 21 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <220> <223> ACTB-R <400> 6 gcatttgcgg tggacgat 18 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> GAPDH-F <400> 7 attccaccca tggcaaattc 20 <210> 8 <211> 22 <212> DNA <213> Artificial sequence <220> <223> GAPDH-R <400> 8 gatgggattt ccattgatga ca 22

Claims

1. A method for screening FGFR inhibitors, characterized in that, It includes the following steps: (1) The FGFR inhibitor to be tested was administered to cell lines with different FGFR1OP2 protein expression levels, and the efficacy of the FGFR inhibitor to be tested was tested. (2) Determine the correlation between the drug efficacy of different cell lines and the expression level of FGFR1OP2 protein in different cell lines; In step (2): Select cell lines in which the expression level of FGFR1OP2 protein is higher than the general level. If the efficacy of the drug is positively correlated with the expression level of FGFR1OP2 protein, then the FGFR inhibitor to be tested is an effective FGFR1 inhibitor. The effective FGFR1 inhibitors are MAX-40279-01, AZD4547, Erdafitinib, Infigratinib, or Pemigatinib.

2. The screening method as described in claim 1, characterized in that, The efficacy is manifested by the IC50 of the FGFR inhibitor to be tested. 50 value; And / or, the cell line is KATOIII, KG1, NCIH716, SKNO1, KASUMI6, SUPB15, OPM2, BDCM, NCIH2009, HGC27, MFE296, NCIH1703, REH, G401, G292CLONEA141B1, AN3CA, A375, HCT116, NCIH460, MFE280, DLD1, NCIH1975, LP1, A498, SNU423, NUGC3, HCC1954, BGC823, CAPAN1, CAMA1, SNU449, or MKN7.

3. The screening method as described in claim 1 or 2, characterized in that, The cell lines mentioned are derived from cell lines from patients with solid tumors or leukemia.

4. A method for screening cell models suitable for specific FGFR inhibitors, characterized in that, It includes the following steps: (1) Detect the expression level of FGFR1OP2 protein in the cell model to be tested; (2) Determine the numerical value of the expression level of the FGFR1OP2 protein; Based on the numerical values ​​of the protein expression levels in step (2), cell models in which the FGFR1OP2 protein expression level is higher than the general level are selected for screening specific FGFR1 inhibitors. The specific FGFR1 inhibitor is MAX-40279-01, AZD4547, Erdafitinib, Infigratinib, or Pemigatinib.

5. The screening method as described in claim 4, characterized in that, The cell model is a cell line derived from patients with solid tumors or leukemia. And / or, the detection method is qPCR detection.

6. The screening method as described in claim 5, characterized in that, The nucleotide sequence of the forward primer for the qPCR detection is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2; or the nucleotide sequence of the forward primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 4.

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