An anti-tumor compound screening system based on glutamine synthetase-lats protein interaction

Abstract

The application discloses an anti-tumor compound screening system based on glutamine synthetase (GS)-LATS protein interaction, and belongs to the field of biotechnology and targeted drug research and development. The application establishes a double-protein interaction verification system of exogenous and endogenous by constructing recombinant plasmids of GS and LATS1 and functional domains thereof, and determines the GS-LATS interaction and key domains. A compound screening system is established taking the targets, small-molecule compounds capable of blocking the GS-LATS combination, stabilizing LATS and inhibiting the YAP pathway are screened, and the synergistic anti-tumor effect can be realized in combination with glutaminase inhibitors or mTOR inhibitors. The system has high specificity and high accuracy, is suitable for drug screening and mechanism research of various tumors such as lung cancer and liver cancer, and provides a key technology and experimental platform for the development of anti-tumor drugs targeting the metabolic-signal interaction interface.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and targeted drug research, specifically to a system for screening antitumor compounds by utilizing the protein interaction between glutamine synthetase (GS) and LATS1 (Large Tumor Suppressor Kinase 1), a key kinase in the Hippo pathway. Background Technology

[0002] The development and progression of tumors is a complex process involving multiple gene mutations, signaling pathway dysregulation, and metabolic reprogramming. Tumor metabolic reprogramming is a core characteristic driving cancer development. It is well known that metabolites in the tumor microenvironment are not only energy and biosynthetic raw materials, but also key molecules regulating tumor-related signaling pathways. Targeted therapy against the "metabolite-key signaling pathway" will provide new strategies for developing combined metabolic checkpoint and pathway blockade therapies, which has social and economic significance for improving the level of precision cancer diagnosis and treatment in my country.

[0003] Glutamine metabolism is the second most common characteristic of tumors after the Warburg effect. Glutamine not only provides carbon and nitrogen sources for rapidly proliferating cancer cells, replenishing the TCA cycle, synthesizing nucleotides, and maintaining redox balance, but also acts as an important signaling molecule regulating multiple growth-related pathways, including mTOR signaling. However, targeted drugs such as rapamycin and its analogues primarily target mTORC1, with limited effects on mTORC2. Clinically, the selectivity of targeted drugs is insufficient to fully elucidate the dual role of mTOR signaling in inducing glutamine homeostasis. Therefore, exploring the regulatory mechanisms of glutamine independent of the classical mTOR pathway is crucial. The key enzymes in glutamine metabolism (glutamine synthase GS, glutaminase GLS) are the only mammalian enzymes capable of resynthesizing / degrading glutamate and ammonia into glutamine, and may be potential therapeutic targets for targeting glutamine homeostasis in cancer. Recent tumor genomic data show that the GS-encoding gene GLUL is highly amplified in liver cancer, lung cancer, and triple-negative breast cancer. Its expression level is positively correlated with the activity of MYC and the Hippo pathway effector YAP, suggesting that GS may transcend its role as a metabolic enzyme and directly participate in the regulation of oncogenic signals. The Hippo pathway mainly consists of the upstream core kinase chains MST1 / 2-SAV1 and LATS1 / 2-MOB1, and the downstream effector YAP and its homolog TAZ. When the Hippo pathway is inactivated, non-phosphorylated YAP / TAZ translocates into the nucleus, binds to transcription factors such as TEAD, and initiates the expression of a series of genes that promote cell proliferation and inhibit apoptosis (such as CTGF, CYR61, and ANKRD1), driving tumorigenesis. Therefore, LATS kinase, as a key tumor suppressor, has its activity and stability as crucial determinants of YAP's oncogenic activity. Stabilizing LATS protein levels is considered a potential therapeutic strategy for inhibiting YAP-driven tumors. In fact, the inventors (Cell Reports, 2025) made a groundbreaking discovery: GS can bind to LATS1 in the form of a "protein scaffold," recruiting the E3 ubiquitin ligase WWP1 / ITCH, catalyzing the polyubiquitination of LATS1, and its degradation by the proteasome. When exogenous glutamine is sufficient, glutamine itself rapidly stabilizes LATS1 by inhibiting GS expression and disrupting the GS-LATS1 complex, triggering YAP phosphorylation and cytoplasmic retention. This discovery redefines GS from a traditional metabolic enzyme as a key upstream regulator of the Hippo signaling pathway, providing a highly attractive new target for anticancer drug development: GS-LATS, namely the protein-protein interaction (PPI) interface.

[0004] Currently, there are no anticancer drugs targeting GS-LATS interactions on the market, and there is a lack of mature technologies and platforms specifically for drug screening targeting this target. Traditional screening methods based on enzyme activity or cytotoxicity are difficult to discover compounds that function by regulating protein interactions. Therefore, developing a system based on GS-LATS protein interactions capable of screening small molecule inhibitors has become an urgent need and a key bottleneck in translating this important basic research discovery into clinical treatments. It also lays the technical and theoretical foundation for the discovery of candidate drugs that synergize with existing GLS or mTOR inhibitors. Summary of the Invention

[0005] The purpose of this invention is to provide a screening system for antitumor compounds that specifically targets the GS-LATS protein interaction. Its core concept is to simulate the physiological conditions of sufficient glutamine in the tumor microenvironment, stabilizing the GS-LATS interaction. Upon biochemical or pharmacological intervention, GS-mediated ubiquitination and degradation of LATS1 are blocked, thereby rapidly increasing LATS1 protein levels, activating the Hippo cascade, promoting YAP phosphorylation and retention in the cytoplasm, leading to a significant reduction in the transcriptional levels of YAP / TEAD-driven proliferative genes (such as CTGF, ANKRD1, CYR61, etc.). Ultimately, from the perspective of glutamine homeostasis imbalance in the tumor cell microenvironment, this system enables rapid and specific screening of small molecule compounds that exert antitumor effects by stabilizing LATS and inhibiting the YAP pathway. Because this system is based on a well-defined pathological mechanism, the screened compounds have high drug development potential and low off-target risk, providing a powerful tool for developing antitumor drugs.

[0006] In a first aspect, the present invention provides an antitumor compound screening system based on GS-LATS protein interactions, characterized in that the system comprises:

[0007] 1. A reporter cell line that stably expresses the following elements:

[0008] (1) The GS protein and its domain fragments that interact with LATS, including the full-length GS protein (FL), the GS grasping domain (β-grasp), and the GS catalytic domain (CD). (As per the appendix of this invention) Figure 1 (As shown).

[0009] (2) The LATS protein and its domain fragments that interact with GS, including the full-length LATS1 protein (FL), the LATS1 ubiquitin-associated domain (UBA domain, UBA), the LATS1 grasping domain (PPxY motif, PY), and the LATS1 catalytic domain (KD). (As per the appendix of this invention) Figure 2 (As shown).

[0010] (3) Negative reporter genes driven by promoters that respond to YAP activity, including genes such as CTGF, ANKRD1, and CYR61. When LATS is stably activated and YAP is inhibited, reporter gene expression is downregulated.

[0011] In summary, cell lines selected included HEK293T (high transfection efficiency), H1299 (lung cancer), H2030 (lung cancer), A549 (lung cancer), and Hep1-6 (liver cancer) cells.

[0012] 2. Screening criteria and detection module: This system includes the following modules:

[0013] (1) Cell culture conditions: The reporter cell line was cultured and screened in a medium containing physiological concentrations of glutamine (RPMI-1640, 2 mM; DMEM, 4 mM) to simulate the glutamine-sufficient state in the tumor microenvironment.

[0014] (2) Detection equipment, including equipment for quantitative detection of interaction strength, gene signal changes and malignant biological behavior of tumor cells, such as chemiluminescence analyzers, multifunctional enzyme-linked immunosorbent assay (ELISA) readers, etc.

[0015] Secondly, the present invention provides a method for constructing the screening system, characterized by comprising the following steps:

[0016] 1. Construction of plasmid fragments containing GS and LATS interaction domains:

[0017] (1) Primer and label design:

[0018] Forward primer: Homologous to the sequence near the start codon (ATG) of the GS / LATS gene, containing protective bases and vector homologous arms or restriction sites.

[0019] Downstream primer (Reverse): This primer is the core of the design and contains the removal of the GS / LATS stop codons themselves. It is placed at the end of the GS / LATS coding sequence but does not contain the original stop codons (such as TAA, TAG). TGA); Tag coding sequence: Following the GS / LATS sequence, directly link the above tag DNA sequence (e.g., FLAG sequence: the DNA sequence encoding the FLAG tag is usually 5'-GACTACAAGGACGACGATGACAAG-3', corresponding amino acid: DYKDDDDK; HA sequence: the DNA sequence encoding the HA tag is usually 5'-TACCCATACGATGTTCCAGATTACGCT-3', corresponding amino acid sequence: YPYDVPDYA; S sequence: the DNA sequence encoding the S tag is usually 5'-AAGGAAACAGCAGCCGCAAAATTCGAACGCCAGCACATGGACTCC-3', corresponding amino acid sequence: KETAAAKFERQHMDS); A new stop codon: Add a stop codon (e.g., TGA) after the tag sequence; Vector homologous arm or restriction enzyme site.

[0020] (2) PCR amplification:

[0021] High-fidelity DNA polymerase was used for PCR to obtain a high-fidelity, high-purity "GS / LATS-No-Stop" gene fragment for subsequent cloning. First, the PCR reaction system was prepared using a high-fidelity DNA polymerase premix to avoid introducing mutations. The DNA polymerase premix, forward and reverse primers, and template DNA (preferably a high-quality plasmid) were added to the reaction system, and the volume was brought up with ddH2O. Next, the reaction program was set, optimized according to the primer characteristics and product length, and included three stages: initial denaturation, cyclic amplification (denaturation, annealing, and extension), and terminal extension. The first stage involved initial denaturation at 98°C for 30-60 seconds to completely denature the template. The second stage consisted of 25-35 cycles: each cycle included denaturation at 98°C for 5-10 seconds, followed by annealing at a temperature 3-5°C higher than the calculated Tm value of the primers for 10-30 seconds (to ensure specific binding), and finally extension at 72°C (calculated at 15-30 seconds per kilobase). The third stage involves a final extension at 72°C for 5-10 minutes to ensure complete product synthesis. Finally, store at 4°C.

[0022] After the reaction, the product needs to be verified and purified. A small amount of product is subjected to agarose gel electrophoresis; a single, clear band should be visible at a position slightly larger than the original gene. Purification is performed using a gel extraction kit: the target band is excised and purified to remove template plasmids, primer dimers, salt ions, etc., obtaining a high-purity target fragment. Finally, the purified DNA is dissolved in ddH2O, and the concentration (ng / μL) and purity (A260 / A280 ratio 1.8-2.0) are measured to obtain a high-quality "GS / LATS-no-stop-codon" fragment for subsequent cloning.

[0023] (3) Cloning and linking:

[0024] Ligation-independent cloning (LIC) was employed to achieve seamless ligation via enzymatic reaction. The vector was double-digested with two restriction endonucleases (BamHI and XhoI) for 30 minutes to 1 hour to ensure complete cleavage into linear molecules. After digestion, the cleavage efficiency was verified by agarose gel electrophoresis, and the target band was recovered from the gel to remove enzymes, buffer, and uncut vector, yielding a pure linearized vector. Next, the purified linearized vector was ligated to the target fragment (the purified DNA fragment obtained in step (2)) in a dedicated buffer system on ice. The key step was the addition of exonuclease III (Exo III), followed by incubation on ice for approximately 1 hour. Furthermore, 0.5 M EDTA (pH=8.0) was added to chelate magnesium ions in the system, rapidly inactivating Exo III and stopping further digestion. The reaction system was then heated in a 58-65°C water bath for 10 minutes to ensure complete enzyme inactivation and prevent residual activity from affecting subsequent experiments. Finally, place the sample on ice to cool and store at -80°C.

[0025] (4) Bacterial transformation, validation, and plasmid extraction:

[0026] The ligation product obtained in step (3) was mixed with DH-5α competent cells, incubated on ice for 30 minutes, then heat-shocked at 42°C for 45 seconds, followed by an ice incubation for 5 minutes. The mixture was then spread onto LB plates with the corresponding antibiotic resistance and incubated upside down at 37°C overnight. Single colonies were picked and inoculated into TB medium, and cultured at 37°C with shaking until turbid. 200 μl of the bacterial culture was sent for sequencing verification. For bacterial cultures with correct sequencing, plasmids were extracted using a plasmid extraction kit, and the concentration (ng / μl) and purity (A260 / A280 = 1.8–2.0) were determined to obtain the recombinant plasmid required for the study.

[0027] 2. Construction of the GS and LATS1 internal / external interaction detection system:

[0028] (1) Construction of the GS and LATS1 exogenous interaction detection system: The full-length GS plasmid carries an HA tag, and the GS functional domain (β-grasp, β-G, Catalytic domain, CD) plasmids carry S tags and FLAG tags, namely SF-GS-β-G and SF-GS-CD; the full-length LATS1 plasmid carries an HA tag, and the LATS1 functional domain (UBA domain, UBA, PPxY motif, PY, Kinase domain, KD) plasmids carry S tags and FLAG tags, namely SF-LATS1-UBA, SF-LATS1-PY, and SF-LATS1-KD. HEK293T tool cells were seeded in 6cm dishes, and transfection with plasmids was prepared after the density reached 70-80%. Forward validation: The full-length HA-GS plasmid and the SF-LATS1 domain segment plasmid were co-transfected using PEI transfection reagent. Reverse validation: The full-length HA-GS plasmid and the SF-LATS1 domain segment plasmid were co-transfected using PEI transfection reagent. Plasmids were coated at a ratio of plasmid (μg): PEI transfection reagent: Opti-MEM serum-free medium = 1:4:100 for 15 minutes and then added to HEK293T cells. Cells were lysed with RIPA lysis buffer after 48 hours. Following the procedure of a commercial FLAG affinity purification kit, the exogenous FLAG-tagged fusion protein in the transfected cell lysate was immunoprecipitated using its accompanying FLAG affinity magnetic beads. The lysate was incubated with well-balanced FLAG beads at 4°C. After washing with wash buffer to remove non-specific binding, the target protein complex was recovered. The resulting product was used for subsequent immunoblotting analysis of protein-protein interaction levels.

[0029] (2) Construction of the GS and LATS endogenous interaction detection system: In order to eliminate artifacts such as overexpression or steric hindrance that may be caused by exogenous tags, this invention establishes an endogenous interaction system in two types of tumor cells, H1299 and H2030. H1299 and H2030 tumor cells were seeded in 10cm dishes. After the density reached 90%, the cells were washed twice with pre-cooled PBS, and mild NETN lysis buffer was added. After incubation on ice for 30 minutes, the cells were centrifuged at 12000g for 15 minutes at 4°C. A small amount of supernatant was taken and an equal volume of 2×SDS loading buffer was added as the input control sample. The remaining supernatant was pre-cleaned with Protein A / G magnetic beads + normal IgG for 1 hour. The precipitate was then washed 4 times with pre-cooled buffer as the IgG group. The supernatant was divided into two parts: forward verification (adding 2 μg of anti-GS antibody and rotating overnight at 4 °C) and reverse verification (adding 2 μg of anti-LAST1 antibody and rotating overnight at 4 °C). The next day, the protein was washed four times with pre-cooled buffer, the supernatant was discarded, and the protein was denatured using 1×SDS loading buffer. The amount of co-precipitated GS and LAST1 in the forward and reverse directions was detected by Western blotting.

[0030] In summary, by amplifying the signal through an exogenous system, the interaction between GS and LATS can be initially screened to exclude the presence of mutants. Furthermore, by eliminating exogenous tag interference through an endogenous system, the dynamics of the GS and LATS complex under physiological conditions can be accurately reflected, providing support for functional confirmation and clinical translation research.

[0031] Thirdly, the present invention provides a method for screening antitumor compounds using the aforementioned screening system, characterized by comprising the following steps:

[0032] 1. Flowchart of an antitumor compound screening system based on GS-LATS protein-protein interaction interface (PPI):

[0033] (1) Establishment and pretreatment of the compound library to be screened: Construct or obtain a diverse small molecule compound library containing known PPI inhibitors, metabolic regulators, kinase inhibitors and natural products. All compounds are pre-dissolved in solvents such as DMSO to prepare a stock solution, which is then aliquoted and stored at -80℃ to avoid repeated freeze-thaw cycles. Before use, the stock solution is diluted in serum-free culture medium to ensure that the final concentration of solvents such as DMSO in the screening system does not exceed 0.1%, and a corresponding solvent control is set up.

[0034] (2) High-throughput primary screening based on reporter cell lines: Cells stably expressing the GS-LATS interaction reporter system were seeded and cultured overnight until adherence. The compounds to be screened were added, along with control wells: positive controls (e.g., known MST inhibitor XMU-MP-1, known LATS inhibitor VT02956, and known GS inhibitor MSO); negative controls (equal volume of solvent); and blank controls (cell-free culture medium). Chemiluminescence assays were used to detect YAP reporter gene activity, and compounds that significantly inhibited YAP transcriptional activity were screened.

[0035] (3) Cellular level antiproliferative activity verification: Compounds with inhibition rates >50% in the initial screening were selected for dose-dependent verification in lung cancer cells such as H1299, H2030, and A549. Seven concentration gradients (0.01-10 μM) were set up, and cell viability was detected by CCK-8 assay after 72 hours of treatment. IC50 was calculated. 50 The drug concentration at which the inhibition rate reaches 50% is selected as the standard value for the next round of mechanism verification.

[0036] (4) Interaction interference verification: Co-IP verification was performed using an exogenous overexpression system (HEK293T+HA / SF-GS+HA / SF-LATS1). After transfecting cells with candidate compounds for 24 hours, the cells were lysed, and immunoprecipitation was performed using FLAG magnetic beads. The co-precipitation level of GS and LATS1 was detected by Western blotting, and compounds that could significantly reduce the binding strength of GS-LATS were selected. Further Co-IP verification was performed using endogenous antibodies in endogenous systems (such as H1299 and H2030 cells) to eliminate the artifact of exogenous overexpression.

[0037] (5) Evaluation of synergistic effect: The screened GS-LATS interaction inhibitors were combined with existing drugs such as glutaminase GLS inhibitors (CB839) or mTOR inhibitors (Rapamycin), and the compound combinations with significant synergistic effects were selected for animal experiments.

[0038] (6) Animal efficacy verification: Establish Hep1-6 or H1299 xenograft tumor models. Set up the following groups: solvent control group, single drug group, and combined drug administration group. Monitor tumor volume, mouse weight and general physiological status. After the experiment, the tumor was removed and weighed, and histopathological and immunohistochemical analyses were performed (detection of markers such as GS, LATS1, p-YAP, and p-S6).

[0039] 2. Changes in the malignant biological behavior of different tumor cell lines by single and combined drug administration:

[0040] (1) Cell seeding: Select lung cancer cells (H1299, H2030, A549) in the logarithmic growth phase and in good condition. After trypsin digestion and termination with complete culture medium, gently pipette to form a uniform single-cell suspension for accurate counting and seeding. Seed the cell suspension into 96-well plates, avoiding the formation of air bubbles during seeding, and fill the peripheral wells with sterile PBS to reduce the evaporation edge effect. After seeding, gently cross-shake the culture plate to ensure uniform cell distribution, and then incubate at 37°C and 5% CO2.

[0041] (2) CCK-8 assay for changes in proliferation of different tumor cell lines by single and combined administration: At pre-set detection time points (e.g., 0, 24, 48, 72, 96 hours), 96-well plates were removed from the incubator. CCK-8 stock solution was mixed with preheated fresh complete culture medium at a volume ratio of 1:9, and thoroughly mixed by inversion to prepare the CCK-8 working solution. Working concentrations for the administration groups: Verteporfin (YAP inhibitor, 10 μM), CB839 (GLS inhibitor, 3 μM), and Rapamycin (mTOR inhibitor, 100 nM); working concentrations for the control group: equal volumes of solvent were administered according to the administration groups. Subsequently, the working solution was added to each well of the culture plate, and the plate was returned to the incubator for further incubation in the dark for 2 hours. After incubation, the 96-well plate was removed, and before detection, the plate was shaken for 10 seconds using the ELISA reader's shaking option. The absorbance (OD value) of each well was measured using the ELISA reader at a wavelength of 450 nm. Using professional statistical analysis software such as GraphPad Prism, cell growth curves were plotted with culture time as the x-axis and corrected OD value as the y-axis, thereby intuitively and quantitatively evaluating the effect of drug treatment on cell proliferation at different time points.

[0042] 3. Effects of combined drug administration on tumor growth in model animals:

[0043] (1) Establishment and grouping of animal models: Five-week-old male nude mice (immunodeficient mice) were used. The control vector (Control) and GS gene knockout (GS KO) Hep1-6 stable cell lines were subcutaneously injected into the right rib area of ​​the nude mice, with 8-10 mice per group. Each mouse received 0.1 mL (1×10⁻⁶) of the injected solution. 7 After implantation of cells (number per ml), the mice were returned to their original cages to allow for spontaneous recovery. Once tumors formed, the mice were randomly divided into four groups and received the following treatments: solvent control group (Vehicle), CB839 monotherapy group (200 mg / kg), rapamycin monotherapy group (2 mg / kg), and CB839 + rapamycin combination group.

[0044] (2) Effect of combined drug administration on tumor growth in animals: Drug administration lasted for 12 days. During the experiment, the long and short diameters of the tumor were measured every two days using vernier calipers, the tumor volume was calculated, and growth curves were plotted. Mice were sacrificed after drug administration, the tumors were completely dissected, and their weight was recorded. Each group had 6 biologically independent tumor samples.

[0045] In summary, compared with existing technologies, this invention has the following advantages: Compared with traditional screening methods based on enzyme activity or cytotoxicity, this invention constructs a screening system targeting GS-LATS protein interactions, achieving effective exploration of this challenging target, protein-protein interactions, with clear mechanism guidance and precise targeting. Simultaneously, this invention employs a ligation-independent cloning (LIC) method to construct expression plasmids for each domain of GS and LATS, and introduces a multi-tag system, combined with a dual verification system of exogenous and endogenous protein interactions, significantly improving the efficiency and reliability of system construction. This system integrates a complete workflow from in vitro initial screening to in vivo verification, applicable to various tumor models such as lung cancer, liver cancer, and breast cancer, and can evaluate the synergistic effects of combined drug therapy, providing research methods and experimental technical support for the clinical development of anti-tumor drugs targeting the "metabolism-signaling" interaction network. Attached Figure Description

[0046] Figure 1 : GS full length and its functional domain pattern diagram.

[0047] Figure 2 : LATS1 full length and its functional domains pattern diagram.

[0048] Figure 3 : Verification diagram of exogenous immunoprecipitation. Forward verification: pull down SF-GS, and use Western blotting to detect the interaction domain between LATS1 and GS; Reverse verification: pull down SF-LATS1, and use Western blotting to detect the interaction domain between GS and LATS1.

[0049] Figure 4 Endogenous immunoprecipitation verification diagram. Forward verification: anti-GS antibody capture, immunoblotting detection of LATS1-GS interaction; Reverse verification: anti-LATS1 antibody capture, immunoblotting detection of GS-LATS1 interaction.

[0050] Figure 5 Tumor cell proliferation activity graph. CCK-8 assay was used to detect changes in the proliferation of H1299, H2030, and A549 tumor cell lines caused by different combination of drug administration regimens. Regimen 1: Verteporfin + CB839; Regimen 2: Rapamycin + Verteporfin; Regimen 3: CB839 + Rapamycin.

[0051] Figure 6 Tumor growth diagram in experimental animals. Changes in tumor size, volume, and wet weight caused by combined drug administration.

[0052] Figure 7 Flowchart of the GS-LATS protein interaction targeted drug screening system. Detailed Implementation

[0053] Example 1: Construction of plasmids containing GS and LATS1 domain fragments

[0054] (1) Vector and tag selection: A mammalian expression vector (pcDNA3.1) containing a strong promoter was used. To facilitate subsequent interaction verification (Co-IP) and protein detection, each plasmid was fused with different affinity tags. The full-length GS plasmid was constructed with an HA tag (HA-GS-FL), and the GS domains (β-G, CD) were constructed with dual S and FLAG tags (SF-GS-β-G, SF-GS-CD). Correspondingly, the full-length LATS1 plasmid was constructed with an HA tag, and the LATS1 domains (UBA, PY, KD) were constructed with dual S and FLAG tags (SF-LATS1-UBA, SF-LATS1-PY, SF-LATS1-KD).

[0055] (2) Primer design and synthesis: Primers were designed and synthesized based on the coding sequences of the human GS gene (GLUL, NM_001033044) and the human LATS1 gene (LATS1, NM_004690). Using GS-FLAG (full-length) or LATS1-FLAG (full-length) as templates, the full-length GS or LATS1 and its functional domains were amplified, respectively. The stop codons of the genes themselves were removed in the primer design. The full-length GS and LATS1 were introduced with the HA tag (5'-TACCCATACGATGTTCCAGATTACGCT-3'), and the coding sequences of S and FLAG double tags (S: 5'-AAGGAAACAGCAGCCGCAAAATTCGAACGCCAGCACATGGACTCC-3'; FLAG: 5'-GACTACAAGGACGACGATGACAAG-3') and BamHI / XhoI restriction sites were introduced into each functional domain.

[0056] (4) PCR amplification: The PCR reaction system (50 μl) included: 25 μl of high-fidelity DNA polymerase premix (TaKaRaPrimeSTAR® HS), 1 μl each of forward and reverse primers (10 μM), 1 μl of cDNA template, and ddH2O to make up the volume. Reaction program: 98℃ pre-denaturation for 30 seconds; followed by 30 cycles (98℃ for 10 seconds, 60℃ for 15 seconds, 72℃ extension depending on fragment length / calculated at 15-30 seconds per kilobase); and a final extension at 72℃ for 5 minutes. The PCR products were verified by 1% agarose gel electrophoresis and purified using a gel extraction kit.

[0057] (5) Vector linearization, ligation, and plasmid acquisition: Following the pre-cloning restriction enzyme digestion protocol for the LIC vector, the expression vector was double-digested with the corresponding restriction endonucleases (BamHI and XhoI) to linearize it and generate ends matching the PCR product, enabling ligation-independent cloning (LIC) with the PCR-amplified and ExoIII-treated insert. The enzyme digestion reaction system (50 μl example) consisted of: 3 μg pcDNA3.1 vector plasmid, 5 μl 10×FastDigest Buffer, 20 U BamHI, 20 U XhoI, and ddH2O to a final volume of 50 μl. The mixture was gently mixed and incubated at 37 °C for 1 hour. Subsequently, 1% agarose gel electrophoresis was performed, the linear band was excised, and purified using a gel extraction kit. A260 / 280 = 1.8–2.0, concentration ≥100 ng / μl. Ligation was performed using the LIC ligase-free method. The ligation reaction system (20 μl example) consisted of: 50 ng of linearized vector, 150 ng of purified PCR insert (vector: fragment = 1:3 molar ratio), 2 μl of 10×NEB Exo III buffer, and ddH2O to a final volume of 20 μl. After mixing, the mixture was pre-cooled on ice for 5 minutes. 1 U of Exo III exonuclease was added, mixed, and incubated on ice for 1 hour for further digestion. Subsequently, 2 μl of 0.5 M EDTA (pH 8.0) was added to terminate the reaction (final concentration 50 mM), the mixture was gently tapped to mix, and the mixture was briefly centrifuged. The mixture was then incubated in a 58-65 °C water bath for 10 minutes to completely denature any remaining enzyme protein. The mixture was immediately transferred to ice for 2 minutes. Finally, 5 μl of the ligation product was directly subjected to DH5α heat shock transformation (30 min on ice, 42°C heat shock for 45 s, then 5 min on ice). The transformed product was then plated on LB agar plates containing 100 μg / ml ampicillin and incubated upside down at 37°C for 16 hours. At least three single colonies were picked, and positive bands were verified by colony PCR. Sequencing confirmed the correct insertion sequence and the absence of mutations. For the correctly sequenced bacterial cultures, plasmids were extracted using a plasmid extraction kit, and the plasmid concentration (ng / μl) and purity (A260 / A280 = 1.8–2.0) were determined using Nanodrop, yielding the recombinant plasmid required for the study.

[0058] Example 2: Establishment of an Intrinsic / Extrinsic Validation System for GS-LATS Protein Interactions

[0059] (1) Construction of exogenous overexpression system:

[0060] The day before the experiment, well-grown HEK293T cells were seeded into 6cm culture dishes at a density of 0.7 × 10⁶ cells per dish. 6For each cell type, 5 ml of DMEM medium containing 10% fetal bovine serum and 1×P / S was added to each dish. The cells were incubated at 37°C with 5% CO2. The next day, when the cell density reached 70-80%, transient co-transfection was performed using the polyethyleneimine (PEI) method. Validation groups were set up for both forward (HA-GS + SF-LATS1 domain plasmids) and reverse (HA-LATS1 + SF-GS domain plasmids). The specific transfection system was as follows: 2 μg of total plasmid (mixed in equal mass ratios to the required co-transfection plasmids) was dissolved in 100 μl of Opti-MEM; in another tube, 8 μl of PEI (1 mg / ml) was dissolved in 100 μl of Opti-MEM. The two tubes were mixed and incubated at room temperature for 15 minutes before adding the complex to HEK293T cells. Forty-eight hours after transfection, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. Add 200 μl of RIPA lysis buffer to each well, lyse on ice for 30 minutes, collect the lysis buffer, centrifuge at 12000g for 15 minutes at 4℃, and take the supernatant. Take a small amount (about 50 μl) and add 50 μl of 2×SDS loading buffer as the input control sample. The remaining protein lysis buffer is used for (2) Co-IP detection.

[0061] (2) Interaction of exogenous co-immunoprecipitation (Co-IP) detection:

[0062] Washing the FLAG beads: Wash 20 μl of FLAG bead suspension three times with 1 ml of pre-cooled buffer (no more than 50 μl of FLAG bead suspension should be washed with each 1 ml of pre-cooled buffer). Discard the supernatant and resuspend. Add the equilibrated Anti-FLAG beads (Sigma) to the lysis buffer (1) and incubate overnight at 4°C using a rotary mixer. The next day, wash the beads four times with pre-cooled buffer. Finally, add 100 μl of 1×SDS loading buffer to the beads and denature the protein at 95°C for 10 minutes. Use Western blotting to detect the interaction between exogenous GS and LATS proteins.

[0063] (3) Construction of endogenous interaction system (taking H1299 lung cancer cells as an example):

[0064] To eliminate exogenous overexpression and tag interference, the interaction between endogenous GS and LATS1 was further verified in H1299 lung cancer cells. The day before the experiment, well-grown H1299 cells were seeded in 10cm culture dishes, with 1×10⁶ cells per dish. 6Cells were added to each dish with 10 ml of RPMI-1640 medium containing 10% fetal bovine serum and 1×P / S, and incubated at 37°C in a 5% CO2 incubator. The next day, when cell density reached 90%, cells were washed twice with pre-chilled PBS, and lysed on ice for 30 min with 1 ml of mild NETN lysis buffer. The lysis buffer was collected, centrifuged at 12000g for 15 min at 4°C, and the supernatant was collected. 50 μl was used as input. The remaining lysis buffer was pre-cleaned with Protein A / G agarose beads and normal IgG for 1 hour. The precipitate was washed four times with pre-chilled buffer, followed by 100 μl of 1×SDS loading buffer and protein denaturation at 95°C for 10 min; this was the IgG sample. The supernatant was transferred to new tubes and divided into two portions: one portion was added with 2 μg of anti-GS antibody (BD Biosciences, catalog number 610517), and the other with 2 μg of anti-LATS1 antibody (CST, catalog number 3477), and incubated overnight at 4°C by rotation. The next day, samples from the Co-IP group were obtained using the same washing method as the IgG group. These samples could be used directly for immunoblotting analysis (4) or stored at -80°C.

[0065] (4) Immunoblotting detection of GS-LATS protein intra- / exogenous interactions:

[0066] The above samples were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk at room temperature for 1 hour, they were incubated overnight at 4°C with primary antibodies against HA (1:2000), FLAG (1:5000), GS (1:1000), and LATS1 (1:1000), respectively. After washing with TBST, the membranes were incubated with the corresponding secondary antibodies (1:5000) at room temperature for 1 hour. After washing with TBST, the membranes were developed using ECL chemiluminescence reagent, and images were acquired. The results showed that GS and LATS1 proteins could be specifically co-precipitated in both exogenous and endogenous systems, and the β-G domain of GS and the UBA domain of LATS1 were key interaction regions, confirming the existence of the GS-LATS complex and laying a crucial molecular biological foundation for the screening system.

[0067] Example 3: Establishment of a screening and validation system for small molecule inhibitors of GS-LATS protein interaction

[0068] (1) GS-LATS Protein Interaction Targeted Drug Screening System Flow: This invention provides a screening scheme for anti-tumor compounds based on GS-LATS protein interactions, realizing a complete chain process of systematic screening, mechanism verification, and in vivo efficacy evaluation of small molecule inhibitors targeting the GS-LATS interaction interface, to ensure that the screened compounds have clear targeting, good druggability, and synergistic therapeutic potential. (See attached flowchart) Figure 7 ).

[0069] (2) Cell line selection and seeding: Three non-small lung cancer cell lines with significant differences in genetic background and metabolic phenotype were selected: H1299, H2030, and A549. Lung cancer cells in logarithmic growth phase and in good condition were selected. After trypsin digestion and termination in complete medium containing 10% FBS, the viability was ensured to be ≥95% using an automated cell counter combined with trypan blue exclusion method. 1500-2000 cells per well for H1299 and H2030, and 1000-1500 cells per well for A549, with a total volume of 100 μl / well, were seeded into 96-well plates. 200 μl of sterile PBS was filled into the peripheral wells to minimize evaporation and temperature gradient. The cells were incubated horizontally at room temperature for 15 minutes, shaken in a crosswise manner, and then incubated at 37 ℃ in a 5% CO2 incubator for 12 hours to ensure cell adhesion.

[0070] (3) Single and combination dosing regimens: Single dosing group: Verteporfin (YAP-TEAD blocker, 10 μM, 0.1% DMSO); CB839 (GLS allosteric inhibitor, 3 μM, 0.05% DMSO); Rapamycin (mTORC1 inhibitor, 100 nM, 0.02% DMSO). Combination dosing regimens: Verteporfin + CB839, Rapamycin + Verteporfin, CB839 + Rapamycin. Solvent control group: consistent with the highest DMSO concentration group, with an additional blank culture medium well (cell-free, with an equal volume of culture medium) for background subtraction.

[0071] (4) CCK-8 detection of proliferation changes: Remove the 96-well plate from the incubator and quickly prepare the CCK-8 working solution in a clean bench: Take the preheated 37 ℃ complete culture medium and CCK-8 stock solution at a ratio of 9:1 and gently invert to mix, avoiding foaming. Use a multi-pipette to slowly add 110 μl / well along the wall, avoiding air bubbles at the edges; always add the solution in the order of control group first and then experimental group to prevent high concentration drug residue. Return the 96-well plate to the incubator and incubate at 37 ℃ and 5% CO2 in the dark for 2 hours. Before detection, use the microplate reader's shaking option to shake for 10 s to make the color uniform, and then read the absorbance OD value at a wavelength of 450 nm. The background is subtracted by the blank culture medium wells of the same plate (cell-free, with an equal volume of culture medium + CCK-8), and the negative control is the same volume of solvent as the drug treatment group. Finally, the OD values ​​at five time points (0, 24, 48, 72, and 96 hours) were imported into GraphPad Prism. With culture time as the x-axis and the corrected OD value as the y-axis, the dynamic effects of single or combined drug administration on the proliferation rate of three types of lung cancer cells were quantified.

[0072] (5) Effect of combined drug administration on tumor growth in experimental animals: Five-week-old male BALB / c nude mice were placed in an SPF barrier system for one week for acclimatization, maintaining a 12-hour light / dark cycle, constant temperature and humidity, and free access to food. Cell suspensions were prepared, and well-grown control vector (Control) and GS gene knockout (GS KO) stable Hep1-6 cell lines were digested with trypsin, centrifuged, and the cells were collected. After resuspending in sterile PBS, the cells were counted, and 100 μl of cell suspension containing 1 × 10⁻⁶ cells was injected into each nude mouse. 7 Cells were counted individually, and a 100 μl suspension (containing 50% Matrigel) was prepared and placed on ice. The cell suspension was aspirated using an insulin injection needle and subcutaneously injected into the right rib area. After needle withdrawal, gentle pressure was applied for 3 seconds to prevent leakage. The day of injection was recorded as Day 0. The tumor was observed daily by touch. When the long diameter of the tumor reached 5-7 mm (approximately day 6-7), the initial tumor volume was precisely measured and recorded using electronic calipers. Animals in the control vector group and the GS gene knockout group were randomly and evenly divided into 4 groups (n=8-10), ensuring that the mean difference in initial tumor volume among the groups was <10%. The groups were: solvent control group; CB839 monotherapy group (200 mg / kg, oral administration); Rapamycin monotherapy group (2 mg / kg, intraperitoneal injection); and combination group CB839 + Rapamycin (CB839 200 mg / kg + Rapamycin 2 mg / kg). The administration cycle was 12 days, completed at the same time each day (±30 min), with standard light and free access to food maintained throughout. Every two days, the long diameter (a) and short diameter (b) of the tumor were measured using calipers, and the volume was calculated as V = 0.5 × a × b². Body weight was also recorded; animals were considered to have reached the human endpoint if their body weight decreased by more than 15% or the single diameter increased by more than 20 mm. After the last administration, the tumor was rapidly and completely removed after carbon dioxide asphyxiation. The capsule and necrotic areas were removed, and the surface blood was blotted dry with filter paper before immediate weighing. Subsequently, some tissue was fixed in 4% paraformaldehyde for immunohistochemistry, and the remaining tissue was flash-frozen in liquid nitrogen at -80 °C, ensuring that at least six biologically independent samples were obtained from each group for subsequent statistical and molecular-level analysis.

Claims

1. A method for constructing an antitumor compound combination drug system based on the interaction between glutamine synthase and LATS proteins, characterized in that, Includes the following sequential steps: (1) Based on the coding sequences of human glutamine synthase (GS, GLUL, NM_001033044) and human LATS1 gene (LATS1, NM_004690), specific primers were designed and synthesized to amplify the full-length and functional domain fragments of GS and LATS1 (GS: β-grasp, CD; LATS1: UBA, PY, KD). BamHI / XhoI restriction sites were introduced at the 5′ end of the primers, and the stop codon was removed at the 3′ end and a tag sequence was fused (HA tag was introduced for the full-length fragment, and S-FLAG double tag was introduced for the functional domain fragment). The target DNA fragments were obtained by PCR amplification. (2) The mammalian expression vector pcDNA3.1 was selected and linearized by double digestion with BamHI and XhoI. The PCR-purified fragments obtained in step (1) were mixed with the linearized vector at a molar ratio of 3:1 and ligated with T4 DNA ligase at room temperature for 1 hour. The mixture was then transformed into DH5α competent cells. Single colonies were picked, and after colony PCR and sequencing verification, endotoxin-free plasmids were extracted to obtain recombinant expression plasmids HA-GS-FL, SF-GS-β-G, SF-GS-CD, HA-LATS1-FL, SF-LATS1-UBA, SF-LATS1-PY, and SF-LATS1-KD. (3) The recombinant plasmids obtained in step (2) were co-transfected into HEK293T cells according to the preset combination, the forward verification group (HA-GS+SF-LATS1 domain plasmids) and the reverse verification group (HA-LATS1+SF-GS domain plasmids). After culturing for 48 hours, the cells were lysed with RIPA lysis buffer, and immunoprecipitation was performed using FLAG affinity magnetic beads. The exogenous interaction between GS and LATS1 protein and each functional domain was positively verified by Western blotting, and the exogenous interaction between LATS1 and GS protein and each functional domain was negatively verified. (4) Using tumor cells such as H1299 and H2030, lyse them with NETN lysis buffer, extract endogenous total protein, take the supernatant, part of which is used as input, and the remaining lysis buffer is pre-cleansed with Protein A / G agarose beads and normal IgG for 1 hour. The precipitate is the IgG group, which is washed 4 times with pre-cooled buffer. The supernatant is discarded on the last wash, and 100 μl of 1×SDS loading buffer is added to form the IgG group (isotype control group). The supernatant is divided into two parts: the positive validation group, with 2 μg of anti-GS antibody added; and the reverse validation group, with 2 μg of anti-LATS1 antibody added. They are incubated overnight at 4℃ to form endogenous antigen-antibody complexes. The next day, they are washed 4 times with pre-cooled buffer. The supernatant is discarded on the last wash, and 100 μl of 1×SDS loading buffer is added to form the IgG group (isotype control group). The CoIP group (experimental group) was prepared by denaturing samples in 1×SDS loading buffer at 95°C in a metal bath for 10 minutes. Western blotting was used to verify the interaction between GS and LATS1 proteins in the forward direction and the interaction between LATS1 and GS proteins in the reverse direction. If LATS1 was detected in the sample precipitated with GS antibody, and GS was also detected in the sample precipitated with LATS1 antibody, while no signal was detected in the normal IgG control, this would confirm a direct interaction between the two proteins under physiological conditions, and this result would not be affected by the expression level of the exogenous tag. (5) Based on the GS-LATS interaction system verified in steps (3) and (4), small molecule compounds that can interfere with GS-LATS interaction were screened. At the cellular level, lung cancer cell lines H1299, H2030, and A549 with representative genetic backgrounds were selected. After trypsin digestion and precise counting, they were seeded in 96-well plates at optimized densities (H1299 / H2030: 1500-2000 cells per well; A549: 1000-1500 cells per well). They were treated with single or combined drugs, including the YAP inhibitor vertepor. Verteporfin (10 μM), the GLS inhibitor CB839 (3 μM), and the mTOR inhibitor rapamycin (100 nM) were administered at multiple time points (0, 24, 48, 72, and 96 hours) after treatment. Cell viability was assessed using the CCK-8 assay, and cell growth curves were plotted to quantitatively evaluate the inhibitory effects of different administration regimens on cell proliferation. Furthermore, at the animal level, a tumor-bearing model was established using 5-week-old male nude mice, and the control vector (Control) and the GS gene knockout vector (GS) were subcutaneously inoculated, respectively. Hep1-6 cells (KO) were used to induce tumor growth. Animals were randomly divided into four groups after tumor growth: a solvent control group, a CB839 monotherapy group (200 mg / kg), a rapamycin monotherapy group (2 mg / kg), and a CB839 + rapamycin combination therapy group. Treatment continued for 12 days. Tumor length and short diameter were measured every two days to calculate tumor volume and plot growth curves. At the end of the experiment, animals were sacrificed, and tumors were dissected and weighed. At least six independent biological samples were collected from each group to systematically and quantitatively evaluate the antitumor activity of the candidate compounds and the synergistic effect of combination therapy at both in vitro and in vivo levels. In summary, this invention constructs a systematic and hierarchical screening strategy for antitumor compounds. Targeting the "GS-LATS protein-protein interaction interface," it integrates multiple steps, including in vitro high-throughput screening, protein-protein interaction interference verification, downstream signaling pathway analysis, synergistic effect assessment, and in vivo efficacy evaluation, to achieve full-chain screening and functional confirmation of potential small molecule inhibitors. This provides a systematic and reproducible experimental platform and evaluation system for the development of antitumor drugs targeting protein-protein interaction interfaces (PPIs) (see Figure 7 for the flowchart).

2. The method according to claim 1, characterized in that: The design, synthesis, and amplification of the full-length GS and LATS1 sequences and their respective domains in step (1) are as follows: (1) The GS functional domain includes a grasping domain (β-grasp, β-G) and a catalytic domain (CD); the LATS1 functional domain includes a ubiquitin-associated domain (UBA domain, UBA), a grasping domain (PPxY motif, PY), and a catalytic domain (Kinase domain, KD). Based on their coding sequences, specific primers were designed and synthesized. Protective bases and BamHI / XhoI restriction sites were introduced at the 5′ end of the primers, and the stop codon was removed at the 3′ end and a tag coding sequence and a new stop codon were directly linked. The full-length GS and LATS1 primers were introduced with a HA tag (5'-TACCCATACGATGTTCCAGATTACGCT-3'), and each functional domain was introduced with S and FLAG double tags (S: 5'-AAGGAAACAGCAGCCGCAAAATTCGAACGCCAGCACATGGACTCC-3'; FLAG: 5'-GACTACAAGGACGACGATGACAAG-3'). (2) PCR amplification: High-fidelity DNA polymerase was used for PCR amplification to ensure the accuracy and integrity of the amplified products. The specific reaction system was 50 μl, including: 25 μl of high-fidelity DNA polymerase premix (TaKaRa PrimeSTAR®HS), 1 μl each of forward and reverse primers (concentration 10 μM), 1 μl of cDNA template, and the remaining volume was supplemented to 50 μl with ddH2O. The PCR reaction program was set as follows: pre-denaturation treatment at 98℃ for 30 seconds to completely dissociate the double-stranded DNA; then 30 cycles of amplification reaction were performed, each cycle including denaturation at 98℃ for 10 seconds, annealing at 60℃ for 15 seconds, and extension at 72℃ (the extension time was calculated according to the length of the target fragment, at a ratio of 15-30 seconds per kilobase); finally, a final extension was performed at 72℃ for 5 minutes to ensure the complete synthesis of all amplified products. After amplification, 5 μl of the product was taken for 1% agarose gel electrophoresis (voltage 130V, time 30 minutes). Using a marker as a reference, observe in a UV gel imaging system whether it is a single, clear target band. After confirming that it is correct, strictly follow the instructions using a commercial gel extraction kit to cut out the target band for purification. The purification steps include: dissolving the gel block containing the target band in binding buffer, adsorbing DNA through a centrifuge column, removing impurities with washing buffer, and finally eluting with low-salt elution buffer to obtain high-purity DNA fragments. The concentration (ng / μl) and purity (A260 / A280 should be between 1.8 and 2.0) are determined using NanoDrop and stored at -20℃ for later use.

3. The method according to claim 1, characterized in that: The LIC connection method described in step (2) includes the following steps: (1) Linearization vector preparation: The expression vector pcDNA3.1, suitable for ligation-independent cloning (LIC), was selected. Double digestion with BamHI and XhoI restriction endonucleases was performed to achieve complete linearization of the vector and provide matching ends for subsequent insertion fragments. A 50 μl digestion reaction system was prepared in a sterile 200 μl EP tube without nucleases: 3 μg of pcDNA3.1 vector plasmid was added to 5 μl of 10×FastDigest Buffer, followed by 20 U each of BamHI and XhoI endonucleases. The total volume was brought to 50 μl with ddH2O. The mixture was gently pipetted to avoid air bubbles. A brief centrifugation was performed to concentrate the liquid at the bottom of the tube. The reaction tube was placed in a constant temperature water bath and incubated at 37°C for 1 hour. After incubation, 5 μl of the digestion product was subjected to 1% agarose gel electrophoresis (130V, 30 minutes). DL5000 DNA was used for verification. Using the marker as a reference, a single, clear linear band should be observed in the UV gel imaging system, while the undigested original plasmid should appear as a supercoiled or open-circular band. After confirming successful linearization, quickly cut the gel block corresponding to the target band under UV light and purify it strictly according to the instructions using a commercial gel extraction kit. The purification steps are the same as above. Store at -20℃ for later use. (2) Ligation reaction (LIC ligase-free method): The LIC method was used for seamless ligation of the linearized vector and the exonuclease-treated insert fragment. A 20 μl ligation reaction system was prepared in a sterile 200 μl EP tube without nuclease: 50 ng of linearized vector DNA, 150 ng of purified PCR insert fragment (molar ratio of vector to insert fragment was 1:3), and 2 μl of 10×NEB Exo III buffer was added. The total volume was brought to 20 μl with ddH2O. The mixture was gently mixed with a pipette, briefly centrifuged, and then pre-chilled on ice for 5 minutes to optimize subsequent enzyme reaction conditions. One unit (U) of Exonuclease III (Exo III) was added to the pre-chilled system, and the mixture was gently pipetted and mixed again. The system was immediately returned to ice and incubated for 1 hour. After incubation, 2 μl of pre-chilled 0.5 M EDTA solution (pH 8.0) was added to chelate magnesium ions in the reaction system, allowing Exo III to ligate into the ligase. The digestion reaction was terminated by rapid inactivation of Exo III. After gentle tapping to mix, the mixture was briefly centrifuged. The reaction tube was then placed in a 58–65°C water bath for 10 minutes to ensure complete heat denaturation of Exo III protein. Immediately afterward, the tube was transferred to an ice bath to cool for 2 minutes to stabilize the DNA complex. The ligation product was then obtained for transformation. (3) Transformation, verification, and plasmid acquisition: Take 5 μl of the above LIC ligation product and add it to 50 μl of DH5α competent cell suspension that is thawed on ice. Gently mix with a pipette tip and let stand on ice for 30 minutes. Then place the EP tube in a 42°C water bath for heat shock for 45 seconds, and quickly return it to the ice bath for 5 minutes. Spread the plating evenly on an LB solid plate containing 100 μg / ml ampicillin using sterile spreader beads. Invert the plate and incubate it in a 37°C incubator for 16 hours until single colonies are clearly visible. Pick at least 3 plump single colonies with a sterile pipette tip and inoculate them into 5 ml of LB liquid medium containing the corresponding antibiotic. Shake vigorously at 37°C (250°F). Incubate overnight at rpm, collect 1 ml of bacterial culture by centrifugation, and verify the presence of the insert fragment using a colony PCR kit: using bacterial cells as a template, amplify using universal vector primers or insert fragment-specific primers, and confirm the presence of a band of the expected size by agarose gel electrophoresis. Send PCR-verified positive bacterial culture samples to Shanghai Sangon Biotech for sequencing, and perform bidirectional sequencing using vector-specific sequencing primers to ensure that the inserted sequence is completely correct and free of any base mutations or frameshifts. For bacterial cultures that have been successfully sequenced, extract plasmids using a commercial plasmid medium-quantity extraction kit according to the instructions, and determine the concentration (ng / μl) and purity (A260 / A280 should be between 1.8 and 2.0) of the obtained plasmids using NanoDrop. The finally obtained high-purity, correctly constructed recombinant plasmids are aliquoted and stored at -80℃ for long-term storage.

4. The method according to claim 1, characterized in that: Step (3) of the external interaction verification includes the following steps: (1) Cell preparation and seeding: HEK293T cells with high transfection efficiency were selected for transfection. One day before the experiment, HEK293T cells in the logarithmic growth phase were seeded at 0.7 × 10⁶ cells per dish. 6 Seed cells at a density of 1000 mcg / mL into 6 cm dishes, with 5 ml of preheated 37°C complete culture medium (DMEM complete medium containing 10% fetal bovine serum and 1× penicillin / streptomycin (P / S)) added to each dish. Incubate overnight at 37°C in a 5% CO2 incubator. Transfect cells when the cell density reaches 70-80%. (2) Preparation of co-transfection plasmid complexes: The GS series plasmids include: HAGSFL (full-length GS fused with HA tag), SF-GS-βG (GS grasping domain fused with SFLAG dual tag), and SFGSCD (GS catalytic domain fused with SFLAG dual tag); the LATS1 series plasmids include: HALATS1FL (full-length LATS1 fused with HA tag), SFLATS1UBA (LATS1 ubiquitin-associated domain fused with SFLAG dual tag), SFLATS1PY (LATS1 PPxY motif domain fused with SFLAG dual tag), and SFLATS1KD (LATS1 kinase domain fused with SFLAG dual tag). According to the experimental design, transfection mixtures for the forward validation group (HA-GS full-length plasmid mixed with each SF-LATS1 domain plasmid in equal mass ratio) and the reverse validation group (HA-LATS1 full-length plasmid mixed with each SF-GS domain plasmid in equal mass ratio) were prepared respectively. The total amount of plasmid for each transfection was 2 μg. The specific operation is as follows: Take a sterile 1.5ml EP tube, add 100μl of Opti-MEM serum-free medium, and add 2μg of total plasmid DNA to the tube. Gently pipette to mix, avoiding vigorous vortexing. Take another sterile 1.5ml EP tube, add 100μl of Opti-MEM serum-free medium, and add 8μL of polyethyleneimine (PEI) transfection reagent (concentration 1mg / mL). Gently mix with a pipette. Combine the two tubes, add the diluted PEI solution to the solution containing plasmid DNA, gently tap the tube wall to mix, and incubate at room temperature for 15 minutes to form a stable DNA-PEI complex. Add the complex to HEK293T cells that have been replaced with fresh complete medium, gently shake the culture dish in a cross shape to distribute evenly, and return to the incubator for further culture. (3) Cell lysis and sample preparation: 48 hours after transfection, remove the culture dishes and place them on ice. Discard the culture medium and gently wash the cells twice with pre-chilled PBS. Thoroughly aspirate the washing solution. Add 200 μl of pre-chilled RIPA lysis buffer (containing 1% NP40, 50 mM TrisHCl pH 7.4, 150 mM NaCl, 0.25% sodium deoxycholate (DOC), 10% glycerol, 1 mM PMSF, 1× protease inhibitor, and 1× phosphatase inhibitor) to each dish. Place the culture dishes on ice for lysis for 30 minutes, gently shaking the dishes every 10 minutes to ensure that the lysis buffer covers all cells. Use a cell scraper to scrape off the cells and transfer the lysis buffer to a pre-chilled 1.5 ml container. Centrifuge at 12000g for 15 minutes in an EP tube at 4°C. Aspirate the supernatant and transfer it to a new pre-chilled EP tube to obtain total protein lysis buffer. Take a small amount (approximately 50 μL) and add an equal volume of 2×SDS loading buffer. Mix well and use as the input control sample. The remaining protein lysis buffer will be used for CoIP. (4) Immunoprecipitation (Co-IP): Immunoprecipitation was performed using a commercially available FLAG affinity magnetic bead kit. 20 μl of FLAG magnetic bead suspension was washed three times with 1 ml of pre-chilled buffer, placed on a magnetic rack, and the supernatant was discarded. The suspension was then resuspended with an equal volume of buffer. Total protein lysis buffer was mixed with the equilibrated FLAG magnetic beads and incubated overnight (12–16 hours) at 4°C to allow for complete binding of the FLAG-tagged proteins. The next day, the centrifuge tube was placed on a magnetic rack, the magnetic beads were adsorbed, and the supernatant was discarded. The magnetic beads were washed four times with 1 ml of pre-chilled buffer, ensuring thorough removal of liquid after each wash to eliminate non-specific binding. Finally, 100 μl of 1×SDS loading buffer was added to the magnetic beads, and the mixture was heated at 95°C for 10 minutes to denature the proteins and dissociate them from the magnetic beads. After centrifugation, the supernatant was collected as the Co-IP sample. This sample can be used directly for immunoblotting analysis or stored at -80°C. (5) Immunoblotting detection of the interaction between exogenous GS and LATS proteins: The Input control group and CoIP group samples were subjected to SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight at 4°C with the following primary antibodies: anti-HA mouse monoclonal antibody (1:2000) to detect HA tag protein; anti-FLAG mouse monoclonal antibody (1:5000) to detect FLAG tag protein. The next day, after washing the membrane with TBST, it was incubated at room temperature for 1 hour with HRP-labeled secondary antibody of the corresponding species (1:5000), and then developed and imaged by ECL chemiluminescence. By comparing the co-precipitation of HA tag protein and FLAG tag protein in CoIP products in different combinations, the interaction between GS and LATS1 and its key structural domains were clarified.

5. The method according to claim 1, characterized in that: Step (4) verification of endogenous interactions includes the following steps: (1) Cell preparation and seeding: One day before the experiment, tumor cells in the logarithmic growth phase and in good condition were seeded at a rate of 1×10⁶ cells per dish. 6 Seed cells at a density of approximately 1000 m² in 10 cm dishes, with 10 ml of preheated 37°C complete culture medium added to each dish. Incubate the dishes in a 37°C, 5% CO₂ incubator until the cell density reaches approximately 90%. At this point, the cells are in an active growth phase, and the expression levels of endogenous proteins are stable. (2) Cell lysis and total protein extraction: Remove the culture dishes from the incubator and place them on ice. Discard the culture medium and gently wash the cells twice with pre-chilled PBS, 5 ml each time, to thoroughly remove residual serum and metabolic waste. Prepare and pre-chill mild NETN lysis buffer in advance (containing 0.5% NP40, 20 mM TrisHCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1× protease inhibitor, and 1× phosphatase inhibitor). Add 1 ml of pre-chilled lysis buffer to each dish and lyse on ice for 30 minutes, gently shaking the culture dish every 10 minutes to ensure that the lysis buffer covers all cells. Then, use a cell scraper to gently and thoroughly scrape the cells off in the same direction and transfer the lysis buffer to 1.5 ml of pre-chilled PBS. Centrifuge at 12000g for 15 minutes in an EP tube at 4°C. Transfer the supernatant to a new pre-chilled EP tube to obtain total protein lysis buffer. Take a small amount (approximately 50 μl) and add an equal volume of 2×SDS loading buffer. Mix well and use as the input control sample. The remaining protein lysis buffer will be used for subsequent immunoprecipitation. (3) Pre-cleaning to reduce non-specific binding: According to the volume of lysis buffer, take an appropriate amount of Protein A / G agarose beads, wash them 3 times with pre-cooled buffer, and resuspend them with an equal volume of lysis buffer. Then, add the washed Protein A / G bead suspension to the total lysis buffer, and add normal IgG (usually mouse or rabbit IgG, with a final concentration of about 1 μg / ml) of the same species as the primary antibody used in subsequent experiments. Place the mixture on a rotary mixer and incubate at room temperature for 1 hour. After incubation, remove the supernatant at 4°C and magnetically attach it for 5 minutes. Transfer the supernatant to a new tube for subsequent specific immunoprecipitation. Wash the precipitate 4 times with pre-cooled buffer, adding 1 ml of pre-cooled buffer each time, and wash at 4°C for 10 minutes. Then discard the supernatant and add 100 μl of 1×SDS loading buffer. This is the IgG group (isotype control group). (4) Specific immunoprecipitation: The pre-cleaned lysis buffer was divided into two equal portions for forward and reverse immunoprecipitation verification, respectively. Experimental group 1 (IP: GS): 2 μg of anti-GS mouse monoclonal antibody (BDBiosciences, catalog number 610517) was added to one portion of the lysis buffer; Experimental group 2 (IP: LATS1): 2 μg of anti-LATS1 rabbit monoclonal antibody (CST, catalog number 3477) was added to the other portion of the lysis buffer. All samples were placed on a 4°C rotary mixer and gently incubated overnight (12-16 hours) to ensure that the antibody and endogenous antigen were fully bound. After overnight incubation, the samples were magnetically adsorbed at 4°C for 5 minutes, the supernatant was discarded, and the precipitate was washed 4 times with pre-cooled buffer, each time with 1 ml of pre-cooled buffer, and washed at 4°C for 10 minutes. The supernatant was then discarded, and 100 μl of 1×SDS loading buffer was added. This was the CoIP group (experimental group). This sample can be used directly for immunoblotting analysis or stored at -80°C. (5) Immunoblotting detection of the interaction between endogenous GS and LATS proteins: The Input control group, IgG group and CoIP group samples were subjected to SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight at 4°C with the following primary antibodies: anti-GS antibody (from a different species than IP antibody, such as rabbit anti-GS, used to verify the precipitation efficiency of IP:GS); anti-LATS1 antibody (from a different species than IP antibody, such as mouse anti-LATS1, used to verify the precipitation efficiency of IP:LATS1) and the corresponding internal control antibody (such as β-Actin). After washing the membrane with TBST, it was incubated with HRP-labeled secondary antibody of the corresponding species at room temperature for 1 hour, and then developed and imaged by ECL chemiluminescence.

6. The method according to claim 1, characterized in that: Step (6) involves screening small molecule compounds that can interfere with GSLATS interactions, which includes the following steps: In vitro cell-level screening and evaluation: (1) Cell line selection and seeding: Three non-small cell lung cancer cell lines with representative genetic backgrounds and metabolic characteristics were selected to cover tumor heterogeneity: H1299 cells (p53 deletion, high basal activation level of YAP pathway), H2030 cells (KRAS mutation, significant glutamine metabolism dependence), and A549 cells (KRAS / p53 double mutation, strong epithelial-mesenchymal transition ability). All cells were taken from a population in logarithmic growth phase and in good condition. After trypsin digestion, the culture medium was terminated with 10% fetal bovine serum to prepare a single-cell suspension. The cells were then counted using an automated cell counter. Precise cell counting was performed using the trypan blue exclusion method to ensure cell viability ≥95%. Based on the optimized seeding density from preliminary experiments, H1299 and H2030 cells were seeded at 1500-2000 cells per well, and A549 cells at 1000-1500 cells per well, with a final volume of 100 μl per well. These were seeded into 96-well cell culture plates. To eliminate edge effects, the peripheral wells were filled with 200 μl of sterile PBS. After seeding, the culture plates were incubated horizontally at room temperature for 15 minutes, followed by gentle shaking using the "cross-shading method" to ensure uniform cell distribution. Finally, the culture plates were placed in a 37°C, 5% CO2 incubator for observation, allowing cells to fully adhere and enter the stable growth phase. (2) Single and combination dosing regimens: Single and combination dosing groups were set up to evaluate synergistic effects. The YAP-TEAD interaction inhibitor verteporfin (10 μM, solvent: 0.1% DMSO), the glutaminase allosteric inhibitor CB839 (3 μM, solvent: 0.05% DMSO), and the mTORC1 inhibitor rapamycin (100 nM, solvent: 0.02% DMSO) were used. The combination dosing groups were designed to explore the effects of pathway cross-inhibition, including the combination of CB839 and verteporfin, the combination of rapamycin and verteporfin, and the combination of CB839 and rapamycin. A strict solvent control group was set up, with the final DMSO concentration consistent with the highest drug group. A blank group containing only complete culture medium and CCK-8 reagent was set up for background value subtraction and normalization in subsequent tests. (3) Cell proliferation and viability detection and data analysis: CCK-8 was used to detect cell viability, and GraphPad Prism software was used to quantify the effect of the drug on lung cancer cell proliferation. Before the start of the preset detection time points (0, 24, 48, 72, 96 hours), the complete culture medium was preheated in a 37°C water bath for at least 30 minutes. The 96-well plate to be tested was taken out of the incubator, and subsequent operations were performed in the biosafety cabinet. CCK-8 working solution was prepared: according to the number of wells to be tested (leaving about 10% redundancy), the required volume of CCK-8 stock solution and preheated complete culture medium was calculated, and the solution was prepared in an accurate ratio of 1:9 (e.g., 1 ml CCK-8 stock solution + 9 ml of preheated complete culture medium). Add 1 ml of culture medium to a sterile centrifuge tube, gently invert and mix 10-15 times, avoiding vortexing to prevent air bubbles. Store the prepared working solution in the dark at room temperature and use within 30 minutes. Next, use an 8-channel or 12-channel pipette for sample addition to ensure operational efficiency and well-to-well consistency. Before adding samples, gently aspirate and mix the working solution several times in the sample well. Hold the pipette tip at an angle to about 1 / 3 of the way down the well wall and slowly add 110 μl of the working solution, allowing the liquid to flow down the well wall. Add the solvent control group (DMSO or PBS) first, then the single-drug group, and finally the combination-drug group. Replace with a new sterile pipette tip after each group is added. During sample addition, closely observe the wells for air bubbles; if any are present, gently puncture them with a sterile fine needle. After adding samples, gently shake the 96-well plate horizontally several times to ensure even distribution of liquid in the wells. Return to a 37°C, 5% sterile environment. Incubate in a CO2 cell culture incubator in the dark for 2 hours. After incubation, remove the culture plate and use the shaking function of the microplate reader to shake at a medium-low speed for 10 seconds to fully dissolve the formazan crystals in the wells and homogenize the color. Immediately use a multi-functional microplate reader to detect the OD value at a wavelength of 450 nm and record the OD value of the blank control group (containing only an equal volume of culture medium and CCK-8 working solution, without cells). Record the OD values ​​from each experimental well. 450 Subtract the OD values ​​from the blank wells containing only culture medium and CCK-8 reagent. 450 The background-corrected OD values ​​were obtained, and then normalized to the background-corrected OD values ​​at 0 hours (before drug treatment) for each group. The relative cell viability at each time point was calculated as follows: Relative cell viability = [(background-corrected value at this time point) / (background-corrected value at 0 hours)] × 100%. Furthermore, the proliferation inhibition rate could be calculated by comparing it with the solvent control group at the same time point: Proliferation inhibition rate = [1 − (relative cell viability of the drug group / relative cell viability of the solvent control group)] × 100%. The processed data were imported into GraphPad Prism software, and the cell growth / inhibition dynamic curves of each experimental group were plotted with culture time (hours) as the x-axis and relative cell viability (%) or proliferation inhibition rate (%) as the y-axis. Each data point was expressed as mean ± standard deviation (n≥3 independent biological replicates). In vivo experimental animal efficacy verification: (1) Preparation of experimental animals and establishment of tumor-bearing model: Five-week-old male BALB / c nude mice were selected as recipient animals to eliminate the interference of the acquired immune system on the growth of transplanted tumors and the evaluation of drug efficacy. All nude mice were placed in an SPF-grade animal room with a barrier system for one week for acclimatization. The environment was strictly controlled under constant temperature and humidity and 12-hour light / dark cycle conditions. Sufficient feed and sterile water were provided for them to eat freely. Before cell inoculation, Hep1-6 liver cancer cell lines with good growth status and in the logarithmic growth phase were selected, including control vector stable transfected cells (Control) and glutamine synthetase gene knockout cells (GS KO). Cell suspension containing 50% Matrigel was prepared. 100 μl of cell suspension was inoculated into each nude mouse, containing 1×10 7 For cell counting, 100 µl of the suspension was drawn up using an insulin syringe and injected subcutaneously into the right rib area of ​​nude mice at a single point. The injection angle was approximately 30 degrees, and the needle depth was approximately 5 mm. After injection, the needle was slowly withdrawn, and a sterile cotton swab was gently pressed at the injection site for 3 seconds to prevent leakage of the cell suspension. The day of inoculation was recorded as day 0 of the experiment. (2) Grouping and administration regimen: Tumor growth was monitored daily by touch. When the long diameter of the subcutaneous tumor nodules reached 5-7 mm, the long diameter (a) and short diameter (b) of the tumor in each mouse were measured and recorded using a high-precision electronic digital caliper. The volume was calculated as V = 0.5 × a × b 2 Calculate the initial tumor volume. Based on the initial volume, use a random grouping method to group the Control group and the GS group. Animals in the KO group were evenly divided into four subgroups (n=8-10) to ensure that the difference in average initial tumor volume between groups was less than 10%, in order to eliminate the interference of initial tumor burden on drug efficacy evaluation. The treatment regimens for each group were as follows: Solvent control group: Administered the same volume of the corresponding solvent as the drug group (CB839 solvent was administered orally by gavage, and rapamycin solvent was administered intraperitoneally); CB839 monotherapy group: Administered once daily by gavage at a dose of 200 mg / kg, with CMC-NA (sodium carboxymethyl cellulose) as the solvent; Rapamycin monotherapy group: Administered once daily by intraperitoneal injection at a dose of 2 mg / kg, with physiological saline solution as the solvent; Combination therapy group: Administered CB839 (200 mg / kg, orally by gavage) and rapamycin (2 mg / kg, intraperitoneally) simultaneously, with an interval of no more than 30 minutes between administrations. The entire dosing cycle lasted for 12 days, with all administration procedures completed within a fixed time window (±30 minutes) each day to minimize the impact of circadian rhythms on drug metabolism. (3) Detection of the effect of combined administration on tumor growth in experimental animals: During the administration period, the long and short diameters of the tumor of each mouse were measured and recorded every two days using vernier calipers. The tumor volume was calculated and growth curves were plotted. At the same time, the weight of the mice was weighed and recorded every two days as an indicator for assessing systemic toxicity. Animal welfare and ethical guidelines were strictly followed during the experiment. The following humane endpoints were set: weight loss of more than 15% compared with the baseline before administration; tumor diameter (any dimension) exceeding 20 mm; or the appearance of severe activity impairment or signs of pain. Mice that met any of the criteria were immediately euthanized and excluded from the study. The relevant data were not included in the final statistical analysis. Within 24 hours after the last administration, all animals were euthanized by progressive carbon dioxide inhalation. They were then quickly dissected, the subcutaneous tumor was completely removed, the fibrous capsule around the tumor was removed, and the bloodstains and body fluids on the surface of the tumor were gently blotted dry with sterile filter paper. The wet weight was weighed and recorded using an analytical balance. At least 6 valid and biologically independent tumor samples were obtained from each experimental group for statistical analysis. The data were imported into GraphPad. Prism software was used to plot dynamic curves of tumor volume changes and wet weight.

7. An in vitro screening and evaluation system based on GS-LATS protein interactions, constructed by any one of claims 1 to 6, characterized in that: This system integrates a platform for verifying the interaction between exogenous and endogenous proteins, a standardized in vitro cell screening model, and a supporting detection and analysis process. It can efficiently evaluate the antitumor activity of single-drug or combination therapy targeting the GS-LATS interaction interface and related signaling pathways (YAP, GLS, mTOR, etc.). The system is suitable for mechanism research, compound screening, drug synergy analysis, and preclinical efficacy prediction for various tumor types such as lung cancer, liver cancer, and breast cancer.

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