Application of Auranofin in the preparation of drugs for mismatch repair-deficient tumors

Abstract

The present invention claims the use of auranofin in the preparation of tumor drugs for mismatch repair deficiency and ARID1A gene deletion, especially in the preparation of drugs for treating ARID1A. ‑ / ‑ and MLH1 ‑ / ‑ Application in oncology medicine. This invention overcomes the side effects and drug resistance limitations of auranofin. It was found that auranofin specifically binds to the TOPBP1 protein, interfering with its interaction with PHF8 and FANCJ, inhibiting the replication stress response. This finding demonstrates enhanced anti-tumor efficacy in ARID1A-deficient or MLH1-deficient tumor cells. This mechanism of action, independent of traditional TrxR inhibition, offers a new perspective for auranofin therapy and opens the door to precision medicine and new uses for established drugs.

Description

Background Art

[0001] Cancer treatment is a major challenge in medicine, and researchers are constantly searching for more effective therapeutic strategies. Auranofin, a platinum-based compound, has shown potential in cancer therapy by binding to DNA, interfering with replication and transcription processes, and inducing apoptosis in cancer cells. However, its clinical application is limited by potential serious side effects, such as kidney, neurotoxicity, and hearing damage, as well as the potential for drug resistance in cancer cells with long-term use. These issues not only affect patients' quality of life but also may reduce therapeutic efficacy, making auranofin a challenge to practical application.

[0002] During tumor development and progression, replication stress has a significant impact on the survival and proliferation of tumor cells. During rapid tumor cell proliferation, the DNA replication machinery faces endogenous damage, leading to the generation of replication stress. This stress is a key factor in maintaining genomic stability. Due to defects in the G1 / S checkpoint, tumor cells are more prone to errors during DNA replication, making them more dependent on effective replication stress coping mechanisms to avoid the accumulation of genomic mutations. If replication stress is not effectively managed, tumor cells may die due to mitotic collapse. Therefore, the molecular mechanisms of replication stress represent a vulnerability in tumor cell survival and provide potential targets for the development of new tumor treatment strategies.

[0003] The initiation, progression, and proliferation of tumor cells are often associated with genetic defects, particularly mismatch repair deficiency (MMRD) or ARID1A (AT-rich interactive domain-containing protein 1A). MMRD is a genetic defect characterized by impaired function of the intracellular mismatch repair (MMR) system, resulting in an inability to promptly correct DNA base pairing errors. MMRD tumors are clinically characterized by high microsatellite instability (MSI-H), which contributes to tumor development and progression. The treatment of MMRD tumors remains challenging, with limited efficacy from conventional therapies. MMRD defects are typically caused by deletions in genes including MLH1, MSH2, MSH6, and PMS2. MLH1 mutations are relatively common, occurring in approximately 1.28% of all cancers, with the highest prevalence in colon cancer, lung cancer, endometrial cancer, invasive ductal breast carcinoma, and conventional glioblastoma multiforme. However, few effective targeted therapeutic strategies are currently available for these MMRD tumors.

[0004] ARID1A is a gene that plays a key role in DNA chromatin remodeling. As a component of the SWI / SNF chromatin remodeling complex, ARID1A is involved in regulating gene expression, DNA repair, and cell cycle control. Mutations in ARID1A often lead to loss of function, impairing chromatin remodeling and gene expression regulation, thereby promoting tumorigenesis. Approximately 6% of human cancers carry inactivating mutations in the ARID1A gene. Several cancer types exhibit the highest frequency of ARID1A inactivating mutations and loss of expression, including ovarian cancer (approximately 50%), endometrial cancer (approximately 37%), gastric cancer (20-30%), bladder cancer (approximately 20%), hepatocellular carcinoma (approximately 14%), melanoma (approximately 12%), colorectal cancer (approximately 9%), and lung cancer (approximately 8%). Therefore, developing more effective targeted therapies for cancer patients harboring ARID1A mutations is a key focus of current clinical research.

[0005] The present invention creatively discovered that auranofin can competitively bind to TOPBP1, inhibiting the activation of the ATR signaling pathway, thereby preventing the replication stress generated by the frequent replication of tumor cells from being effectively alleviated, ultimately leading to tumor cell death. The present invention creatively discovered that in tumor cells with ARID1A deficiency or mismatch repair pathway deficiency, the use of auranofin treatment increases tumor sensitivity. Auranofin can kill tumor cells at lower doses, increasing patient survival, overcoming the shortcomings of auranofin, such as its significant side effects and drug resistance. Summary of the Invention

[0006] The present invention provides a use of a competitive inhibitor of TOPBP1 in the preparation of a pharmaceutical composition for treating and / or improving tumors, wherein the tumor is selected from:

[0007] i) Mismatch repair deficient (MMRD) tumors;

[0008] or ii) ARID1A-deficient tumors.

[0009] Furthermore, the mismatch repair-deficient tumor is an MLH1-deficient tumor.

[0010] Furthermore, the binding site of the competitive inhibitor of TOPBP1 is the BRCT7-8 pocket of TOPBP1.

[0011] Furthermore, the competitive inhibitor of TOPBP1 has the effect of disrupting the binding of PHF8-TOPBP1 and FANCJ-TOPBP1.

[0012] Furthermore, the competitive inhibitor of TOPBP1 is auranofin.

[0013] A second aspect of the present invention provides a therapeutic device, characterized in that it comprises module i) and module ii):

[0014] Module i) is a detection module, and the module i) includes detection reagents required for detecting whether the in vitro sample is ARID1A-deficient or MLH1-deficient;

[0015] Module ii) is an identification and administration module, wherein the competitive inhibitor of TOPBP1 is administered to the ex vivo sample of the patient identified as ARID1A-deficient or MLH1-deficient in module i).

[0016] Furthermore, the detection reagent in module i) of the device is a biomolecule that specifically hybridizes with the ARID1A or MLH1 gene, an expression product of the ARID1A or MLH1 gene, and / or a detection reagent that uses the ARID1A or MLH1 gene as a detection target.

[0017] Furthermore, in module ii of the device, the competitive inhibitor of TOPBP1 has the function of binding to the BRCT7-8 pocket of TOPBP1 or the competitive inhibitor of TOPBP1 has the function of disrupting the binding of PHF8-TOPBP1, FANCJ-TOPBP1; preferably, the competitive inhibitor of TOPBP1 is auranofin.

[0018] The third aspect of the present invention provides the use of a reagent for detecting a biomarker in preparing a kit for predicting the anti-tumor efficacy of auranofin, wherein the biomarker is:

[0019] i) ARID1A or MLH1 gene; or

[0020] ii) ARID1A or MLH1 gene expression products.

[0021] Furthermore, the tumor includes colon cancer, lung cancer, endometrial cancer, invasive ductal carcinoma of the breast, conventional glioblastoma multiforme, ovarian cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, melanoma, and / or colorectal cancer.

[0022] The fourth aspect of the present invention provides a pharmaceutical composition comprising component 1 and component 2,

[0023] The component 1 is auranofin;

[0024] The component 2 is an MLH1 inhibitor or an ARID1A inhibitor.

[0025] A fifth aspect of the present invention provides the use of auranofin and an MLH1 inhibitor and / or an ARID1A inhibitor in the preparation of an anti-tumor drug, wherein the tumor is an ARID1A-deficient or MLH1-deficient tumor.

[0026] The present invention has the following advantages:

[0027] 1. Discovery of a novel mechanism of action of auranofin: This invention breaks the traditional limitation that auranofin exerts its pharmacological effects by inhibiting thioredoxin reductase (TrxR). It reveals that auranofin, as a small molecule compound, can bind to the BRCT7-8 pocket of TOPBP1, occupying the key binding site of PHF8 and FANCJ, thereby inhibiting the formation of PHF8-TOPBP1 and FANCJ-TOPBP1 complexes.

[0028] 2. It was discovered that auranofin affects the replication stress response: Auranofin inhibits the activation of ATR signaling and the accumulation of RPA on damaged chromatin by interfering with the function of TOPBP1, which is crucial for the regulation of replication stress and the cell's response to DNA damage.

[0029] 3. It was found that auranofin has enhanced anti-tumor activity in specific genetic backgrounds: Auranofin showed a synthetic lethal effect on mismatch repair deficiencies, such as MLH1-deficient colorectal cancer or ARID1A-deficient colorectal cancer.

[0030] 4. Auranofin exhibits enhanced tumor inhibitory effects in the treatment of ARID1A-deficient or MLH1-deficient tumors: In cell experiments and xenograft animal models, when auranofin was co-treated with ARID1A-deficient or MLH1-deficient colorectal cancer cells, it showed a more significant tumor growth inhibitory effect than when either treatment was performed alone.

[0031] 5. The present invention creatively discovered that auranofin, which has high side effects and drug resistance in treatment, has good sensitivity for tumor patients carrying ARID1A or MLH1 mutations. Therefore, the development of auranofin's low-dose anti-tumor effect will not only achieve precision medicine, but also realize the new use of old drugs, promoting the therapeutic potential of chemotherapy drugs with high efficacy and low side effects.

[0032] 6. Clinical application potential: Since auranofin is already an FDA-approved drug for the treatment of rheumatoid arthritis, the application of this drug developed in the preparation of tumor drugs may accelerate the clinical translation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A Shown is the use of biolayer interferometry (BLI) to quantify the binding affinity of auranofin to His-tagged recombinant BRCT7-8 purified from Escherichia coli cells; the figure includes BLI sensorgrams and dissociation constant (Kd) determination results; the black line represents the fitted curve, and the colored lines represent the raw data.

[0034] Figure 1B A flow chart showing the BLI assay used to examine the inhibitory effects of auranofin on BRCT7-8 binding to PHF8 / APS peptide and FANCJ peptide containing phosphorylated Thr1133 (pFANCJ).

[0035] Figure 1C A diagram of the chemical structure of auranofin is shown.

[0036] Figure 1D BLI analysis demonstrated the inhibitory effect of auranofin on the binding of BRCT7-8 to PHF8 / APS peptide and pFANCJ. The figure shows the incubation results of His-tagged BRCT7-8 at different auranofin concentrations, with the black line representing the fitted curve and the colored line representing the original data.

[0037] Figure 2A Shown are immunoprecipitation (IP) and immunoblotting (IB) experiments using cell lysates from HeLa cells expressing FLAG-TOPBP1 and treated with auranofin at different doses for 2 hours.

[0038] Figure 2B Shown is a Co-IP assay detecting changes in TOPBP1-PHF8 and TOPBP1-FANCJ interactions in HeLa cell lysates treated with auranofin (2 μM, 2 hours).

[0039] Figure 2C Co-IP analysis was performed to compare the differences in TOPBP1-PHF8 and TOPBP1-FANCJ interactions in HeLa cells treated with auranofin, PMX464, and piperlongumine.

[0040] Figure 2D Shown are the results of BLI analysis of auranofin binding to recombinant BRCT7-8 / Wt and BRCT7-8 / F1411A, including BLI sensorgrams and dissociation constants (Kd).

[0041] Figure 2E A schematic diagram of the prime editing system and representative sequencing results of the edited genome of HeLa cells F1411A are shown, including the sequence of the key elements of epegRNA.

[0042] Figure 2F Co-IP assays were performed to examine the binding of TOPBP1 to PHF8 and FANCJ in wild-type and F1411A endogenously edited HeLa cells treated with DMSO or auranofin.

[0043] Figure 3AWe report the detection of TOPBP1 foci formation under physiological conditions by immunostaining and confocal microscopy analysis. Cells were pre-extracted and fixed 3 h after treatment with auranofin, PMX464, and piperlongumine in the presence or absence of NAC (50 μM).

[0044] Figure 3B Figure 3: TOPBP1 foci formation under conditions of replication stress (CPT) analyzed by immunostaining and confocal microscopy. Cells were treated with auranofin, PMX464, and piperlongumine for 3 hours and then stimulated with CPT (2 μM) for 1 hour.

[0045] Figure 3C Shown Figure 3A -B, Experimental flow chart and quantification results of TOPBP1 foci intensity in EdU-positive cells.

[0046] Figure 3D Shown are immunoblotting analyses of changes in ATR kinase activity in U2OS cells treated with the indicated concentrations of auranofin.

[0047] Figure 4A Figure 5. RPA2 pS33 and RPA2 foci formation were detected by immunostaining and confocal microscopy analysis under physiological conditions. Cells were pre-extracted and fixed 3 h after treatment with auranofin, PMX464, and piperlongumine in the presence or absence of NAC (50 μM).

[0048] Figure 4B Figure 3 shows the formation of RPA2pS33 and RPA2 foci detected by immunostaining and confocal microscopy analysis under conditions of replication stress (CPT). Cells were treated with auranofin, PMX464, and piperlongumine for 3 hours and then stimulated with CPT (2 μM) for 1 hour.

[0049] Figure 4C Shown Figure 4A -B Quantification of RPA2 pS33 foci intensity in EdU-positive cells.

[0050] Figure 4D Shown Figure 4A -B Quantification of RPA2 foci intensity in EdU-positive cells.

[0051] Figure 4E A schematic diagram showing the operational flow of the SIRF experiment is shown.

[0052] Figure 4FThe interaction of RPA1 with nascent DNA (biotinylated-EdU) in HeLa cells under different treatment conditions was examined by SIRF analysis.

[0053] Figure 4G The number of PLA foci per cell was quantified.

[0054] Figure 5A The effects of different doses of auranofin on ARID1A were shown. + / + and ARID1A - / - The effects of the assay on the viability of HCT116 cells were investigated, and the half-maximal inhibitory concentration (IC50) was calculated.

[0055] Figure 5B The results show that different doses of auranofin can inhibit ARID1A in SW48 cells. + / + and ARID1A - / - The effects on cell viability were evaluated, and the half-maximal inhibitory concentration (IC50) was calculated and displayed.

[0056] Figure 5C The results show that different doses of auranofin can inhibit ARID1A in OVCAR-3 cells. + / + and ARID1A - / - The effects on cell viability were investigated, and IC50 was calculated and displayed.

[0057] Figure 6A The results show that different doses of auranofin can inhibit the expression of MLH1 in HCT116 cells. + / + and MLH1 - / - The effects on cell viability were investigated, and IC50 was calculated and displayed.

[0058] Figure 6B The results show that different doses of auranofin can inhibit the expression of MLH1 in SW48 cells. + / + and MLH1 - / - The effects on cell viability were investigated, and IC50 was calculated and displayed.

[0059] Figure 7A ARID1A + / + and ARID1A - / - Tumor weight changes of HCT116 cell xenografts in NOD / SCID mice treated with auranofin every two days.

[0060] Figure 7B Shown Figure 7A The quantitative results of the data shown are shown in the graph, which clearly demonstrates the statistical analysis of tumor weight under different treatment conditions.

[0061] Figure 8A MLH1 + / +and MLH1 - / - Figure 3. Changes in tumor weight of HCT116 cell xenografts in NOD / SCID mice. Mice were treated with auranofin every two days.

[0062] Figure 8B Shown Figure 8A The quantitative results of the data shown are shown in the graph, which clearly demonstrates the statistical analysis of tumor weight under different treatment conditions. DETAILED DESCRIPTION

[0063] The present invention will be further described below in conjunction with specific examples. The described embodiments are some embodiments of the present invention, rather than all embodiments. It should be understood that the following examples are provided to provide a complete disclosure and description of how to utilize the methods and compositions of the present invention to those skilled in the art, and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0064] Example 1: Study on the mechanism and pathway of auranofin's tumor inhibition

[0065] 1.1 In vitro identification of auranofin binding to TOPBP1 BRCT7-8

[0066] Experimental Methods: Biolayer interferometry (BLI) was used to investigate the tumor inhibition mechanism and pathway of auranofin. BLI was performed using an Octet RED96 instrument (ForteBio). In binary binding experiments, all purified proteins, including TOPBP1 BRCT7-8 / Wt and TOPBP1 BRCT7-8 / F1411A, were first biotinylated using EZ-Link NHS-Biotin (Thermo Fisher Scientific) according to the manufacturer's instructions and then added to SSA biosensors (ForteBio). All proteins reached an average saturation response level of 15 nM within 15 minutes. The loaded sensors were washed for 10 minutes in assay buffer containing PBS, 0.1% BSA, 0.02% Tween-20, and 1% DMSO to establish a stable baseline. Binding-dissociation cycles were then initiated with the test compound and DMSO as a reference. Raw kinetic data were processed using the manufacturer's data analysis software using a double reference subtraction method, whereby a DMSO-only reference and an inactive reference were subtracted. The association and dissociation rates were fitted using a 1:1 binding model. off With K on The equilibrium dissociation constant (Kd) was calculated from the ratio of

[0067] In the in vitro competition assay, an assay buffer containing PBS, 0.1% BSA, 0.02% Tween-20, and 1% DMSO was used. Biotin-PHF8_APS (GACFKDAEYIYPSLESDDDDPA) and biotin-pFANCJ (EAEDESIYF(pT)PELYDPEDT) were immobilized on the biosensor surface. During the binding process, TOPBP1 BRCT7-8 / Wt was preincubated with auranofin concentration gradient of 0, 1.875, 3.75, 7.5, 15, and 30 μM (this concentration gradient targets the ability of auranofin to competitively inhibit TOPBP1 BRCT7-8 / Wt binding to PHF8_APS) or 0, 3.125, 6.25, 12.5, 25, and 50 μM (this concentration gradient targets the ability of auranofin to competitively inhibit TOPBP1 BRCT7-8 / Wt binding to pFANCJ). Dissociation was then allowed to proceed for 2 minutes in the assay buffer. Kinetic parameters were determined using a 1:1 binding model and local fitting.

[0068] Experimental results: The researchers purified TOPBP1 BRCT7-8 in vitro and verified the ability of auranofin to bind to it using biofilm interferometry (BLI). The results showed that auranofin has the ability to bind to BRCT7-8 (see Figure 1A Furthermore, the results of in vitro competitive experiments showed that as the concentration of auranofin increased, the binding ability of TOPBP1 BRCT7-8 to PHF8-APS and pFANCJ gradually weakened, indicating that auranofin can inhibit the binding of PHF8-TOPBP1 and FANCJ-TOPBP1 in vitro ( Figures 1B-1D ).

[0069] 1.2 In vivo experiments verify the ability of auranofin to disrupt PHF8-TOPBP1 and FANCJ-TOPBP1 binding

[0070] 1.2.1 Plasmid transfection experiments

[0071] Experimental Method: Linearized polyisocyanate (PEI) was used for transfection. When HeLa cells (purchased from the ATCC cell bank) reached 70% confluency, a mixture of plasmid and PEI was added at a ratio of 1:5:100:plasmid (μg):PEI:OPTI-MEM.

[0072] Protein expression and purification experiments

[0073] Experimental Methods: A cDNA fragment encoding the BRCT 7-8 domain (amino acids 1264 to 1493) of TOPBP1 (Gene ID: 11073, Protein Data Bank (PDB) ID: 7CMZ) was cloned into a modified pET-28a-smt vector. These His-SUMO-tagged proteins were expressed in Escherichia coli BL21(DE3) strains by induction with 0.15 mM isopropyl-β-D-thiogalactopyranoside overnight at 16°C. Cells were harvested by centrifugation and sonicated in buffer A (25 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole, 1 mM EDTA, and 1 mM β-mercaptoethanol). Cell debris was removed by centrifugation at 4,000 g for 40 minutes at 4°C. The supernatant was applied to a Ni Sepharose Excel column (GE Healthcare), and the beads were eluted with buffer A. The fusion protein was eluted with buffer B (25 mM Tris-HCl (pH 8.0), 500 mM NaCl, 200 mM imidazole, 1 mM EDTA, and 1 mM β-mercaptoethanol). The His-tagged protein was subjected to SUMO protease to remove the SUMO tag. The detagged protein was added to a pre-equilibrated HiTrap SP HP column (GE Healthcare) and eluted with buffer C (20 mM MES (pH 6.0), 100 mM NaCl, 1 mM EDTA, and 1 mM β-mercaptoethanol) using a linear gradient from 0.1 to 1.0 M NaCl. The eluted protein was concentrated by ultrafiltration and further purified using a HiLoad Superdex 200 16 / 60 gel exclusion column (GE Healthcare) in a buffer containing 20 mM MES (pH 6.0), 300 mM NaCl, and 1 mM β-mercaptoethanol. High-purity fractions were collected and concentrated to approximately 40 mg / ml and stored in PBS buffer.

[0074] In order to identify the site where auranofin simultaneously disrupts the binding of TOPBP1 to PHF8 and FANCJ, a mutant (F1411A) BRCT 7-8 mutant protein (F1411A) was constructed. The expression and purification procedures of the mutant protein were the same as those of the wild-type protein.

[0075] 1.2.2 Co-immunoprecipitation experiments

[0076] Experimental Methods: Cell lysates were prepared by incubating cells in NETN buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2% Nonidet P-40, 2 mM EDTA) with a protease inhibitor cocktail (Roche) at 4°C for 20 minutes. Subsequently, the cells were centrifuged at 14,000 g for 15 minutes at 4°C. For immunoprecipitation, approximately 500 μg of protein was incubated with control or specific antibodies (1–2 μg) at 4°C for 12 hours with rotation. Then, 50 μl of 50% protein G magnetic beads (Invitrogen) were added and incubated for an additional 2 hours. The beads were then washed five times with lysis buffer, pelleted between washes using a magnetic stand (Invitrogen). Precipitated proteins were eluted from the beads by resuspending them in 2× SDS-PAGE buffer and boiling them at 95°C for 10 minutes. Proteins were then separated by SDS-PAGE electrophoresis and subjected to immunoblotting.

[0077] Experimental results:

[0078] The present invention transfected HeLa cells with FLAG-tagged TOPBP1 plasmid and treated them with 0 μM, 1 μM, and 2 μM auranofin respectively; the effect of auranofin on the binding ability of TOPBP1 to PHF8 and FANCJ was detected by immunoprecipitation technique. The experimental results showed (see Figure 2A ), with the increase of auranofin concentration, the binding ability of TOPBP1 to PHF8 and FANCJ weakened.

[0079] At the same time, immunoprecipitation experiments were performed using endogenous TOPBP1 antibodies, and the experimental results showed (see Figure 2B ) The addition of auranofin disrupts the binding of TOPBP1 to PHF8 and FANCJ.

[0080] Two well-known TrxR inhibitors, piperlongumine (Selleck, S7551) and PMX464 (MCE, HY-108534), were used as controls. HeLa cells were treated with both of these well-known TrxR inhibitors at a concentration of 10 μM for 4 hours to detect whether the interaction between TOPBP1 and PHF8 and FANCJ was inhibited. The results showed that only auranofin could affect the binding between TOPBP1 and PHF8 and FANCJ, while piperlongumine and PMX464 could not (see Figure 2CLanes 4-5). This leads to the conclusion that auranofin does not rely on the traditional mode of action, that is, it does not regulate the binding of TOPBP1 to PHF8 and FANCJ by inhibiting thioredoxin reductase (TrxR).

[0081] 1.2.3 Molecular docking experiment

[0082] Experimental Methods: Auranofin was used as the ligand for molecular docking. All docking simulations were performed using AutoDock 4.2. The crystal structure of TOPBP1 BRCT 7-8 was used as the target conformation for docking (PDB: 7CMZ). The docking grid was centered on the geometric center of the APS. The grid diameter was set to approximately 1.8 nm, the grid size was 60 × 60 × 60, and the grid spacing was set to 0.375. The docking box was large enough to encompass the potential binding activation pocket. The docked ligand was considered conformationally flexible, and its torsion bond was defined by AutoDock 4.2 based on its chemical characteristics. The final ligand docking mode with the receptor was determined by selecting the position with the lowest binding free energy.

[0083] Experimental results: Molecular docking revealed that auranofin binds to the F1411 site of TOPBP1. Therefore, in order to determine whether auranofin binds to the F1411 site and thereby disrupts the binding of TOPBP1 to PHF8 and FANCJ, this experiment constructed wild-type (Wt) and mutant (F1411A) BRCT 7-8 and confirmed this using BLI technology. The results showed that the F1411 mutation weakened the binding of TOPBP1 to auranofin, indicating that the F1411 site is one of the key sites for the interaction between TOPBP1 and auranofin (see Figure 2D ).

[0084] 1.2.4 Prime editing experiments

[0085] Experimental Methods: HeLa cells were transfected with 3 μg of PE3, 1 μg of epegRNA, and 0.4 μg of nicking sgRNA plasmid at approximately 60% confluency. Cells were cultured for 3 days after transfection, and individual colonies were sorted by flow cytometry. When cells reached sufficient confluency, genomic DNA was extracted, followed by PCR amplification and DNA sequencing.

[0086] epegRNA sequence: caccGTGGATCAGTCTCGAGAAGCgttttAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGgtgcGTTTGGCGCCTGCTTCTCGAGACTAAATTTGT

[0087] Nicking sgRNA sequence:

[0088] caccgAGAACTCATCATAGCAATCT

[0089] Experimental results: Prime editing was used to mutate F1411 on the genome into A, achieving amino acid mutation on the endogenous genome (see Figure 2E Through immunoprecipitation experiments, researchers found that compared with the DMSO group (control group), under the action of 2μM auranofin (experimental group), the binding ability of endogenous wild-type TOPBP1 to PHF8 and FANCJ decreased (see Figure 2F Lanes 2-3); In endogenous F1411A cells, compared with the DMSO control group, the addition of auranofin did not further weaken the binding of TOPBP1 to PHF8 and FANCJ (see Figure 2F Lanes 4-5). This further demonstrates that TOPBP1 F1411 is the key site for auranofin binding.

[0090] Based on these experimental results, the researchers concluded that auranofin disrupts the protein-protein interaction between TOPBP1, PHF8, and FANCJ by specifically binding to the F1411 site within the BRCT7-8 domain of TOPBP1. This effect, independent of the traditional TrxR inhibition mechanism, provides a new perspective for understanding the pharmacology of auranofin and may offer a potential target for developing new therapeutic strategies. These findings are of great significance for a deeper understanding of the molecular mechanisms of auranofin and its application in disease treatment.

[0091] Example 2: Auranofin inhibits the activation of ATR signaling and the accumulation of RPA at damaged replication forks

[0092] This example used immunofluorescence to examine the effect of auranofin on TOPBP1 accumulation at injury sites. The study investigated whether auranofin affects TOPBP1 accumulation by increasing the oxidized form of TrxR and ROS levels. The antioxidant N-acetylcysteine ​​(NAC) and known TrxR inhibitors, piperlongumine and PMX464, were used to assess their effects on TOPBP1 foci formation. Activation of the ATR signaling pathway was assessed by treatment with varying concentrations of auranofin.

[0093] 2.1 Auranofin inhibits TOPBP1 recruitment and ATR signaling activation under replication stress

[0094] Experimental Methods: For immunofluorescence assays, cells were plated on glass slides (BD Biosciences), fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Samples were blocked in 5% donkey serum and stained with appropriate primary and secondary antibodies conjugated to Alexa Fluor 488, 594, or 647 (Invitrogen). To avoid bleed-through effects in double-staining experiments, each dye was scanned independently in multi-tracking mode. To detect nuclear RPA2, RPA1, TOPBP1, or RPA2 pS33 foci, cells were pretreated with 0.5% Triton X-100 on ice for 5 minutes and fixed with 3% paraformaldehyde and 2% sucrose for 15 minutes at room temperature. Cells were then permeabilized with 0.5% Triton X-100 on ice for 5 minutes and blocked in 5% donkey serum for 1 hour at room temperature. For S-phase differentiation, cells were treated with 10 μM EdU for 1 hour in a 37°C incubator and then fixed. S phase labeling was performed using keyFluor 647-azide (Keygen Technologies) according to the manufacturer's instructions. Two parallel models were set up: a blank control group without 2μM camptothecin (CPT) treatment, also known as the DMSO model (this group primarily investigates changes in cellular localization of TOPBP1 under physiological conditions in response to drugs such as auranofin); and a replication stress model group with 2μM camptothecin (CPT), also known as the CPT model (this group primarily investigates changes in TOPBP1 recruitment to replication stress sites under replication stress conditions in response to drugs such as auranofin). Within each model, experimental groups were set up: auranofin group, piperlongumine-treated group, PMX464-treated group, and a solvent control group, DMSO group.

[0095] Experimental results:

[0096] Based on the information disclosed before the filing date of this application, PHF8 regulates the accumulation of TOPBP1 at the damage site and the activation of ATR signal by binding to TOPBP1. In this example, immunofluorescence experiments were used to detect the accumulation of TOPBP1 at the site after the replication stress induced by 2 μM CPT after auranofin treatment. Figure 3B Column 2 compared to column 1 and Figure 3C Comparison between column 7 and column 6 shows that the addition of auranofin severely inhibits the accumulation of TOPBP1 signals at the damage site.

[0097] To further verify whether the addition of auranofin severely inhibited the accumulation of TOPBP1 signals at the damage site, the researchers simultaneously set up an experimental group with the addition of the antioxidant N-acetylcysteine ​​(NAC) (see figure). The antioxidant N-acetylcysteine ​​(NAC) was used to clear ROS in the cells, and the results were shown in Figure 3B Column 3 compared to column 1 and Figure 3C Comparing column 8 with column 6, it can be seen from the results that the fluorescence signal of TOPBP1 was not restored by the addition of NAC. Therefore, the result that auranofin inhibited the accumulation of TOPBP1 signal at the damage site was not related to its increase in the oxidized form of TrxR and thus the increase in ROS levels;

[0098] At the same time, the researchers set up two experimental groups treated with TrxR inhibitors (10 μM piperlongumine and 10 μM PMX464) for 4 hours. Figure 3B Columns 4-5 compared to column 1 and Figure 3C Comparison of columns 9-10 with column 6 shows that 4 hours of treatment did not affect the formation of TOPBP1 foci.

[0099] The researchers induced replication stress by adding 2 μM camptothecin (CPT) for 1 hour, and then treated with different concentrations of auranofin. They found that the activation of ATR signaling (CHK1 pS345, RPA2 pS33 and RPA2 pS4 / 8) was inhibited in a concentration-dependent manner by the addition of auranofin. Figure 3D ).

[0100] 2.2 Auranofin inhibits RPA binding to damaged chromatin regions

[0101] Experimental methods: The same immunofluorescence assay and in situ analysis of protein interactions at DNA replication forks (SIRF) assay as in section 2.1 were used to further investigate the ability of auranofin to inhibit the binding of RPA to damaged chromatin regions.

[0102] SIRF Experimental Procedure

[0103] 1. EdU Incubation and Washing: U2OS cells (purchased from ATCC Cell Bank) were incubated with high-glucose medium containing 125 μM EdU for 8 minutes to label active DNA synthesis during S phase. After incubation, the EdU medium was removed and the slides were washed twice with PBS to remove unincorporated EdU. The slides were then incubated with medium containing 1 mM HU for 4 hours in the presence or absence of auranofin or NAC.

[0104] Step 2. Fixation, Permeabilization, and Click Reaction: Cells were first washed with cold PBS and then fixed with 4% paraformaldehyde for 5 minutes to preserve cellular structure. Next, cells were permeabilized with 0.25% Triton X-100 for 15 minutes to enhance the penetration of subsequent reagents. After permeabilization, cells were washed again with PBS to remove the permeabilizing agent. Freshly prepared Click reaction mixture was then added and incubated at room temperature for 1 hour to detect EdU-labeled DNA.

[0105] Step 3. Blocking, Antibody Incubation, PLA, and Imaging: Block the cells with blocking buffer containing 10% FBS at 37°C for 1 hour to reduce nonspecific binding. After blocking, incubate the cells with the primary antibody against the target protein overnight at 4°C. Perform PLA according to the Duolink In Situ Detection Kit instructions to detect protein-protein interactions. Finally, mount the slides with Fluoroshield containing DAPI and perform nuclear staining. Images were captured using a Zeiss LSM 900 microscope and quantitatively analyzed using ImageJ software. Antibody specificity was verified by knocking down RPA1 to ensure robustness of the results.

[0106] Experimental results: Based on the information previously disclosed before the filing date of this application, it is known that FANCJ promotes the accumulation of RPA at stalled replication forks by binding to TOPBP1 BRCT7-8. The researchers used the same immunofluorescence assay as in Section 2.1 to detect the changes in RPA2 recruitment at replication stress sites after treatment with 2 μM auranofin. The experimental results showed that the addition of auranofin significantly reduced the ability of RPA2 to form foci ( Figure 4B Comparing column 2 to column 1 and Figure 4DCompare the 7th column with the 6th column). Two parallel models were set up in this experiment. One model was the blank control group without 2μM camptothecin (CPT) treatment, namely the DMSO model group (this group mainly explored the changes in the cellular localization of RPA2 under physiological conditions under the treatment of drugs such as auranofin), and the other was the replication stress model group with 2μM camptothecin (CPT), namely the CPT model group (this group mainly explored the changes in the ability of RPA2 to recruit to replication stress sites under replication stress conditions under the treatment of drugs such as auranofin). Experimental groups were set up in each model group: auranofin group, piperlongumine treatment group, PMX464 treatment group, and solvent control group DMSO group. Figure 3B -C conclusion is similar, NAC clearing ROS does not lead to the disappearance of the effect of auranofin in inhibiting RPA recruitment ( Figure 4B Column 3 compared to column 1 and Figure 4D Compare column 8 with column 6). Meanwhile, TrxR inhibitors such as piperlongumine and PMX464 do not produce the effect of auranofin in inhibiting RPA recruitment ( Figure 4B Columns 4-5 compared to column 1 and Figure 4D Compare columns 9-10 with column 6).

[0107] The in situ analysis of protein interactions at DNA replication forks (SIRF) experiment provides in situ spatial resolution, allowing scientists to observe and analyze the dynamic changes and collaborative working mechanisms of proteins in the natural state of cells, thereby gaining a deeper understanding of the accuracy and efficiency of DNA replication. Based on this experiment, the researchers found that the ability of RPA1 to bind to nascent DNA was reduced after auranofin treatment ( Figure 4F Compare column 6 with column 5 and Figure 4G Compare column 6 with column 5). It can be inferred that the addition of auranofin reduces the ability of tumor cells to cope with replication stress, increasing the probability of genomic instability in tumor cells. Once relevant gene mutations or gene deletions occur in the tumor cell DNA damage response system, such as ARID1A or MLH1 deletions, the tumor cells will be unable to repair DNA damage and ultimately die.

[0108] Example 3: Auranofin enhances ARID1A at the cellular level - / - and MLH1 - / - Tumor cell killing ability

[0109] 3.1 Auranofin enhances inhibition of ARID1A in xenografts - / - Tumor growth

[0110] Experimental methods:

[0111] 3.1.1 Preparation of ARID1A - / -and MLH1 - / - Stable Expression Cell Lines: Virus was prepared using linearized polyisocyanate (PEI) transfection. 293T cells were cultured in 10 cm dishes. When the cells reached 70% confluency, a mixture of plasmids (μg): PEI: OPTI-MEM was added at a ratio of 1:5:100. Plasmids included packaging plasmids (pMDLg / pRRE, pRSV-REV, and pVSVG) and target plasmids (shControl, shARID1A, Control, and sgMLH1). Viral supernatants were collected at 48 and 72 hours. Cell lines to be infected (colorectal cancer cell lines HCT116 and SW48, and ovarian cancer cell line OVCAR-3) were prepared in advance. Blank controls were maintained. After 24 hours of virus addition, the culture medium was replaced with normal medium for selection using puromycin or flow cytometry.

[0112] 3.1.2. Auranofin Enhances ARID1A - / - Tumor cell killing effect

[0113] HCT116 cells expressing shControl and shARID1A constructed in Section 3.1.1 were cultured in 96-well plates at 1500 cells / well. After 24 hours, each group of cells was treated with 0, 100 nM, 200 nM, 500 nM, 750 nM, or 1000 nM auranofin and cultured for an additional 72 hours. Cell Titer Aqueous One Solution Reagent (G3582, Promega) was then added to each well, and the cells were cultured in a 37°C incubator for another hour. Cell viability was determined by measuring the absorbance at 490 nm using a 550 BioRad plate-reader (Bio-Rad, Hertfordshire, UK), and survival curves were plotted. The results are shown in Figure 2. Figure 5A As shown in the figure, the survival rate of the shARID1A group (IC50: 306.4 nM) was significantly lower than that of the control group shControl cells (IC50: 1395 nM), that is, the concentration of auranofin that reached the half-lethal dose of the control group cells was 1395 nM, while the concentration of auranofin that reached the half-lethal dose of the experimental group cells was 306.4 nM. We can conclude that compared with the control group, the survival rate of the shARID1A group was significantly lower than that of the control group shControl cells (IC50: 1395 nM). - / -Using a low dose of auranofin in HCT116 tumor cells can achieve the effect of tumor inhibition. The experimental method is the same as above. In SW48 cells and OVCAR-3 cells, researchers reached the same conclusion, that is, the survival rate of the shARID1A group in SW48 cells (IC50: 451.8 nM) was significantly lower than that of the control group shControl cells (IC50: 1788 nM), and the survival rate of the shARID1A group in OVCAR-3 cells (IC50: 622.3 nM) was significantly lower than that of the control group shControl cells (IC50: 3353 nM), indicating that the use of a low dose of auranofin in these two cell lines can also effectively kill ARID1A. - / - The ability of cells. Figures 5B-5C ).

[0114] 3.2 Auranofin enhances MLH1 - / - Tumor cell killing effect

[0115] Control and sgMLH1 HCT116 cells were cultured in 96-well plates at 1500 cells / well. After 24 hours, the cells were treated with 0, 100 nM, 200 nM, 500 nM, 750 nM, and 1000 nM auranofin, respectively. After 72 hours, Cell Titer Aqueous One Solution Reagent (G3582, Promega) was added to each well and the cells were cultured in a 37°C incubator for 1 hour. The cell viability was determined by measuring the absorbance at 490 nm using a 550 BioRad plate-reader (Bio-Rad, Hertfordshire, UK) and plotted as a survival curve. The results are shown in Figure 2. Figure 6A As shown, the survival rate of the sgMLH1 group was significantly lower than that of the control group: the survival rate of the sgMLH1 group (IC50: 436.6 nM) was significantly lower than that of the control group (IC50: 1413 nM), that is, the concentration of auranofin that reached the half-lethal dose of the control group cells was 1413 nM, while the concentration of auranofin that reached the half-lethal dose of the experimental group cells was 436.6 nM. We can conclude that compared with the control group, the use of a low dose of auranofin in sgMLH1 HCT116 tumor cells can achieve the effect of tumor inhibition ( Figure 6AThe experimental method was the same as above. The researchers reached the same conclusion in SW48 cells, that is, the survival rate of the sgMLH1 group (IC50: 467.5 nM) in SW48 cells was significantly lower than that of the control group shControl cells (IC50: 1775 nM), indicating that low doses of auranofin can effectively kill MLH1 in this cell line. - / - The ability of cells ( Figure 6B ).

[0116] Example 4: Using animal models to verify the enhanced effect of auranofin on ARID1A - / - and MLH1 - / - Tumor-killing effect

[0117] 1. Auranofin Enhances Inhibition of ARID1A in Xenografts - / - Tumor growth

[0118] HCT116 cells stably expressing shControl and shARID1A constructed in Example 3 (1.5×10 6 ), resuspended in 200 μL PBS, and orthotopically transplanted into the subcutaneous tissues of 6-week-old immunocompromised (SCID) female mice. The tumor volume reached 40 mm 3 Afterwards, the shControl control group and the shARID1A experimental group were intraperitoneally injected with auranofin (5 mg / kg, 2% v / v dimethyl sulfoxide, 10% v / v HPBCD, 2-hydroxypropyl-β-cyclodextrin, PBS) every two days. Five to six animals were used per group. Tumor volume was measured every two days with a vernier caliper and calculated as 0.5 × length × width. 2 .

[0119] The experimental results are shown in Figures 7A-7B As can be seen from the figure, after treatment with the same dose and the same cycle of auranofin, the tumor weight of mice transplanted with shARID1A was significantly smaller than that of mice transplanted with shControl, which means that the tumors of mice transplanted with shARID1A have a higher therapeutic response to auranofin. In the future, it will be possible to identify the genotype of the tumor and use low doses of auranofin to treat ARID1A. - / - tumor.

[0120] 2. Auranofin Enhances Inhibition of MLH1 in Xenografts - / - Tumor growth

[0121] 1. Use HCT116 cells (1.5×10 6), resuspended in 200 μL PBS, and orthotopically transplanted into the subcutaneous tissue of 6-week-old immunocompromised (SCID) female mice. The tumor volume reached 40 mm 3 Afterwards, the control and experimental groups received intraperitoneal injections of auranofin (5 mg / kg, 2% v / v dimethyl sulfoxide, 10% v / v HPBCD, 2-hydroxypropyl-β-cyclodextrin, PBS) every two days. Five to six animals were used in each group. Tumor volume was measured every two days with a vernier caliper and calculated as 0.5 × length × width. 2 .

[0122] 2. Experimental results are shown in Figures 8A-8B We found that the tumor weight of mice transplanted with sgMLH1 was significantly smaller than that of mice transplanted with Control. This means that the tumors of mice transplanted with sgMLH1 have a higher response to treatment with auranofin. In the future, we can identify the genotype of the tumor and use low doses of auranofin to treat MLH1 - / - tumor.

Claims

1. Use of a competitive inhibitor of TOPBP1 in the preparation of a pharmaceutical composition for treating mismatch repair-deficient colorectal cancer, characterized in that: The mismatch repair-deficient colorectal cancer is MLH1 gene-deficient, and the competitive inhibitor of TOPBP1 is auranofin.

2. A therapeutic device, characterized in that: Contains modules i) and ii): Module i) is a detection module, and the module i) includes a detection reagent required for detecting whether the in vitro sample is MLH1 deficient; Module ii) is an identification and administration module, wherein the module ii) administers a competitive inhibitor of TOPBP1 to an ex vivo sample of a patient identified as having MLH1 deficiency in module i), wherein the patient is a colorectal cancer patient; the competitive inhibitor of TOPBP1 is auranofin.

3. The device according to claim 2, characterized in that The detection reagent described in module i) is a detection reagent that uses the MLH1 gene as the detection target.

4. The device according to claim 2, characterized in that The detection reagent described in module i) is a biological molecule that specifically hybridizes with the MLH1 gene or the expression product of the MLH1 gene.

5. Use of a reagent for detecting the MLH1 gene or its expression product in the preparation of a kit for predicting the efficacy of a competitive inhibitor of TOPBP1 against colorectal cancer, characterized in that: The competitive inhibitor of TOPBP1 is auranofin.

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