Application of medicine capable of simultaneously targeting tyrosine kinase activity of EGFR (Epidermal Growth Factor Receptor) and GEF activity

Abstract

The invention provides application of an EGFR (epidermal growth factor receptor) inhibitor in preparation of a medicine for treating diseases mediated by the activity of a guanylate exchange factor (GEF) or jointly mediated by the activity of the guanylate exchange factor (GEF) and the activity of EGFR tyrosine kinase. The diseases are cancers selected from the following groups: lung cancer, colon cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, prostate cancer, melanoma, leukemia and glioblastoma. Specifically, the invention discovers that EGFR has a GEF function for promoting activation of Ras homologous protein (Rhb) besides traditional tyrosine kinase activity, the function does not depend on the tyrosine kinase activity and plays a key role by depending on a glutamic acid residue at the 804th site on an EGFR molecule, the Rhb-GEF activity of EGFR causes allosteric activation of a mammal rapamycin target protein complex 1 (mTORC1), and the activity of the EGFR causes allosteric activation of the mammal rapamycin target protein complex 1 (mTORC1). The tumor growth is finally promoted.

Description

Technical Field

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

[0002] Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein whose intracellular tyrosine kinase domain plays a crucial role in cell signaling. EGFR binds to growth factors, undergoes dimerization and autophosphorylation, and subsequently activates multiple downstream signaling pathways, regulating fundamental biological processes such as cell proliferation, differentiation, survival, and migration. Under normal physiological conditions, EGFR signaling is finely regulated to maintain cellular homeostasis.

[0003] In various malignant tumors (such as lung cancer, colon cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, and prostate cancer), EGFR is often abnormally activated due to overexpression, gene amplification, or specific mutations. This abnormal activation leads to continuous signal transmission by EGFR, thereby causing sustained activation of downstream signaling pathways. For example, continuous EGFR activation can indirectly and continuously stimulate the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, promoting protein synthesis, cell proliferation, and inhibiting autophagy, thus playing a key role in tumorigenesis and progression.

[0004] Current EGFR inhibitors (such as gefitinib and osimertinib) only target its intracellular kinase domain, leading to a high incidence of drug resistance. Studies have found that drug-resistant tumors may escape inhibition by activating the non-kinase-dependent EGFR pathway, but research on the non-kinase-dependent pathway is limited, failing to effectively address the problem of high EGFR resistance.

[0005] Therefore, there is an urgent need in this field to explore in depth the molecular mechanisms of tumors driven by EGFR, especially the mechanisms by which tumor cells develop resistance to drugs through the non-kinase-dependent pathway of EGFR. Revealing its potential regulatory mechanisms will not only help explain the reasons for the limited efficacy of existing treatments, but also provide a theoretical basis and new application prospects for developing more precise, efficient and safe combination anti-tumor therapy strategies. Summary of the Invention

[0006] To address the above problems, this invention provides the use of a drug that can simultaneously regulate EGFR tyrosine kinase activity and GEF activity.

[0007] In a first aspect of the invention, there is provided the use of an EGFR inhibitor for preparing a pharmaceutical composition for treating diseases mediated by guanylate exchange factor (GEF) activity or by a combination of guanylate exchange factor (GEF) activity and EGFR tyrosine kinase activity.

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

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

[0010] In a preferred embodiment, 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 embodiment, the pharmaceutical composition is also used to regulate abnormal nucleotide exchange of Rheb; preferably, the regulation is independent of regulation of abnormal EGFR kinase activity or expression.

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

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

[0014] In another preferred embodiment, the pharmaceutical composition regulates the nucleotide exchange of Rheb by modulating GEF activity, independent of the regulation of EGFR kinase activity.

[0015] In another preferred embodiment, the pharmaceutical composition modulates GEF activity by acting on the intracellular tyrosine kinase domain of EGFR, independent of the regulation of EGFR kinase activity.

[0016] In another preferred embodiment, the pharmaceutical composition regulates GEF activity by acting on the 804th glutamate residue of EGFR, independent of the regulation of EGFR kinase activity.

[0017] In another preferred embodiment, the EGFR inhibitor can cause a mutation in the 804th glutamate residue of EGFR.

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

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

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

[0021] In another preferred embodiment, the EGFR inhibitor acts simultaneously on the EGFR tyrosine kinase domain and the 804th glutamate residue of EGFR.

[0022] In another preferred embodiment, the action includes modifying gene regions associated with EGFR tyrosine kinase activity and gene regions associated with GEF activity of the 804th glutamate residue of EGFR.

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

[0024] In another preferred embodiment, the nanomaterial drug is a liposomal drug.

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

[0026] In another preferred embodiment, the enzyme drug is selected from the group consisting of: DNA enzymes, ribozymes, or combinations thereof.

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

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

[0029] In a preferred embodiment, the EGFR inhibitor is a small molecule compound represented by the following formula:

[0030]

[0031] A second aspect of the present invention provides a drug combination comprising an EGFR tyrosine kinase inhibitor and a GEF activity inhibitor, or a dual-target inhibitor of EGFR tyrosine kinase and GEF activity.

[0032] In another preferred embodiment, the drug combination can modulate the nucleotide exchange of Rheb.

[0033] A third aspect of the invention provides the use of a pharmaceutical combination as described in the second aspect of the invention for preparing a pharmaceutical composition for treating tumors driven by EGFR or resistant to a single EGFR tyrosine kinase inhibitor.

[0034] 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.

[0035] A fourth aspect of the present invention provides a method for designing a dual-target inhibitor of EGFR tyrosine kinase and GEF activity, comprising the steps of:

[0036] (a) Provide a candidate molecule that can act simultaneously on the kinase domain of EGFR and the 804th glutamate residue of EGFR;

[0037] (b) Determine the ability of the candidate molecules to inhibit EGFR kinase and GEF activity.

[0038] A fifth aspect of the present invention provides a method for screening for the treatment of diseases mediated by GEF activity or by a combination of GEF activity and EGFR tyrosine kinase activity, comprising the following steps:

[0039] (A) Administering the candidate drug to an animal model of a disease associated with EGFR tyrosine kinase activity and GEF activity;

[0040] (B) Detect the nucleotide exchange capacity of Rheb in the above animal models, and the drug that reduces the nucleotide exchange capacity of Rheb is the target drug.

[0041] A sixth aspect of the invention provides a method for treating a disease mediated by GEF activity or by a combination of GEF activity and EGFR tyrosine kinase activity, the method comprising the step of administering to a subject in need a combination of drugs comprising an EGFR tyrosine kinase inhibitor and a GEF activity inhibitor or a dual-target inhibitor of EGFR tyrosine kinase / GEF activity.

[0042] The subjects were selected from the following groups: humans, mice, rats, guinea pigs, pigs, dogs, monkeys, and orangutans.

[0043] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0044] Figure 1 The study showed that preventing activated EGFR from localizing to the lysosomal surface significantly inhibits mTORC1 activation. Figure 1In the study, A: Activated EGFR can be localized on the lysosomal surface; B: After using endocytosis inhibitors or cathepsin S inhibitors (late endosome-lysosomal transport inhibitors), mTORC1 activation was significantly inhibited, while EGFR kinase activity was unaffected.

[0045] Figure 2 This demonstrates a direct interaction between EGFR and Rheb, and that this interaction is independent of EGFR kinase activity. Figure 2 In the diagram, A: The interaction between EGFR and Rheb is independent of the kinase activity of EGFR; B: Schematic diagram of different intracellular truncated forms of EGFR; C: EGFR mainly interacts with Rheb through its intracellular tyrosine kinase domain; D: The intracellular tyrosine kinase domain of EGFR can bind directly to Rheb.

[0046] Figure 3 The results showed that EGFR-TKD promotes nucleotide exchange of Rheb.

[0047] Figure 4 The diagram shows a schematic of the structural model obtained by predicting the binding of the EGFR-Rheb complex based on AlphaFold2.

[0048] Figure 5 The effect of the glutamate site at position 804 of EGFR on its GEF activity was shown. Figure 5 In the above, A: the EGFR-E804K mutant loses its ability to interact with Rheb; B: the EGFR-TKD-E804K mutant cannot promote nucleotide exchange with Rheb; C: the lysosomal localized LAMP2-V5-EGFR-TKD-E804K fusion protein cannot activate mTORC1, while wild-type (WT) or kinase-inactive (KD) EGFR-TKD can effectively activate mTORC1.

[0049] Figure 6 This study demonstrates the effects of CRISPR-Cas9-mediated gene editing to knock in the EGFR-E804K mutation on the mTORC1 signaling pathway and cell proliferation in PC9 lung cancer cells. Figure 6In Figure E, A: EGFR-E804K knock-in PC9 cells cannot activate mTORC1, but their EGFR kinase activity is unaffected; B: CCK8 assay to assess the proliferation capacity of parental PC9 cells and EGFR-E804K knock-in cells; C: Clonogenic assay to assess the clonogenic capacity of parental PC9 cells and EGFR-E804K knock-in cells; D: EdU assay to detect the DNA synthesis capacity of parental PC9 cells and EGFR-E804K knock-in cells; E: Schematic diagram of parental PC9 cells and EGFR-E804K knock-in cells in mouse subcutaneous tumors; F: Volume changes of subcutaneous tumors in Figure E; G: Weight changes of subcutaneous tumors in Figure E.

[0050] Figure 7 The combined inhibitory effect of the small molecule compound BIEGe-1 on EGFR kinase and GEF function was demonstrated. Figure 7 In the diagram, A: Schematic diagram of the chemical structure of the small molecule compound BIEGe-1; B: BIEGe-1 inhibits EGFR-mediated Rheb nucleotide exchange; C: BIEGe-1 can disrupt the interaction between EGFR and Rheb; D: In PC9 cells, BIEGe-1 can inhibit the activation of mTORC1; E: In NCI-H1975 cells, BIEGe-1 can inhibit the activation of mTORC1; F: BIEGe-1 can inhibit the proliferation of tumor cells driven by EGFR. Detailed Implementation

[0051] Through long-term and in-depth research and extensive screening, the inventors discovered for the first time that EGFR, in addition to its traditional kinase activity, also possesses GEF activity dependent on the 804th glutamate site on its protein, thereby promoting nucleotide exchange of Rheb (Ras homolog), which in turn activates mTORC1 and promotes tumor growth. Furthermore, simultaneously targeting and inhibiting both the tyrosine kinase activity and GEF activity of EGFR can effectively suppress EGFR-driven tumor growth. Based on this, the inventors completed this invention.

[0052] Treatment strategies that simultaneously target EGFR kinase activity and GEF activity

[0053] The inventors discovered that regulating EGFR can simultaneously modulate EGFR kinase activity and GEF activity, and that the GEF activity of EGFR depends on its 804th glutamate residue, which activates mTORC1 by promoting nucleotide exchange of Rheb. Based on the above findings, this invention further proposes a combined therapeutic strategy that simultaneously inhibits EGFR kinase activity and GEF activity to achieve more effective treatment of tumors at least partially driven by EGFR.

[0054] The EGFR kinase activity is a sustained signal transduction caused by EGFR overexpression, gene amplification, or specific mutations.

[0055] The specific mutations include, but are not limited to, EGFRvIII mutants, L858R point mutations, or in- or out-of-frame deletion mutations of part-19.

[0056] This invention provides a novel strategy for treating tumors, the core of which lies in simultaneously inhibiting EGFR kinase activity and GEF activity. The tumor is at least partially driven by EGFR, preferably including, but not limited to, lung cancer, colon cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, and prostate cancer.

[0057] The specific implementation scheme of the novel strategy for treating tumors described in this invention includes: using one or more treatment methods, such as inhibitors, small molecule compounds, nanomaterial drugs (e.g., liposomal drugs), and gene editing technology, to achieve simultaneous targeted inhibition of EGFR kinase activity and GEF activity.

[0058] The small molecule compound has the ability to penetrate cell membranes and can interact with both the kinase domain of EGFR and the active binding site of GEF, thereby improving the specificity and efficacy of treatment.

[0059] Among them, the nanomaterial drugs, after surface modification, can specifically target the tumor microenvironment, enhancing their affinity and selectivity for diseased tissues.

[0060] The nanomaterial-based drugs bind drugs to liposomes, albumin, and polymer micelles, allowing them to cross biological barriers and continuously release drugs to maintain appropriate blood drug concentrations.

[0061] The gene editing technology (such as the CRISPR / Cas9 system) can fundamentally regulate related pathological processes by knocking out or modifying gene regions related to EGFR kinase activity and GEF activity.

[0062] This invention also provides the clinical or laboratory application of the above-described strategy of simultaneously targeting EGFR kinase activity and GEF activity. The application includes administering one or more of the above-described treatments to a subject (e.g., a mammalian or tumor cell line) to inhibit EGFR kinase activity and GEF activity, further inhibiting the mTORC1 signaling pathway, thereby blocking tumor cell proliferation and ultimately inhibiting tumor growth.

[0063] The subjects may include humans, mice, rats, guinea pigs, pigs, dogs, monkeys, orangutans, etc.

[0064] The tumor cells include lung cancer, colon cancer, and glioma, which are driven by abnormal EGFR activity.

[0065] Through a series of experiments, the inventors demonstrated that EGFR can interact directly with Rheb, and that this interaction mainly depends on the glutamate residue at position 804 of EGFR and is unrelated to its kinase activity.

[0066] Further experiments showed that combining gene knock-in of the EGFR-E804K mutant or the application of small molecule compounds to achieve inhibition can effectively suppress nucleotide exchange of Rheb and mTORC1 activation, thereby inhibiting tumor cell proliferation.

[0067] In summary, this invention reveals for the first time that EGFR, in addition to its traditional kinase activity, also possesses a GEF function that regulates Rheb nucleotide exchange, and identifies glutamic acid at position 804 as an important functional site for its GEF activity.

[0068] Based on this discovery, this invention proposes a therapeutic strategy that simultaneously targets EGFR kinase activity and GEF activity, providing a new theoretical basis and technical means for developing highly effective treatments for tumors that are at least partially driven by EGFR.

[0069] The main advantages of this invention are:

[0070] 1. This invention expands our understanding of the molecular mechanism of action of EGFR, namely, in addition to its traditional tyrosine kinase activity, EGFR also has a GEF function that promotes the activation of Ras homolog (Rheb). In other words, EGFR can simultaneously inhibit the kinase activity and GEF activity of EGFR, and the GEF activity of EGFR depends on its 804th glutamate residue, thereby activating mTORC1 by promoting nucleotide exchange of Rheb, and ultimately promoting tumor growth.

[0071] 2. This invention provides new targets for cancer treatment. For example, existing EGFR inhibitors mainly target the kinase domain, while drug-resistant tumors may rely on the GEF function of EGFR to survive. Therefore, developing inhibitors targeting the GEF domain could provide alternative treatment strategies.

[0072] 3. This invention is beneficial for the development of novel drugs. For example, based on the GEF exchange experiment, a high-throughput screening platform for Rheb-GEF activity against EGFR can be developed to accelerate drug discovery.

[0073] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, were generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0074] Example 1. Preventing activated EGFR from localizing on the lysosomal surface can significantly inhibit mTORC1 activation.

[0075] I. Experimental Methods

[0076] 1. Cell lysis and lysosomal immunoprecipitation (Lyso-IP)

[0077] Lysosomes were isolated from PC9 non-small cell lung cancer cell lines that stably expressed the HA tag TMEM192 (a lysosomal marker protein) and PC9 cells that stably expressed the FLAG tag TMEM192, respectively.

[0078] First, cells were washed with PBS, scraped, and centrifuged to collect the cell pellet. The pellet 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 homogenized. The homogenate was centrifuged at 1,000 g for 3 minutes at 4°C, and the supernatant was collected. The supernatant was incubated with 20 μL of anti-HA magnetic beads (Beyotime) under rotating conditions for 1 hour. Finally, the magnetic beads were washed 6 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 Western blotting. The results are as follows: Figure 1 As shown in Figure A.

[0079] 2. Cell lysis and Western blot (WB) of proteins

[0080] Non-small cell lung cancer cell lines PC9 and HCC827 carrying EGFR exon 19 deletion mutations, and human lung adenocarcinoma cell lines NCI-H1975 carrying EGFR T790M (drug-resistant mutation) and L858R mutations, were treated with solvent DMSO, EGFR endocytosis inhibitor Dyngo-4a, or cathepsin C inhibitor (late endosome-lysosome transport inhibitor) LY3000328, respectively.

[0081] Cells treated according to the above methods were collected and lysed for 20 minutes using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. The cells were then centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was collected. Protein concentration was quantified using the Coomassie Brilliant Blue method. The obtained protein samples were first separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After membrane blocking, the membrane was incubated with primary and secondary antibodies sequentially. Finally, a chromogenic substrate was added, and the expression level of the target protein was detected under a suitable imaging system. The results are shown below. Figure 1 As shown in B.

[0082] This experiment was used to detect the expression of target proteins and their phosphorylation levels in samples, with S6K1 phosphorylation (p-T389-S6K1) level used as an indicator of mTORC1 activation.

[0083] II. Experimental Results

[0084] like Figure 1 As shown in Figure A, the Lyso-IP results showed that EGFR and the lysosomal marker protein LAMP1 could be detected simultaneously in the PC9 cell line of TMEM192 that stably expressed the HA tag, indicating that activated EGFR can be localized on the lysosomal surface.

[0085] like Figure 2 As shown in Figure B, compared with the DMSO-treated group, the expression of phosphorylated S6K1 (p-T389-S6K1) was significantly reduced in cells treated with the EGFR endocytosis inhibitor Dyngo-4a or the cathepsin C inhibitor (late endosome-lysosome transport inhibitor) LY3000328, but the expression of phosphorylated EGFR (p-Y1068-EGFR) was not different. This indicates that inhibiting EGFR endocytosis or inhibiting EGFR localization on the lysosomal surface can effectively inhibit mTORC1 activation, but does not affect the activity of EGFR kinase.

[0086] Existing research indicates that abnormally activated EGFR enhances its endocytosis. In normal cells, EGFR enters lysosomes after endocytosis and is degraded, thereby terminating signal transduction. However, in tumor cells, abnormal endocytosis not only inhibits EGFR degradation but may also retain some active EGFR in specific intracellular regions, thus forming a continuous signaling "signaling platform" that indirectly stimulates the mTORC1 signaling pathway.

[0087] The experimental results of this application show that preventing EGFR endocytosis or preventing EGFR localization on the lysosomal surface can inhibit mTORC1 activation without changing EGFR kinase activity, suggesting that there are other non-kinase-dependent regulatory mechanisms between EGFR and mTORC1.

[0088] Example 2. EGFR and Rheb interact directly, and the kinase activity is independent of EGFR.

[0089] I. Experimental Methods

[0090] 1. Immunoprecipitation (IP)

[0091] 1.1 Cell Culture and Preparation:

[0092] 1.1.1 HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 humidified incubator.

[0093] 1.1.2. One day before transfection, HEK293T cells were seeded at an appropriate density in 6-well cell culture plates to ensure that the cells reached the appropriate degree of confluence at the time of transfection.

[0094] 1.2 Construction of the carrier

[0095] The pSIN-EGFR-WT-HA plasmid was constructed using conventional PCR amplification techniques. Additionally, using the wild-type gene plasmid as a PCR template, appropriate mutation primers were designed and amplified according to experimental requirements. The PCR products were recovered by agarose gel electrophoresis, then ligated and transformed. Finally, sequencing was used to verify the success of the mutation.

[0096] 1.3 Instantaneous transfection

[0097] Following the instructions for Lipofectamine 3000, transfect the plasmid into HEK293T cells as follows:

[0098] Vector group: transfected pSIN plasmid;

[0099] WT group: transfected with pSIN-EGFR-WT-HA plasmid;

[0100] KD (EGFR kinase inactivation type) group: Following the site-directed mutagenesis method, the 745th lysine in the EGFR-WT-HA plasmid was mutated to alanine (K745A) to construct the EGFR-KD-HA plasmid, which was then transfected.

[0101] 1.4 Experimental Procedure

[0102] The cells were lysed with an appropriate amount of IP lysis buffer for 30 minutes, centrifuged, and the supernatant was collected. 20 μL of agarose beads containing HA antibody was added, and the mixture was incubated overnight at 4°C. Subsequently, the agarose bead-antigen-antibody complex was collected, the supernatant was discarded, and the cells were washed three times with pre-chilled 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, Western blotting was performed, and the results were as follows: Figure 2 As shown in Figure A.

[0103] 2. Truncation and IP Construction

[0104] 2.1 Using conventional genetic engineering techniques, construct the following truncated somatic plasmids (e.g. Figure 2 As shown in B), that is:

[0105] The WT-ICD group: pSIN-EGFR-WT-ICD-V5, containing the wild-type EGFR intracellular domain;

[0106] KD-ICD group: pSIN-EGFR-KD-ICD-V5, containing the intracellular domain of EGFR with K745A mutation;

[0107] The WT-TKD group: pSIN-EGFR-WT-TKD-V5, containing the wild-type EGFR intracellular tyrosine kinase domain;

[0108] KD-TKD group: pSIN-EGFR-KD-TKD-V5, containing the K745A-mutated EGFR intracellular tyrosine kinase domain;

[0109] RD group: pSIN-EGFR-RD-V5, containing the EGFR regulatory domain but not the EGFR intracellular tyrosine kinase domain.

[0110] 2.3 Construction of stable strains

[0111] HEK293T cell lines overexpressing SFB-Rheb were constructed using conventional lentiviral infection methods. Following successful construction, the HEK293T cells overexpressing SFB-Rheb were seeded at an appropriate density in 6-well cell culture plates one day before transfection to ensure adequate cell confluence at transfection.

[0112] 2.4 Instantaneous Transfection and IP

[0113] Following the Lipofectamine 3000 instructions, the above plasmids were transfected into HEK293T cells overexpressing SFB-Rheb. Thirty-six hours after transfection, the cells were lysed for 30 minutes with an appropriate amount of IP lysis buffer, centrifuged, and the supernatant was collected. 20 μL of agarose beads containing V5 antibody was added, and the cells were incubated overnight at 4°C. Subsequently, the agarose bead-antigen-antibody complex was collected, the supernatant was discarded, and the cells were washed three times with pre-chilled 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, Western blotting was performed to detect the binding of EGFR to Rheb. The results are as follows: Figure 2 As shown in C.

[0114] 3. Protein purification and pull-down assay

[0115] 3.1 Protein purification

[0116] GST and GST-Rheb proteins were induced to express using a prokaryotic system; EGFR protein was induced to express using an insect cell system.

[0117] The specific procedures include: constructing recombinant plasmids and then transferring them into the corresponding host cells; lysing the cells by high-pressure or ultrasonic disruption; separating the supernatant and cell debris by centrifugation; and then purifying the protein solution using an affinity chromatography column.

[0118] 3.2 Pull-down assay

[0119] The groups are as follows:

[0120] GST group: GST protein (tag protein) purified via a prokaryotic system.

[0121] GST-Rheb group: GST-Rheb protein purified via a prokaryotic system.

[0122] EGFR-TKD-WT group: EGFR-TKD-WT protein purified using an insect cell system.

[0123] Take the pre-washed and equilibrated GST-beads and add the purified protein solution to be detected as shown in the figure. Incubate the mixture at 4°C for 2 hours to ensure sufficient binding of the protein to the GST-beads. After incubation, centrifuge at 5000 rpm for 30 seconds, discard the supernatant, and repeat the centrifugation and washing twice to collect the beads. Wash the beads three times with pre-cooled IP elution buffer to remove non-specific binders. After adding an appropriate amount of loading buffer, boil the sample at 100°C for 10 minutes to fully denature it. Subsequently, stain the boiled sample with Coomassie Brilliant Blue to detect the target protein. The results are shown in the figure. Figure 2 As shown in D.

[0124] II. Experimental Results

[0125] Existing research has found that Rheb (Ras Homolog Enriched in Brain) on lysosomes is a key protein for activating mTORC1, but whether there is a link between EGFR and Rheb is unknown.

[0126] like Figure 2 As shown in Figure A, in HEK293T cells, exogenous overexpression of EGFR (WT group) or EGFR kinase inactivation (KD group) can both bind to endogenous Rheb, indicating that the interaction between EGFR and Rheb does not depend on the kinase activity of EGFR.

[0127] like Figure 2As shown in B and 2C, in HEK293T cells overexpressing SFB-Rheb, all cells containing the EGFR intracellular tyrosine kinase domain (WT-ICD group, KD-ICD group, WT-TKD group, and KD-TKD group) can interact with SFB-Rheb, while cells containing only the EGFR regulatory domain but not the EGFR intracellular tyrosine kinase domain (RD group) cannot bind. This indicates that EGFR mainly binds to Rheb through the intracellular tyrosine kinase domain (TKD).

[0128] like Figure 2 As shown in Figure D, in the in vitro pull-down experiment, 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.

[0129] Based on the above data, it is confirmed that EGFR mainly interacts with Rheb directly through TKD, and this process does not depend on the kinase activity of EGFR.

[0130] Example 3. EGFR can act as a GEF for Rheb (i.e., EGFR has a GEF function that regulates Rheb activity).

[0131] I. Experimental Methods: In vitro nucleotide exchange experiment

[0132] Rheb is a small G protein whose activity is regulated by guanylate exchange factor (GEF). Specifically, Rheb is in an activated state when it binds to GTP, which can activate downstream molecules. It becomes inactive when it is hydrolyzed to GDP. GEF can promote the transition of Rheb from the GDP state (inactivated state) to the GTP state (activated state). In other words, GEF can promote the nucleotide exchange of Rheb.

[0133] To determine whether EGFR possesses the ability to regulate the guanylate exchange factor (GEF) activity of Rheb, the applicant conducted in vitro nucleotide exchange experiments using the RhoGEF Exchange Assay kit (Cytoskeleton, BK100). The groupings are as follows:

[0134] RhoA group: RhoA protein alone; RhoA is also a small G protein, and its activity is regulated by GEF;

[0135] RhoA+hDbs group: RhoA protein + hDbs protein; hDbs can promote the transition of RhoA from GDPase (inactive state) to GTPase (activated state), therefore the GEF domain of hDbs can be used as a positive control GEF;

[0136] RhoA+EGFR-TKD group: RhoA protein + EGFR-TKD protein; detection of whether EGFR has the ability to regulate the GEF activity of RhoA;

[0137] Rheb group: Rheb protein alone;

[0138] Rheb+hDbs group: Rheb protein + hDbs protein; detection of whether hDbs has the ability to regulate the GEF activity of Rheb;

[0139] Rheb+EGFR-TKD group: Rheb protein + EGFR-TKD protein; detection of whether EGFR has the ability to regulate the GEF activity of Rheb.

[0140] The kit contains the GEF domains of human RhoA protein and human Dbs (hDbs). hDbs can promote the transition of RhoA from the GDPase (inactive state) to the GTPase (activated state). Therefore, GEF activity is assessed by detecting the binding of the fluorescent nucleotide analog mant-GTP to the activated RhoA state. Once 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) will significantly increase. In other words, 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 proteins.

[0141] II. Experimental Results

[0142] like Figure 3 As shown, the relative fluorescence intensity of bound mant-GTP and free mant-GTP in the Rheb+EGFR-TKD group was similar to that in the positive control group (RhoA+hDbs group), indicating that EGFR-TKD can promote the nucleotide exchange of Rheb in vitro, that is, EGFR-TKD has the GEF function of regulating Rheb activity.

[0143] However, the relative fluorescence intensity of bound mant-GTP and free mant-GTP was weak in the RhoA+EGFR-TKD group and the Rheb+hDbs group, indicating that EGFR-TKD cannot promote nucleotide exchange of RhoA, and hDbs cannot promote nucleotide exchange of Rheb. That is, EGFR-TKD does not have the GEF function to regulate RhoA activity.

[0144] Therefore, EGFR can act as the GEF of Rheb, meaning that EGFR exhibits GEF activity.

[0145] Example 4. Predicting the binding of EGFR and Rheb using AlphaFold2

[0146] I. Experimental Methods

[0147] The structure of the EGFR-TKD / Rheb complex was modeled using AlphaFold2-multimer on Google Labs servers, and then... The company's PyMOL molecular graphics system is used to analyze and display prediction models.

[0148] II. Experimental Results

[0149] The pLDDT score was approximately 84.4 points, as shown in the following figures. Figure 4 As shown, the model obtained by AlphaFold2-multimer demonstrates that EGFR-TKD and Rheb have good docking.

[0150] Based on the evaluation of the docking model, it is speculated that glutamic acid at position 804 on EGFR (E804) may be the key amino acid site for EGFR to exert GEF activity.

[0151] To verify this hypothesis, subsequent studies were conducted to confirm that glutamic acid at position 804 on EGFR is a key site for GEF activity.

[0152] Example 5. Glutamic acid at position 804 on EGFR is the key GEF active site.

[0153] I. Experimental Methods

[0154] 1. Construction of the EGFR-E804K mutant

[0155] The glutamic acid (E) at position 804 of EGFR was mutated to lysine (K) using site-directed mutagenesis technology. The specific steps are as described in the site-directed mutagenesis method in Example 2.

[0156] 2. Construction of fusion protein particles

[0157] Different EGFR-TKD mutants (WT, KD, or E804K) are linked to the C-terminus of the LAMP2-V5 fusion protein to achieve targeted localization of the fusion protein to the lysosome.

[0158] The specific method was as follows: the sequences of LAMP2 and different EGFR-TKD (WT, KD, E804K) were cloned respectively, and the LAMP2-V5-EGFR-TKD recombinant plasmid was constructed using the V5 tag as a linker. After all constructions were completed, the sequence correctness was confirmed by nucleotide sequencing.

[0159] 3. IP Experiment

[0160] As described in Example 2 regarding the IP method, in short, in HEK293T cells stably overexpressing SFB-Rheb, the plasmids pSIN or pSIN-EGFR-WT / KD / E804K-HA, as shown in the figure, were transiently transfected for IP experiments to detect the binding of different forms of EGFR (WT, KD, or E804K) to Rheb. The results are as follows: Figure 5 As shown in Figure A.

[0161] 4. In vitro nucleotide exchange experiment

[0162] The experimental method was the same as the in vitro nucleotide exchange experiment in Example 3. The RhoGEF Exchange Assay kit (Cytoskeleton, BK100) was used to detect the ability of different forms of EGFR (WT or E804K) to promote nucleotide exchange of Rheb, i.e., GEF activity. The results are as follows: Figure 5 As shown in B.

[0163] 5. Western blot (WB) experiment

[0164] In HEK293T cells, LAMP2-V5 or LAM2-V5-EGFR-TKD-WT / KD / E804K was stably overexpressed. After confirming successful expression, the cells were subjected to serum starvation for 24 hours to detect mTORC1 activation. The results are as follows: Figure 5 As shown in C.

[0165] II. Experimental Results

[0166] like Figure 5 As shown in Figure A, no protein band was detected in the EGFR-E804K group compared to the EGFR-WT and EGFR-KD groups, indicating that the EGFR with the glutamate mutation at position 804 cannot interact with Rheb. Therefore, glutamate position 804 (E804) on EGFR is a key site for the interaction between EGFR and Rheb.

[0167] like Figure 5 As shown in Figure B, the relative fluorescence intensity of the EGFR-TKD-E804K group was weaker than that of the Rheb+EGFR-TKD-WT group, indicating that the EGFR with the glutamate mutation at position 804 cannot promote the in vitro nucleotide exchange of Rheb. In other words, the glutamate at position 804 (E804) on EGFR is the key site for EGFR to have GEF activity.

[0168] like Figure 5As shown in C, both LAMP2-V5-EGFR-TKD-WT and LAMP2-V5-EGFR-TKD-KD can effectively activate mTORC1 after being targeted to the lysosomal surface, while LAMP2-V5-EGFR-TKD-E804K cannot activate mTORC1 after being targeted to the lysosomal surface, further indicating that glutamate at position 804 on EGFR (E804) is a key site for EGFR regulation of mTORC1.

[0169] The above results fully demonstrate that glutamate at position 804 on EGFR (E804) is the key site for it to exert GEF activity and thereby regulate mTORC1 activation.

[0170] Example 6. PC9 cells with endogenous EGFR-E804K knock-in failed to activate mTORC1 and inhibit tumor growth.

[0171] I. Experimental Methods

[0172] 1. Construction of PC9 cells with endogenous EGFR-E804K knock-in

[0173] We designed sgRNAs targeting EGFR and screened for highly efficient sequences, while simultaneously constructing donor DNA templates containing the E804K mutation. The donor DNA template was constructed in a pSIN vector (excluding the EF1α promoter) and integrated with encoding EGFP and the upstream internal ribosome entry site (IRES) to improve the screening efficiency of positive clones.

[0174] sgRNA and donor DNA template were co-transfected into parental PC9 cells. About 3 days after transfection, Alexa Fluor-488 positive single cells were sorted using a MoFloAstrios EQ cell sorter (Beckman Co., Ltd.), and positive clones were verified by PCR and Sanger sequencing, confirming the successful construction of PC9 cells with endogenous gene knock-in EGFR-E804K.

[0175] 2. Tumor cell proliferation detection

[0176] The proliferation capacity of parental PC9 cells (parental group) and EGFR-E804K knock-in PC9 cells (E804K group) was evaluated using the CCK8 assay, clonogenic assay, and EdU (Beyotime, C0071) assay, respectively.

[0177] 3. Subcutaneous tumor-bearing experiment in nude mice

[0178] The animal experiments of this invention have been approved by the Animal Research Committee of Sun Yat-sen University Cancer Center (Approval No. L025501202410005).

[0179] Female BALB / c nude mice weighing 18-20 grams and 4 weeks old were purchased from Zhuhai Best Biotechnology Co., Ltd. (Zhuhai, China).

[0180] Mice were randomly assigned to two groups of six each. For tumor growth testing, 5 × 10⁶ mice were injected into each group. 6 Parental PC9 cells or EGFR-E804K knock-in PC9 cells were subcutaneously injected into the axilla of mice, designated as the parental group and the E804K group, respectively. Starting one week post-injection, tumor volume was measured every three days using calipers. The formula for calculating volume was: V (volume) = (width) / (width) 2 ×length×π) / 6. After 5 weeks of growth, mice were euthanized with 100% CO2 at a flow rate of 30-70% of the chamber volume per minute. Tumors were collected, and their volume and weight were measured using calipers and an electronic balance, respectively. The results are as follows: Figure 6 As shown in E, 6F, and 6G.

[0181] II. Experimental Results

[0182] like Figure 6 A showed that, compared with the parental PC9 cells (parental group), the phosphorylated EGFR (p-Y1068-EGFR) in EGFR-E804K knock-in PC9 cells (E804K group) was not different, but the expression of phosphorylated S6K1 (p-T389-S6K1) was significantly reduced. This indicates that although EGFR-E804K knock-in PC9 cells (E804K group) can maintain normal EGFR kinase activity, they cannot activate mTORC1. This further proves that glutamate at position 804 on EGFR (E804) is a key site for EGFR regulation of mTORC1.

[0183] like Figure 6 As shown in B (CCK8 assay), 6C (clonal formation assay), and 6D (EdU assay), the proliferation capacity of PC9 cells with EGFR-E804K gene knock-in (E804K group) was significantly reduced compared with the parental PC9 cells (parental group), indicating that inhibiting glutamate at position 804 of EGFR (E804) (i.e., inactivating EGFR) can effectively inhibit tumor growth.

[0184] like Figure 6 E, 6F, and 6G showed that, compared with subcutaneously injected parental PC9 cells (parental group), subcutaneously injected EGFR-E804K gene knock-in PC9 cells (E804K group) significantly reduced tumor volume and weight in a nude mouse subcutaneous tumor-bearing model, further demonstrating that inhibiting glutamate at position 804 (E804) on EGFR can effectively inhibit tumor growth.

[0185] In summary, PC9 cells with endogenous EGFR-E804K knock-in (i.e., EGFR-inactivated PC9 cells) cannot activate mTORC1, thereby inhibiting tumor growth.

[0186] Example 7. Combined inhibitory effect of the small molecule compound BIEGe-1 on EGFR kinase and GEF function

[0187] I. Experimental Methods

[0188] 1. Screening and identification of small molecule compounds that inhibit EGFR kinase activity

[0189] Studies have reported that 7-oxopyridopyrimidine compounds are a novel type of EGFR tyrosine kinase inhibitor, with the potential to overcome the problems of poor selectivity and toxic side effects of traditional EGFR inhibitors on wild-type cells.

[0190] As described in the in vitro nucleotide exchange experiment in Example 3, the above-mentioned 7-oxopyridopyrimidine compounds were screened using the RhoGEF Exchange Assay kit (Cytoskeleton, BK100), and the results are as follows. Figure 7 As shown in A and 7B.

[0191] The groups are as follows:

[0192] Rheb group: Rheb protein alone.

[0193] DMSO(Rheb+EGFR-TKD) group: Rheb+EGFR-TKD, with the addition of DMSO, i.e., control group.

[0194] BIEGi-1 (Rheb+EGFR-TKD) group: Rheb+EGFR-TKD, with the addition of BIEGi-1, i.e., the drug-added group.

[0195] 2. IP Experiment

[0196] As described in the IP method of Example 2, an immunoprecipitation experiment was performed in PC9 cells. Cells from the control group and the drug-treated group were lysed with an appropriate amount of IP lysis buffer for 30 minutes, centrifuged, and the supernatant was collected. 1 μg of the corresponding antibody and 50 μL of Protein A / G beads were added, and the mixture was incubated overnight at 4°C. Subsequently, the agarose bead-antigen antibody complex was collected, the supernatant was discarded, and the cells were washed three times with pre-chilled IP elution buffer. After boiling at 100°C for 10 minutes and cooling, Western blotting was performed. The ability of BIEGI-1 to inhibit EGFR-Rheb binding was detected. The results are as follows: Figure 7 As shown in C.

[0197] 3. Drug addition experiment.

[0198] PC9 and NCI-H1975 cells were treated with either DMSO or BIEGI-1, respectively, and the activation of mTORC1 was detected. The results are as follows: Figure 7 As shown in D and 7E.

[0199] 4. IC 50 Determination methods

[0200] A suitable amount of target cells were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. A series of test drug solutions of different concentrations were prepared, with at least three parallel wells for each concentration. A control group with only an equal volume of culture medium was also included. After 72 hours of drug treatment, 10 μL of CCK8 reagent was added to each well, and the plates were incubated for 1 hour. The absorbance (OD) of each well was measured at 450 nm using a microplate reader. Data processing was performed using GraphPad Prism 9.0 software to calculate the cell viability corresponding to each concentration (formula: viability = [(treatment group OD – blank OD) / (control group OD – blank OD)] × 100%). Nonlinear regression analysis was used to determine the IC50. 50 Value, i.e., the drug concentration required to inhibit cell proliferation by 50%, the results are as follows Figure 7 As shown in F.

[0201] II. Experimental Results

[0202] like Figure 7 As shown in B, compared to DMSO, adding Figure 7 The small molecule compound (BIEGi-1) shown in Figure A exhibits weaker relative fluorescence between bound mant-GTP and free mant-GTP, indicating that BIEGI-1 can significantly inhibit EGFR-mediated nucleotide exchange of Rheb. In other words, BIEGI-1 can inhibit the GEF activity of EGFR.

[0203] like Figure 7 As shown in Figure C, compared with the DMSO group, the protein expression of Rheb in the BIEGI-1 group was significantly reduced, indicating that BIEGI-1 can disrupt the interaction between EGFR and Rheb.

[0204] like Figure 7 As shown in Figure D, in PC9 cells, compared with the DMSO group, the expression of phosphorylated S6K1 (p-T389-S6K1) protein was significantly reduced in the BIEGI-1 group, indicating that BIEGI-1 can inhibit mTORC1 activation.

[0205] like Figure 7As shown in F, in NCI-H1975 cells, compared with the DMSO group, the expression of phosphorylated S6K1 (p-T389-S6K1) protein was significantly reduced in the BIEGI-1 group, indicating that non-small cell lung cancer cell lines carrying the EGFR-T790M resistance mutation (T790M mutation is one of the important mechanisms of EGFR kinase inhibitor resistance) are still sensitive to BIEGI-1, that is, BIEGI-1 can inhibit mTORC1 activation in EGFR kinase resistant cell lines.

[0206] like Figure 7 As shown in E, in non-small cell lung cancer cell lines PC9, HCC827, and NCI-H1975 carrying EGFR mutations, the IC50 of BIEGei-1... 50 The values ​​were 17 nM, 20 nM and 24 nM, respectively, indicating that BIEGI-1 has excellent ability to inhibit EGFR-driven tumor cell proliferation.

[0207] In summary, in addition to its known regulation of EGFR tyrosine kinase activity, EGFR also has a GEF function that promotes Rheb nucleotide exchange, which mainly depends on the 804th glutamate site on EGFR, thus further linking EGFR to the mTORC1 signaling pathway.

[0208] Based on the above findings, the combined targeted inhibition of EGFR kinase activity and GEF activity using inhibitors, small molecule compounds, nanomaterials, or gene editing technology can become a new strategy for treating tumors at least partially driven by EGFR. This provides new technical ideas and methods for treating tumors primarily driven by EGFR, and lays a solid experimental foundation for the development of highly effective anti-tumor drugs, with significant application prospects and important clinical significance.

[0209] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an EGFR inhibitor, characterized in that, This is used to prepare pharmaceutical compositions for treating diseases mediated by guanylate exchange factor (GEF) activity or by a combination of guanylate exchange factor (GEF) activity and EGFR tyrosine kinase activity.

2. The use according to claim 1, characterized in that, The diseases mentioned are cancers selected from the following group: lung cancer, colorectal cancer, breast cancer, endometrial cancer, thyroid cancer, glioma, squamous cell carcinoma, prostate cancer, melanoma, leukemia, and glioblastoma.

3. The use according to claim 1, characterized in that, The pharmaceutical composition is also used to regulate abnormal nucleotide exchange of Rheb; preferably, the regulation is independent of regulation of abnormal EGFR kinase activity or expression.

4. The use according to claim 1, characterized in that, The pharmaceutical composition simultaneously regulates EGFR tyrosine kinase activity and GEF activity, and the regulation of GEF activity is independent of the regulation of EGFR tyrosine kinase activity.

5. The use according to claim 1, characterized in that, The EGFR inhibitor is selected from the group consisting of: small molecule compounds, nanomaterial drugs, gene editing technology, protein antibodies, nucleic acid drugs, enzyme drugs, expression vectors, cell therapy, hormone drugs, peptide drugs, or combinations thereof.

6. The use according to claim 5, characterized in that, The EGFR inhibitor is a small molecule compound.

7. The use according to claim 6, characterized in that, The EGFR inhibitor is a small molecule compound as shown in the following formula:

8. A drug combination, characterized in that, It includes EGFR tyrosine kinase inhibitors and GEF activity inhibitors, or dual-target inhibitors of EGFR tyrosine kinase and GEF activity.

9. Use of the pharmaceutical combination as claimed in claim 8, characterized in that, Pharmaceutical compositions for the preparation of treatments for tumors driven by EGFR or resistant to single 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.

10. A method for designing a dual-target inhibitor of EGFR tyrosine kinase and GEF activity, characterized in that, Including the following steps: (a) Provide a candidate molecule that can act simultaneously on the kinase domain of EGFR and the 804th glutamate residue of EGFR; (b) Determine the ability of the candidate molecules to inhibit EGFR kinase and GEF activity.

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