Applications of drugs that simultaneously target EGFR tyrosine kinase activity and GEF activity

Abstract

This invention provides applications for EGFR inhibitors in the preparation of drugs for treating diseases mediated by guanylate exchange factor (GEF) activity, or diseases co-mediated by GEF activity and EGFR tyrosine kinase activity. [Solution] The invention provides an application of an EGFR inhibitor, characterized in that it is used in the preparation of a pharmaceutical composition for treating a disease mediated by GEF activity or a disease co-mediated by GEF activity and EGFR tyrosine kinase activity. The disease is characterized in that it is a cancer selected from the group consisting of lung cancer, colorectal cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, prostate cancer, melanoma, leukemia, and glioblastoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically relates to the use of a drug that simultaneously targets the tyrosine kinase activity and GEF activity of EGFR.

Background Art

[0002] Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein, and its intracellular tyrosine kinase domain plays a very important role in cell signal transduction. After binding to a growth factor, EGFR undergoes dimerization and autophosphorylation, thereby activating multiple downstream signal transduction pathways, and regulating and controlling basic biological processes such as cell proliferation, differentiation, survival and migration. Under normal physiological conditions, EGFR signal transduction is precisely regulated and controlled to maintain cellular homeostasis.

[0003] In multiple types of malignant tumors (such as lung cancer, colon cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma and prostate cancer, etc.), EGFR is frequently abnormally activated due to overexpression, gene amplification or specific mutations. Such abnormal activation continuously triggers EGFR signal transduction, thereby causing continuous activation of downstream signal transduction pathways. For example, sustained EGFR activation indirectly and continuously stimulates the signal transduction pathway of mammalian target of rapamycin complex 1 (mTORC1), promotes protein synthesis and cell proliferation, and inhibits the autophagy process, thus playing an important role in tumor occurrence and progression.

[0004] Current EGFR inhibitors (such as gefitinib and osimertinib) only target the intracellular kinase domain thereof, thus causing a high incidence of drug resistance. Studies have shown that drug-resistant tumors may evade inhibition by activating EGFR non-kinase-dependent pathways, but there are fewer studies on non-kinase-dependent pathways, and there is a problem that the high incidence of EGFR drug resistance cannot be effectively solved.

[0005] Therefore, in this field, elucidating the tumor molecular mechanisms driven by EGFR, particularly the mechanisms by which tumor cells acquire drug resistance via EGFR non-kinase-dependent pathways, is an urgent task. Unraveling its potential regulatory mechanisms will not only help explain the limitations of existing therapeutic measures, but will also provide a theoretical foundation and new application prospects for developing more precise, efficient, and safe combination antitumor therapy strategies. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] To solve the above problems, the present invention provides a drug application that can simultaneously regulate EGFR tyrosine kinase activity and GEF activity. [Means for solving the problem]

[0007] A first aspect of the present invention provides an application for EGFR inhibitors and is used in the preparation of pharmaceutical compositions for treating diseases mediated by guanylate exchange factor (GEF) activity, or diseases co-mediated by guanylate exchange factor (GEF) activity and EGFR tyrosine kinase activity.

[0008] In another preferred example, the EGFR inhibitor inhibits GEF activity.

[0009] In another preferred example, the EGFR inhibitor simultaneously inhibits both EGFR tyrosine kinase activity and GEF activity.

[0010] In a preferred example, the disease is a cancer selected from the group consisting of lung cancer, colorectal cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, prostate cancer, melanoma, leukemia, and glioblastoma.

[0011] In a preferred example, the pharmaceutical composition is also used to regulate abnormal nucleotide exchanges of Rheb, preferably without relying on the regulation of EGFR kinase activity or expression abnormalities.

[0012] In another preferred example, the pharmaceutical composition can simultaneously regulate EGFR kinase activity and Rheb nucleotide exchange capacity, and the regulation of Rheb nucleotide exchange capacity is independent of the regulation of EGFR kinase activity.

[0013] In another preferred example, the pharmaceutical composition inhibits mTORC1 activation by regulating Rheb nucleotide exchange.

[0014] In another preferred example, the pharmaceutical composition modulates Rheb nucleotide exchange by regulating GEF activity and is independent of the regulation of EGFR kinase activity.

[0015] In another preferred example, the pharmaceutical composition modulates GEF activity by acting on the tyrosine kinase domain within EGFR cells, and is independent of the modulation of EGFR kinase activity.

[0016] In another preferred example, the pharmaceutical composition modulates GEF activity by acting on the glutamic acid residue at position 804 of EGFR, and is independent of the modulation of EGFR kinase activity.

[0017] In another preferred example, the EGFR inhibitor can induce a mutation in the glutamate residue at position 804 of EGFR.

[0018] In a preferred example, the pharmaceutical composition simultaneously modulates EGFR tyrosine kinase activity and GEF activity, and the modulation of GEF activity is independent of the regulated control of EGFR tyrosine kinase activity.

[0019] In another preferred example, the EGFR tyrosine kinase activity is caused by EGFR overexpression, EGFR gene amplification or a specific mutation of EGFR.

[0020] In another preferred example, the specific mutation of EGFR is the EGFRvIII mutant, the L858R point mutation or the in-frame exon 19 deletion mutation.

[0021] In another preferred example, the EGFR inhibitor acts simultaneously on the EGFR tyrosine kinase domain and the glutamic acid residue at position 804 of EGFR.

[0022] In another preferred example, the action comprises modifying the gene region associated with EGFR tyrosine kinase activity and the gene region associated with the GEF activity of the glutamic acid residue at position 804 of EGFR.

[0023] In a preferred example, the EGFR inhibitor is selected from the group consisting of small molecule compounds, nanomaterial-based drugs, gene editing technologies, protein antibodies, nucleic acid-based drugs, enzyme-based drugs, expression vectors, cell therapy, hormone-based drugs, peptide-based drugs, or combinations thereof.

[0024] In another preferred example, the nanomaterial-based drug is a liposome drug.

[0025] In another preferred example, the nucleic acid-based drug is selected from the group consisting of mRNA, siRNA, shRNA, microRNA, oligonucleotides, polynucleotides, aptamers, or combinations thereof.

[0026] In another preferred example, the enzyme-based drug is selected from the group consisting of DNase, ribozyme, or a combination thereof.

[0027] In another preferred example, the cell therapy is CAR-T cell therapy.

[0028] In a preferred example, the EGFR inhibitor is a small molecule compound.

[0029] In a preferred example, the EGFR inhibitor is a small molecule compound represented by the following formula. [ka]

[0030] A second aspect of the present invention provides a pharmaceutical composition comprising an EGFR tyrosine kinase inhibitor and a GEF activity inhibitor, or an EGFR tyrosine kinase / GEF activity dual-target inhibitor.

[0031] In another preferred example, the pharmaceutical composition can regulate Rheb nucleotide exchange.

[0032] A third aspect of the present invention provides the use of the pharmaceutical composition described in the second aspect of the present invention, which is used in the preparation of a pharmaceutical composition for treating EGFR-driven tumors or tumors that exhibit drug resistance to independent EGFR tyrosine kinase inhibitors.

[0033] Preferably, the tumor is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, prostate cancer, melanoma, leukemia, and glioblastoma.

[0034] A fourth aspect of the present invention provides a method for designing EGFR tyrosine kinase and GEF activity dual-target inhibitors, comprising the following steps: (a) Provide a candidate molecule that can act simultaneously on the kinase domain of EGFR and the glutamic acid residue at position 804 of EGFR, (b) The ability of the candidate molecule to inhibit EGFR kinase and GEF activity is measured.

[0035] A fifth aspect of the present invention provides a screening method for treating diseases mediated by GEF activity or diseases co-mediated by GEF activity and EGFR tyrosine kinase activity, comprising the following steps: (A) Candidate drugs are administered to animal models of diseases related to EGFR tyrosine kinase activity and GEF activity, (B) In the above animal model, drugs that detect and reduce the in vivo Rheb nucleotide exchange capacity are targeted drugs.

[0036] A sixth aspect of the present invention provides a method for treating a disease mediated by GEF activity or a disease co-mediated by GEF activity and EGFR tyrosine kinase activity, the method comprising administering to a subject in need a pharmaceutical composition comprising an EGFR tyrosine kinase inhibitor and a GEF activity inhibitor or an EGFR tyrosine kinase / GEF activity dual-target inhibitor.

[0037] The subjects are selected from a group consisting of humans, mice, rats, guinea pigs, pigs, dogs, monkeys, and chimpanzees. [Effects of the Invention]

[0038] It should be understood that, within the scope of the present invention, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0039] [Figure 1A] This demonstrates that inhibiting the localization of activated EGFR to the lysosome surface significantly inhibits mTORC1 activation. Figure 1A shows that activated EGFR can be localized to the lysosome surface. [Figure 1B]This demonstrates that inhibiting the localization of activated EGFR to the lysosome surface significantly inhibits mTORC1 activation. Figure 1B shows that after using an endocytosis inhibitor or a cathepsin S inhibitor (late endosomal-lysosome transport inhibitor), mTORC1 activation is significantly inhibited, but EGFR kinase activity remains unaffected. [Figure 2A] This shows that EGFR and Rheb interact directly, and that this interaction is independent of EGFR kinase activity. Figure 2A shows that the interaction between EGFR and Rheb is independent of EGFR kinase activity. [Figure 2B] This shows that EGFR and Rheb interact directly, and that this interaction is independent of EGFR kinase activity. Figure 2B is a schematic diagram of different intracellular cleavage products of EGFR. [Figure 2C] This shows that EGFR and Rheb interact directly, and that this interaction is independent of EGFR kinase activity. Figure 2C shows that EGFR interacts with Rheb primarily through its intracellular tyrosine kinase domain. [Figure 2D] This demonstrates that EGFR and Rheb interact directly, and that this interaction is independent of EGFR kinase activity. Figure 2D shows that the intracellular tyrosine kinase domain of EGFR can directly bind to Rheb. [Figure 3] This study demonstrates that EGFR-TKD promotes Rheb nucleotide exchange. [Figure 4] A schematic diagram of the structural model obtained by predicting the binding status of the EGFR-Rheb complex based on AlphaFold2 is shown. [Figure 5A] This shows the effect of the glutamate moiety at position 804 of EGFR on its GEF activity. Figure 5A shows that the EGFR-E804K mutant loses its ability to interact with Rheb. [Figure 5B] Figure 5B shows the effect of the glutamate moiety at position 804 of EGFR on its GEF activity. The EGFR-TKD-E804K mutant is unable to promote Rheb nucleotide exchange. [Figure 5C]Figure 5C shows the effect of the glutamate site at position 804 of EGFR on its GEF activity. The LAMP2-V5-EGFR-TKD-E804K fusion protein localized by lysosomes cannot activate mTORC1, but wild-type (WT) or kinase-inactivated (KD) EGFR-TKD can all effectively activate mTORC1. [Figure 6A] This study shows the effects on the mTORC1 signaling pathway and cell proliferation after knocking in the EGFR-E804K mutation via CRISPR-Cas9-mediated gene editing in PC9 lung cancer cells. Figure 6A shows that EGFR-E804K gene knock-in PC9 cells cannot activate mTORC1, but their EGFR kinase activity remains unaffected. [Figure 6B] This figure shows the effects on the mTORC1 signaling pathway and cell proliferation after knocking in the EGFR-E804K mutation via CRISPR-Cas9-mediated gene editing in PC9 lung cancer cells. Figure 6B shows the CCK8 experiment evaluating the proliferative capacity of parental PC9 cells and EGFR-E804K gene knock-in cells. [Figure 6C] This figure shows the effects on the mTORC1 signaling pathway and cell proliferation after knocking in the EGFR-E804K mutation via CRISPR-Cas9-mediated gene editing in PC9 lung cancer cells. Figure 6C evaluates the cloning ability of parental PC9 cells and EGFR-E804K gene knock-in cells in cloning experiments. [Figure 6D] This study demonstrates the effects on the mTORC1 signaling pathway and cell proliferation after knocking in the EGFR-E804K mutation via CRISPR-Cas9-mediated gene editing in PC9 lung cancer cells. Figure 6D shows that the EdU experiment detects the DNA synthesis capacity of both parental PC9 cells and EGFR-E804K gene knock-in cells. [Figure 6E]This paper demonstrates the effects on the mTORC1 signaling pathway and cell proliferation after knocking in the EGFR-E804K mutation via CRISPR-Cas9-mediated gene editing in PC9 lung cancer cells. Figure 6E is a schematic diagram of parental PC9 cells and EGFR-E804K gene knock-in cells in mouse subcutaneous tumors. [Figure 6F] This study shows the effects on the mTORC1 signaling pathway and cell proliferation after knocking in the EGFR-E804K mutation via CRISPR-Cas9-mediated gene editing in PC9 lung cancer cells. Figure 6F shows the volume change of subcutaneous tumors in Figure E. [Figure 6G] This study shows the effects on the mTORC1 signaling pathway and cell proliferation after knocking in the EGFR-E804K mutation via CRISPR-Cas9-mediated gene editing in PC9 lung cancer cells. Figure 6G shows the weight change of subcutaneous tumors in Figure E. [Figure 7A] This shows the combined inhibitory effect of the small molecule compound BIE Gi-1 on EGFR kinase and GEF function. Figure 7A is a schematic diagram of the chemical structure of the small molecule compound BIE Gi-1. [Figure 7B] This shows the combined inhibitory effect of the small molecule compound BIE Gi-1 on EGFR kinase and GEF function. Figure 7B shows that BIE Gi-1 inhibits EGFR-mediated Rheb nucleotide exchange. [Figure 7C] This shows the combined inhibitory effect of the small molecule compound BIE Gi-1 on EGFR kinase and GEF function. Figure 7C shows that BIE Gi-1 can inhibit the interaction between EGFR and Rheb. [Figure 7D] This demonstrates the combined inhibitory effect of the small molecule compound BIE Gi-1 on EGFR kinase and GEF function. Figure 7D shows that BIE Gi-1 can inhibit mTORC1 activation in PC9 cells. [Figure 7E] This shows the combined inhibitory effect of the small molecule compound BIE Gi-1 on EGFR kinase and GEF function. Figure 7E shows that BIE Gi-1 can inhibit mTORC1 activation in NCI-H1975 cells. [Figure 7F] This shows the combined inhibitory effect of the small molecule compound BIE Gi-1 on EGFR kinase and GEF function. Figure 7F shows that BIE Gi-1 can inhibit the proliferation of tumor cells driven by EGFR. [Modes for carrying out the invention]

[0040] Through extensive and meticulous research and large-scale screening, the inventors discovered for the first time that, in addition to its conventional kinase activity, EGFR exerts GEF activity depending on the glutamate site at position 804 on its protein. This promotes nucleotide exchange of Rheb (Ras homologous protein), thereby activating mTORC1 and promoting tumor growth. Furthermore, by simultaneously targeting and inhibiting EGFR-mediated tyrosine kinase activity and GEF activity, EGFR-driven tumor growth can be effectively inhibited. Based on this, the inventors completed the present invention.

[0041] Therapeutic strategies that simultaneously target EGFR kinase activity and GEF activity The inventors have discovered for the first time that EGFR kinase activity and GEF activity can be simultaneously regulated by regulating EGFR, and that the GEF activity of EGFR depends on the glutamate residue at position 804, which activates mTORC1 by promoting Rheb nucleotide exchange. Based on these findings, the present invention further proposes a combination therapy strategy that simultaneously inhibits EGFR kinase activity and GEF activity, thereby achieving more effective treatment of at least some tumors driven by EGFR.

[0042] The aforementioned EGFR kinase activity is a sustained signaling pathway induced by EGFR overexpression, gene amplification, or specific mutations.

[0043] The aforementioned specific mutations include, but are not limited to, EGFRvIII mutants, L858R point mutations, or intra-frame exon 19 deletion mutations.

[0044] The present invention provides a novel strategy for tumor treatment centered on simultaneously inhibiting EGFR kinase activity and GEF activity. The tumors are at least some tumors driven by EGFR, and preferably include, but are not limited to, lung cancer, colon cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, and prostate cancer.

[0045] A specific embodiment of the novel strategy for tumor treatment described in the present invention includes simultaneously targeting and inhibiting EGFR kinase activity and GEF activity using one or more therapeutic means such as inhibitors, small molecule compounds, nanomaterial drugs (e.g., liposomal drugs), and gene editing technologies.

[0046] Here, the small molecule compound has the ability to permeate the cell membrane and to simultaneously interact with the EGFR kinase domain and the GEF active binding site, thereby improving therapeutic specificity and efficacy.

[0047] Here, the nanomaterial-based drug can specifically target the tumor microenvironment after surface modification, and its affinity and selectivity for lesion tissue can be enhanced.

[0048] Here, the nanomaterial-based drug penetrates the biological barrier by binding the drug to liposomes, albumin, and polymer micelles, thereby continuously releasing the drug and maintaining an appropriate blood drug concentration.

[0049] Here, the gene editing technology (e.g., the CRISPR / Cas9 system) can fundamentally regulate and control related pathological processes by knocking out or modifying gene regions associated with EGFR kinase activity and GEF activity.

[0050] The present invention also provides clinical or laboratory applications of strategies that simultaneously target the above-mentioned EGFR kinase activity and GEF activity. These applications include inhibiting EGFR kinase activity and GEF activity by administering one or more of the above-mentioned therapeutic means to a subject (e.g., a mammal or tumor cell line), further inhibiting the mTORC1 signaling pathway to block tumor cell proliferation, and ultimately inhibiting tumor growth.

[0051] The subjects may include humans, mice, rats, guinea pigs, pigs, dogs, monkeys, chimpanzees, etc. The aforementioned tumor cells include lung cancer, colon cancer, glioma, etc., which are driven by abnormal EGFR activity.

[0052] Through a series of experiments, the inventors demonstrate that EGFR directly interacts with Rheb, and that this interaction is primarily dependent on the glutamate residue at position 804 of EGFR and is independent of its kinase activity.

[0053] Further experiments demonstrate that tumor cell proliferation can be inhibited by achieving combined inhibition through gene knock-in of the EGFR-E804K mutant or by applying small molecule compounds, thereby effectively inhibiting Rheb nucleotide exchange and mTORC1 activation.

[0054] In summary, this invention is the first to reveal that EGFR, in addition to its conventional kinase activity, also possesses a GEF function that regulates Rheb nucleotide exchange, and identifies the important functional site at position 804 where glutamate is responsible for this GEF activity.

[0055] Based on this discovery, the present invention proposes a therapeutic strategy that simultaneously targets EGFR kinase activity and GEF activity, providing a new theoretical foundation and technical means for the development of efficient treatments for at least some tumors driven by EGFR.

[0056] The main advantages of this invention are as follows:

[0057] 1. The present invention deepens our understanding of the molecular mechanism of action of EGFR, specifically by discovering that in addition to its conventional tyrosine kinase activity, EGFR also possesses a GEF function that promotes the activation of Ras homologous protein (Rheb). In other words, EGFR can simultaneously inhibit both its kinase activity and GEF activity, and the GEF activity of EGFR is dependent on its glutamic acid residue at position 804, activating mTORC1 by promoting Rheb nucleotide exchange, and ultimately promoting tumor growth.

[0058] 2. The present invention provides novel targets for cancer treatment. For example, existing EGFR inhibitors primarily target the kinase domain, but drug-resistant tumors may survive depending on the GEF function of EGFR. Therefore, the development of inhibitors that target the GEF domain may provide a therapeutic strategy.

[0059] 3. The present invention is advantageous for the development of novel drugs, and for example, a high-throughput screening platform targeting the Rheb-GEF activity of EGFR based on GEF exchange experiments can be developed, thereby accelerating drug discovery.

[0060] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are used solely for illustrative purposes and do not limit the scope of the invention. In the following embodiments, experimental methods that do not specify conditions are generally carried out under conventional conditions or conditions proposed by the manufacturer. Unless otherwise specified, percentages and parts are calculated as weight percentages and weight parts, respectively.

[0061] Example 1. By localizing to the lysosome surface, it inhibits EGFR activation and significantly inhibits mTORC1 activation. I. Experimental Method 1. Cell lysis and lysosomal immunoprecipitation (Lyso-IP) Lysosomes were isolated from PC9 cells, a non-small cell lung cancer cell line that stably expresses TMEM192 (lysosomal marker protein) with an HA tag, and from PC9 cells that stably express TMEM192 with a FLAG tag.

[0062] First, the cells were washed with PBS, scraped off, and centrifuged to collect the cell precipitate. The precipitate was then resuspended in KPBS buffer (composition: 136 mM KCl, 10 mM KH2PO4, a mixture of protease inhibitor and phosphatase inhibitor, pH 7.25), and homogenization was performed. The homogenized solution was centrifuged at 1000 g for 3 minutes at 4°C, and the supernatant was collected. The supernatant and 20 μL of anti-HA magnetic beads (Beyotime) were incubated under rotational conditions for 1 hour. Finally, the magnetic beads were washed six times with KPBS buffer, and an appropriate amount of 5× loading buffer was added. The mixture was boiled in a metal bath at 100°C for 10 minutes, cooled, and then subjected to WB detection. The results are shown in Figure 1A.

[0063] 2. Cell lysis and Western blotting (WB) Non-small cell lung cancer cell lines PC9 and HCC827, which have EGFR exon 19 deletion mutations, and human lung adenocarcinoma cell line NCI-H1975, which has EGFR T790M (drug resistance mutation) and L858R mutations, were treated with the solvent DMSO, the EGFR endocytosis inhibitor Dyngo-4a, or the cathepsin C inhibitor (late endosome-lysosome transport inhibitor) LY3000328, respectively.

[0064] Cells treated by the above-described method were collected and lysed for 20 minutes using RIPA lysis solution containing protease inhibitors and phosphatase inhibitors. The cells were then centrifuged at 12000 rpm at 4°C for 20 minutes, the supernatant was collected, and the protein concentration was quantified using the Coomassie Brilliant Blue method. The obtained protein samples were first separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After sealing the membrane, the primary antibody and corresponding secondary antibody were incubated sequentially, and finally, a chromogenic substrate was added. The expression level of the target protein was detected under an appropriate imaging system, and the results are shown in Figure 1B.

[0065] This experiment was used to detect the expression of target proteins and their phosphorylation levels in the sample, with the S6K1 phosphorylation (p-T389-S6K1) level used as an indicator of mTORC1 activation.

[0066] 2. Experimental Results As shown in Figure 1A, Lyso-IP results showed simultaneous detection of EGFR and lysosomal marker protein LAMP1 expression in PC9 cell lines that stably express HA-tagged TMEM192, suggesting that activated EGFR is localized to the lysosomal surface.

[0067] As shown in Figure 2B, compared to cells treated with DMSO, cells treated with the EGFR endocytosis inhibitor Dyngo-4a or the cathepsin C inhibitor (late endosomal-lysosome transport inhibitor) LY3000328 showed a significant decrease in the expression of phosphorylated S6K1 (p-T389-S6K1), but no change was observed in phosphorylated EGFR (p-Y1068-EGFR). This indicates that inhibition of EGFR endocytosis or inhibition of EGFR localization to the lysosome surface can effectively inhibit mTORC1-like activation, but does not affect the activity of EGFR kinase.

[0068] Existing studies have shown that abnormally activated EGFR enhances its endocytic activity. In normal cells, EGFR terminates signaling by entering lysosomes and degrading after endocytosis. In tumor cells, abnormal endocytosis not only inhibits EGFR degradation but also forms a persistent signaling "signaling platform" by retaining some activated EGFR in specific intracellular regions, thereby continuously and indirectly stimulating the mTORC1 signaling pathway.

[0069] According to the experimental results of this application, inhibiting EGFR endocytosis or inhibiting EGFR localization to the lysosome surface inhibits mTORC1 activation without altering EGFR kinase activity, suggesting the existence of other kinase-independent regulatory mechanisms between EGFR and mTORC1.

[0070] Example 2. Direct interaction between EGFR and Rheb, and EGFR-independent kinase activity. I. Experimental Method 1. Immunoprecipitation (IP) 1.1. Cell culture and preparation: 1.1.1. HEK293T cells are cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 humidified incubator. 1.1.2. The day before transfection, HEK293T cells are inoculated into a 6-well cell culture plate at an appropriate density to ensure that the cells reach appropriate confluence during transfection.

[0071] 1.2. Vector Construction The pSIN-EGFR-WT-HA plasmid is constructed using conventional PCR amplification techniques. Furthermore, the wild-type gene plasmid is used as a PCR template, and corresponding mutant primers are designed and amplified according to the experimental requirements. After the PCR products are recovered by agarose gel electrophoresis, they are ligated and transformed, and finally, the success of the mutation is verified by sequencing.

[0072] 1.3. Transient transfection Following the instructions for Lipofectamine 3000, each plasmid was transfected into HEK293T, i.e., Vector group: Transfect with pSIN plasmid, WT group: Transfected with the pSIN-EGFR-WT-HA plasmid. KD (EGFR kinase inactivated) group: Following a site-directed mutagenesis method, the EGFR-KD-HA plasmid is constructed by mutating lysine at position 745 of the EGFR-WT-HA plasmid to alanine (K745A), and then transfected.

[0073] 1.4. Experimental Stage The above cells were lysed with an appropriate amount of IP lysis buffer for 30 minutes, and the supernatant was collected after centrifugation. 20 μL of agarose beads containing HA antibody were added, and the mixture was incubated overnight at 4°C. The agarose bead-antigen-antibody complex was then collected, the supernatant was discarded, and the mixture was washed three times with pre-cooled IP elution buffer. An appropriate amount of 5× loading buffer was added, and the mixture was boiled in a metal bath at 100°C for 10 minutes. After cooling, WB detection was performed, and the results are shown in Figure 2A.

[0074] 2. Construction of the cut body and IP 2.1. Using conventional genetic engineering techniques, the following cleaved plasmids were constructed (as shown in Figure 2B), namely, WT-ICD group: pSIN-EGFR-WT-ICD-V5, containing the wild-type EGFR intracellular domain, KD-ICD group: includes pSIN-EGFR-KD-ICD-V5 and the K745A mutant EGFR intracellular domain. WT-TKD group: pSIN-EGFR-WT-TKD-V5, contains the wild-type EGFR intracellular tyrosine kinase domain, KD-TKD group: Contains pSIN-EGFR-KD-TKD-V5 and K745A mutant EGFR intracellular tyrosine kinase domains. RD group: pSIN-EGFR-RD-V5, contains an EGFR regulatory domain but does not contain an EGFR intracellular tyrosine kinase domain.

[0075] 2.3. Building a stable stock portfolio Using a conventional lentiviral infection method, a HEK293T cell line overexpressing SFB-Rheb is constructed. After successful construction, the day before transfection, the SFB-Rheb-overexpressing HEK293T cells are inoculated into a 6-well cell culture plate at an appropriate density to ensure that the cells reach appropriate confluence during transfection.

[0076] 2.4 Transient transfection and IP The above plasmids were transfected into HEK293T cells overexpressing SFB-Rheb according to the Lipofectamine 3000 instructions. 36 hours after transfection, the cells were lysed in an appropriate amount of IP lysis buffer for 30 minutes, and the supernatant was collected after centrifugation. 20 μL of agarose beads containing V5 antibody were added, and the cells were incubated overnight at 4°C. The agarose bead-antigen-antibody complex was then collected, the supernatant was discarded, and the cells were washed three times with pre-cooled IP elution buffer. An appropriate amount of 5× loading buffer was added, and the cells were boiled in a metal bath at 100°C for 10 minutes. After cooling, WB detection was performed to detect the binding status of EGFR and Rheb. The results are shown in Figure 2C.

[0077] 3. Protein purification and pull-down assay 3.1. Protein Purification GST and GST-Rheb proteins are expressed using a prokaryotic system, while EGFR protein expression is induced using an insect cell system.

[0078] The specific procedure involves constructing a recombinant plasmid, transfecting it into the corresponding host cells, lysing the cells by high-pressure disruption or sonication, separating the supernatant and cell debris by centrifugation, purifying them using affinity chromatography, and obtaining a purified protein solution.

[0079] 3.2. Pull-down assay The groups are divided as follows: GST group: GST proteins (tag proteins) purified using a prokaryotic system. GST-Rheb group: GST-Rheb proteins purified using a prokaryotic system. EGFR-TKD-WT group: EGFR-TKD-WT proteins purified using insect cell lines.

[0080] Pre-washed and equilibrated GST beads are taken, and the purified protein solution to be detected is added to each, as shown in the figure. The mixture is incubated at 4°C for 2 hours to ensure sufficient binding between the protein and the GST beads. After incubation, the mixture is centrifuged at 5000 rpm for 30 seconds, the supernatant is discarded, and the beads are collected by repeating centrifugation and washing twice. The beads are washed three times with pre-cooled IP eluate to remove nonspecific conjugates. After adding an appropriate amount of loading buffer, the sample is boiled at 100°C for 10 minutes to allow it to denature completely. The boiled sample is then stained with Coomassie brilliant blue to detect the target protein, and the results are shown in Figure 2D.

[0081] 2. Experimental Results Existing research has revealed that Rheb (Ras Homolog Enriched in Brain) on lysosomes is an important protein that activates mTORC1, but the relationship between EGFR and Rheb remains unclear.

[0082] As shown in Figure 2A, in HEK293T cells, both exogenous EGFR overexpression (WT group) and EGFR kinase inactivation (KD group) bound to endogenous Rheb, indicating that the interaction between EGFR and Rheb is independent of EGFR kinase activity.

[0083] As shown in Figures 2B and 2C, in HEK293T cells overexpressing SFB-Rheb, domains containing the EGFR intracellular tyrosine kinase domain (WT-ICD group, KD-ICD group, WT-TKD group, and KD-TKD group) all interact with SFB-Rheb, but domains containing only the EGFR regulatory domain and not the EGFR intracellular tyrosine kinase domain (RD group) cannot bind. This indicates that EGFR binds to Rheb mainly via the intracellular tyrosine kinase domain (TKD).

[0084] As shown in Figure 2D, in in vitro pull-down experiments, EGFR-TKD can interact with GST-Rheb but not with GST, indicating that the intracellular tyrosine kinase domain of EGFR (EGFR-TKD) can directly bind to Rheb.

[0085] In summary, the data above demonstrates that EGFR interacts directly with Rheb primarily via TKD, and that this process is independent of EGFR kinase activity.

[0086] Example 3. EGFR capable of functioning as a GEF for Rheb (i.e., EGFR has a GEF function that modulates and controls Rheb activity). 1. Experimental Method: In vitro nucleotide exchange experiment Rheb is a small G protein whose activity is regulated by guanylate exchange factors (GEFs). Specifically, Rheb is activated when it binds to GTP, can activate neighboring and downstream molecules, and is inactivated when hydrolyzed to GDP. Here, GEFs facilitate the conversion of Rheb from the GDP state (inactivated state) to the GTP state (activated state). In other words, GEFs can facilitate nucleotide exchange in Rheb.

[0087] To detect whether EGFR has the ability to regulate and control the guanylate exchange factor (GEF) activity of Rheb, the applicant conducted in vitro nucleotide exchange experiments using the RhoGEF Exchange Assay Kit (Cytoskeleton, BK100), with the following groupings: RhoA group: RhoA protein alone; RhoA is also a small G protein, and its activity is regulated by GEFs. RhoA+hDbs group: RhoA protein + hDbs protein. hDbs converts RhoA from GDPase (inactivated state) to GTPase (activated state). Therefore, the GEF domain of hDbs can function as a positive control GEF. RhoA+EGFR-TKD group: RhoA protein + EGFR-TKD protein. EGFR was detected to determine whether or not it possesses GEF activity that regulates RhoA. Rheb group: individual Rheb proteins, Rheb+hDbs group: Rheb protein + hDbs protein. The presence or absence of GEF activity that regulates Rheb was detected. Rheb+EGFR-TKD group: Rheb protein + EGFR-TKD protein. The EGFR group detects whether or not it has GEF activity that regulates Rheb.

[0088] The kit contains the GEF domain of human RhoA protein and human Dbs (hDbs), where hDbs can facilitate the conversion of RhoA from GDPase (inactive state) to GTPase (active state). Therefore, GEF activity can be evaluated by detecting the binding status of the fluorescent nucleotide analog mant-GTP to the activated state of RhoA. When mant-GTP binds to the activated GTPase, the fluorescence intensity of mant-GTP (excitation wavelength Ex: 485±20 nm, emission wavelength Em: 535±25 nm) increases significantly. That is, the stronger the relative fluorescence between bound mant-GTP and free mant-GTP, the stronger the GEF activity. Therefore, this method can be used to reflect the GEF activity of a protein.

[0089] 2. Experimental Results As shown in Figure 3, the relative fluorescence intensities of bound mant-GTP and free mant-GTP in the Rheb+EGFR-TKD group were similar to those of the positive control group (RhoA+hDbs group), indicating that EGFR-TKD can promote Rheb nucleotide exchange in vitro, that is, that EGFR-TKD has a GEF function that modulates and controls Rheb activity. However, the relative fluorescence intensity between bound mant-GTP and free mant-GTP in the RhoA+EGFR-TKD group and the Rheb+hDbs group was weak. This indicates that EGFR-TKD could not promote nucleotide exchange of RhoA, and hDbs could not promote nucleotide exchange of Rheb. In other words, EGFR-TKD does not have a GEF function to regulate and control RhoA activity.

[0090] Therefore, EGFR can function as a GEF for Rheb, meaning that EGFR exhibits GEF activity.

[0091] Example 4. Prediction of EGFR-Rheb binding using AlphaFold2 I. Experimental Method We will use AlphaFold2-multimer on Google's laboratory servers to model the structure of the EGFR-TKD / Rheb complex, and then analyze and display the predictive model using the PyMOL molecular graphics system provided by Schrodinger.

[0092] 2. Experimental Results The pLDDT score was approximately 84.4, and the results are shown in Figure 4, indicating that the model obtained by AlphaFold2-multimer shows good docking between EGFR-TKD and Rheb.

[0093] Based on the evaluation of the docking model, it is suggested that glutamate at position 804 on EGFR (E804) may be an important amino acid site through which EGFR exerts GEF activity.

[0094] To test this hypothesis, subsequent related studies were conducted to confirm that glutamate at position 804 on EGFR is a crucial site for GEF activity.

[0095] Example 5. Glutamate at position 804 on EGFR, an important GEF active site. I. Experimental Method 1. Construction of the EGFR-E804K mutant Site-directed mutagenesis technology is used to mutate glutamate (E) at position 804 on EGFR to lysine (K). For specific steps, refer to the site-directed mutagenesis method in Example 2.

[0096] 2. Construction of Fusion Protein Plasmids We achieved targeting of the lysosome-localized fusion protein by affixing different EGFR-TKD mutants (WT, KD, or E804K) to the C-terminus of the LAMP2-V5 fusion protein.

[0097] The specific procedure is as follows: The sequences of LAMP2 and different EGFR-TKD (WT, KD, E804K) are cloned, and a LAMP2-V5-EGFR-TKD recombinant plasmid is constructed using the V5 tag as the connecting segment. After all plasmids are constructed, the accuracy of the sequences is confirmed by nucleotide sequencing.

[0098] 3. IP Experiment As described in the IP method in Example 2, in short, HEK293T cells that stably overexpress SFB-Rheb were transiently transfected with either pSIN or pSIN-EGFR-WT / KD / E804K-HA plasmids as shown in the figure, and IP experiments were performed to detect the binding status of different forms of EGFR (WT, KD, or E804K) to Rheb. The results are shown in Figure 5A.

[0099] 4. In vitro nucleotide exchange experiment The test method was the same as the in vitro nucleotide exchange experiment in Example 3. An in vitro nucleotide exchange experiment was performed using the RhoGEF Exchange Assay Kit (Cytoskeleton, BK100) to detect the ability of different forms of EGFR (WT or E804K) to promote Rheb nucleotide exchange, i.e., GEF activity. The results are shown in Figure 5B.

[0100] 5. WB experiment In HEK293T cells, LAMP2-V5 or LAM2-V5-EGFR-TKD-WT / KD / E804K was stably overexpressed, and after confirming successful expression, the cells were subjected to 24-hour serum starvation, and the activation status of mTORC1 was detected. The results are shown in Figure 5C.

[0101] 2. Experimental Results As shown in Figure 5A, compared to the EGFR-WT and EGFR-KD groups, no protein band was detected in the EGFR-E804K group, indicating that EGFR with a glutamate mutation at position 804 cannot interact with Rheb. This suggests that the glutamate at position 804 (E804) on EGFR is a crucial site for interaction between EGFR and Rheb.

[0102] As shown in Figure 5B, the relative fluorescence intensity of the EGFR-TKD-E804K group was weaker compared to the Rheb+EGFR-TKD-WT group. This indicates that EGFR with a glutamate mutation at position 804 could not promote in vitro nucleotide exchange of Rheb. In other words, the glutamate at position 804 (E804) on EGFR is an important site for EGFR's GEF activity.

[0103] As shown in Figure 5C, LAMP2-V5-EGFR-TKD-WT and LAMP2-V5-EGFR-TKD-KD both effectively activate mTORC1 after target localization to the lysosome surface, but LAMP2-V5-EGFR-TKD-E804K fails to activate mTORC1 after target localization to the lysosome surface. This further indicates that glutamate at position 804 on EGFR (E804) is an important site for EGFR to regulate mTORC1.

[0104] The results above clearly demonstrate that glutamate at position 804 on EGFR (E804) is an important site that exerts GEF activity and, consequently, regulates mTORC1 activation.

[0105] Example 6. PC9 cells that cannot activate mTORC1 and inhibit tumor growth by knocking in the endogenous EGFR-E804K gene. I. Experimental Method 1. Construction of PC9 cells by gene knock-in of endogenous EGFR-E804K. We design EGFR-targeting sgRNAs and screen for highly efficient sequences, while simultaneously constructing donor DNA templates containing the E804K mutation. By constructing the donor DNA templates in a pSIN vector (without the EF1α promoter) and integrating and encoding EGFP and the upstream internal ribosome entry site (IRES), we improve the efficiency of positive cloning screening.

[0106] sgRNA and donor DNA templates were co-transfected into parental PC9 cells. Approximately three days after transfection, Alexa Fluor-488-positive single cells were sorted using a MoFloAstrios EQ cell sorter (Beckman), and positive cloning was verified by PCR and Sanger sequencing to confirm the construction of PC9 cells by endogenous EGFR-E804K gene knock-in.

[0107] 2. Detection of tumor cell proliferation The proliferation capacity of parental PC9 cells (parental group) and EGFR-E804K gene knock-in PC9 cells (E804K group) will be evaluated using CCK8 experiments, cloning experiments, and EdU (Beyotime, C0071) experiments, respectively.

[0108] 3. Subcutaneous cancer-bearing experiments in nude mice The animal experiments of this invention have been approved by the Animal Research Committee of the Center for Tumor Prevention and Treatment, Sun Yat-sen University (Approval Number L025501202410005).

[0109] We purchased 4-week-old female BALB / c nude mice weighing 18-20g, originating from Zhuhai BesTest Bio-Tech Co., Ltd. (Zhuhai City, China).

[0110] The mice were randomly divided into two groups of 6 each. To conduct a tumor growth test, each group was divided into 5 × 10⁻¹⁴⁻¹ 6These parental PC9 cells or EGFR-E804K gene knock-in PC9 cells were subcutaneously injected into the axilla of mice, and these were designated as the parental group and the E804K group, respectively. Starting one week after injection, the tumor volume was measured once every three days using a caliper, and the volume was calculated using the formula V(volume) = (width). 2 The formula is (x length × π) / 6. After growing for 5 weeks, the mice were euthanized with 100% CO2 at a flow rate of 30-70% of the chamber volume per minute. The tumors were collected, and their volume and weight were measured using calipers and an electronic balance, respectively. The results are shown in Figures 6E, 6F, and 6G.

[0111] 2. Experimental Results As shown in Figure 6A, compared to parental PC9 cells (parental group), the expression of phosphorylated EGFR (p-Y1068-EGFR) in EGFR-E804K knock-in PC9 cells (E804K group) remained unchanged, but the expression of phosphorylated S6K1 (p-T389-S6K1) was significantly reduced. This indicates that EGFR-E804K knock-in PC9 cells (E804K group) maintain normal EGFR kinase activity but are unable to activate mTORC1. Furthermore, it is demonstrated that glutamate at position 804 on EGFR (E804) is an important site for EGFR to regulate mTORC1.

[0112] As shown in Figures 6B (CCK8 experiment), 6C (cloning experiment), and 6D (EdU experiment), the proliferative capacity of EGFR-E804K gene knock-in PC9 cells (E804K group) was significantly reduced compared to the parental PC9 cells (parental group). This indicates that inhibition of glutamate at position 804 on EGFR (E804) (i.e., inactivated EGFR) can effectively inhibit tumor growth.

[0113] As shown in Figures 6E, 6F, and 6G, compared to a nude mouse subcutaneous tumor model injected with parental PC9 cells (parental group), the tumor volume and weight formed in the nude mouse subcutaneous tumor model injected with EGFR-E804K gene knock-in PC9 cells (E804K group) were all significantly reduced. Furthermore, this demonstrates that inhibition of glutamate at position 804 on EGFR (E804) can effectively inhibit tumor growth.

[0114] In summary, PC9 cells in which endogenous EGFR-E804K has been gene-knocked in (i.e., EGFR-inactivated PC9 cells) cannot activate mTORC1, and therefore tumor growth is inhibited.

[0115] Example 7. Combined inhibitory effect of the small molecule compound BIE Gi-1 on EGFR kinase and GEF function I. Experimental Method 1. Screening and determination of small molecule compounds that inhibit EGFR kinase activity. According to research reports, 7-oxopyridonopyrimidine compounds are novel EGFR tyrosine kinase inhibitors that have the potential to overcome the problems of low selectivity for wild-type cells and toxic side effects that conventional EGFR inhibitors have.

[0116] As described in the in vitro nucleotide exchange experiment in Example 3, the above 7-oxopyridonopyrimidine compounds were screened using the RhoGEF Exchange Assay Kit (Cytoskeleton, BK100), and the results are shown in Figures 7A and 7B.

[0117] The group divisions are as follows: Rheb group: Individual Rheb proteins. DMSO (Rheb + EGFR-TKD) group: Rheb + EGFR-TKD, with DMSO added; i.e., the control group. BIE Gi-1 (Rheb + EGFR-TKD) group: Rheb + EGFR-TKD, BIE Gi-1 added, i.e., drug-added group.

[0118] 2. IP Experiment The IP method is the same as in Example 2. An immunoprecipitation experiment is performed on PC9 cells. Cells from the control group and the drug-treated group are lysed with an appropriate amount of IP lysis buffer for 30 minutes, centrifuged, and the supernatant is collected. 1 μg of the corresponding antibody and 50 μL of Protein A / G beads are added, and the mixture is incubated overnight at 4°C. The agarose bead-antigen-antibody complex is then collected, the supernatant is discarded, the mixture is washed three times with pre-cooled IP elution buffer, boiled at 100°C for 10 minutes, cooled, and Western blotting detection is performed. This detects whether BIE Gi-1 can inhibit EGFR-Rheb binding. The results are shown in Figure 7C.

[0119] 3. Drug addition experiment. PC9 and NCI-H1975 cells were treated with drugs, specifically DMSO or BIE Gi-1, and then the activation status of mTORC1 was detected. The results are shown in Figures 7D and 7E.

[0120] 4. IC 50 Measurement method A suitable amount of target cells was inoculated into a 96-well plate and incubated at 37°C and 5% CO2 for 24 hours to prepare a series of drug solutions of different concentrations. At least three parallel wells were prepared for each concentration, and a control group containing only the same amount of culture medium was simultaneously prepared. 72 hours after drug treatment, 10 μL of CCK8 reagent was added to each well and incubated in an incubator for 1 hour. The absorbance (OD) of each well was measured at 450 nm using a microplate reader, and the data was processed using GraphPad Prism 9.0 software to calculate the cell viability at each concentration (formula: viability = [(treatment group OD - blank OD) / (control group OD - blank OD)] × 100%). Nonlinear regression analysis was performed to calculate IC. 50 The value, i.e., the drug concentration required to inhibit cell proliferation by 50%, was determined, and the results are shown in Figure 7F.

[0121] 2. Experimental Results As shown in Figure 7B, compared to DMSO, the relative fluorescence of bound mant-GTP and free mant-GTP was weaker after adding the small molecule compound (BIEGi-1) shown in Figure 7A. This indicates that BIE Gi-1 can significantly inhibit the Rheb nucleotide exchange capacity mediated by EGFR, or in other words, BIE Gi-1 can inhibit the GEF activity of EGFR.

[0122] As shown in Figure 7C, the expression of Rheb-like proteins was significantly reduced in the BIE Gi-1 group compared to the DMSO group, indicating that BIE Gi-1 can block the interaction between EGFR and Rheb.

[0123] As shown in Figure 7D, in PC9 cells, the expression of phosphorylated S6K1 (p-T389-S6K1) protein was significantly reduced in the BIE Gi-1 group compared to the DMSO group, indicating that BIE Gi-1 can inhibit mTORC1 activation.

[0124] As shown in Figure 7F, in NCI-H1975 cells, the expression of phosphorylated S6K1 (p-T389-S6K1) protein was significantly reduced in the BIE Gi-1 group compared to the DMSO group. This indicates that non-small cell lung cancer cell lines with the EGFR-T790M drug resistance mutation (the T790M mutation is one of the important mechanisms of EGFR kinase inhibitor drug resistance) still maintain sensitivity to BIE Gi-1. In other words, BIE Gi-1 can inhibit mTORC1 activation in EGFR kinase drug-resistant cell lines.

[0125] As shown in Figure 7E, in non-small cell lung cancer cell lines PC9, HCC827, and NCI-H1975, which have EGFR mutations, BIE Gi-1 IC 50 The values ​​were 17 nM, 20 nM, and 24 nM, respectively, indicating that BIE Gi-1 has excellent ability to inhibit EGFR-driven tumor cell proliferation.

[0126] In summary, in addition to its known regulating EGFR tyrosine kinase activity, EGFR also possesses a GEF function that promotes Rheb nucleotide exchange, which is primarily dependent on the glutamate site at position 804 on EGFR, thus further strengthening the association between EGFR and the mTORC1 signaling pathway.

[0127] Based on the above findings, combined targeted inhibition of EGFR kinase activity and GEF activity using inhibitors, small molecule compounds, nanomaterials, or gene editing technologies can be implemented as a novel strategy for treating at least some EGFR-driven tumors. The treatment of EGFR-driven tumors offers new technological ideas and methods, while simultaneously laying a solid experimental foundation for the development of highly effective antitumor drugs, possessing remarkable applicability and important clinical significance.

[0128] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, persons skilled in the art can make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. Uses of EGFR inhibitors, The use is characterized by being used in the preparation of pharmaceutical compositions for treating diseases mediated by guanylate exchange factor (GEF) activity or diseases co-mediated by guanylate exchange factor (GEF) activity and EGFR tyrosine kinase activity.

2. The aforementioned disease is characterized by being a cancer selected from the group consisting of lung cancer, colorectal cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, prostate cancer, melanoma, leukemia, and glioblastoma. The use described in claim 1.

3. The pharmaceutical composition is used to regulate abnormalities in Rheb nucleotide exchange, and preferably, the regulation is independent of regulating EGFR kinase activity or expression levels. The use described in claim 1.

4. The pharmaceutical composition is characterized by simultaneously regulating EGFR tyrosine kinase activity and GEF activity, and the regulation of GEF activity is independent of the regulatory control of EGFR tyrosine kinase activity. The use described in claim 1.

5. The EGFR inhibitor is characterized by being selected from the group consisting of small molecule compounds, nanomaterial drugs, gene editing technologies, protein antibodies, nucleic acid drugs, enzyme drugs, expression vectors, cell therapies, hormone drugs, peptide drugs, or combinations thereof. The use described in claim 1.

6. The EGFR inhibitor is characterized by being a small molecule compound. The use described in claim 5.

7. The EGFR inhibitor is characterized by being a small molecule compound represented by the following formula. The use described in claim 6. 【Chemistry 2】

8. A pharmaceutical composition, The pharmaceutical composition is characterized by comprising an EGFR tyrosine kinase inhibitor and a GEF activity inhibitor, or an EGFR tyrosine kinase / GEF activity dual-target inhibitor.

9. Uses of the pharmaceutical composition according to claim 8, It is used in the preparation of pharmaceutical compositions for treating tumors driven by EGFR or tumors that exhibit drug resistance to independent EGFR tyrosine kinase inhibitors. Preferably, the tumor is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, prostate cancer, melanoma, leukemia, and glioblastoma, and the use of the pharmaceutical composition according to claim 8.

10. A method for designing EGFR tyrosine kinase and GEF activity dual-target inhibitors, Including the following stage, (a) Provide a candidate molecule that can act simultaneously on the kinase domain of EGFR and the glutamic acid residue at position 804 of EGFR. (b) A method for designing a dual-target inhibitor of EGFR tyrosine kinase and GEF activity, characterized by measuring the ability of the candidate molecule to inhibit EGFR kinase and GEF activity.

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