Use of auranofin in the preparation of medicaments for the treatment of pituitary adrenocorticotroph tumours

Abstract

The application relates to the application of auranofin in the preparation of a medicine for treating pituitary adrenocorticotropic hormone tumors. The application verifies that auranofin can enhance the TRIM65-TPIT interaction, reduce the TPIT protein level, and reduce the ACTH level in a cell culture solution / serum at the cell level and the animal level.

Description

Technical Field

[0001] This invention belongs to the field of intracranial tumor technology, and specifically relates to the application of aurinol in the preparation of drugs for treating pituitary adrenocorticotropic hormone tumors. Background Technology

[0002] Pituitary neuroendocrine adenomas (PitNETs) are common intracranial tumors. Based on transcription factors related to cell differentiation origin, PitNETs can be divided into four main categories: the PIT1 lineage, the TPIT lineage, the SF1 lineage, and PitNETs without a definitive cell lineage. Among them, the TPIT lineage PitNETs are characterized by expression of the upstream transcription factor TPIT of the POMC gene, and are positive for TPIT staining on immunohistochemistry. Based on whether they secrete adrenocorticotropic hormone (ACTH), they can be divided into functional ACTH adenomas and silent ACTH adenomas. Pituitary ACTH adenomas account for 15% of all PitNETs, ​​with an incidence of 1.6 cases per million people. Approximately 40% of TPIT lineage PitNETs are silent, accounting for 3-6% of all PitNETs. Functional ACTH adenomas are mainly pituitary ACTH microadenomas (average 6 mm), and their symptoms are not due to the mass effect of the tumor, but mainly caused by abnormal hormone secretion. The clinical manifestation is persistent hypercortisolism, leading to a series of metabolic disorders and complications such as hypertension, diabetes, hyperlipidemia, osteoporosis, hypokalemia, depression, anxiety, and cognitive impairment, severely impacting patients' quality of life and even causing disability or endangering their lives. Currently, for the vast majority of ACTH adenoma patients, transsphenoidal resection is the preferred first-line treatment. Radiotherapy and medications such as dopamine receptor agonists, somatostatin analogs, ketoconazole, gefitinib, and mifepristone are used as adjuvant therapies after surgery for refractory ACTH adenomas. The remission and recurrence rates vary considerably, with an average remission rate of 77.8%, an average recurrence rate of 13.2%, and an average recurrence time of 13.6-66 months. Recurrence patients have a poor prognosis and high mortality rate. Therefore, controlling abnormal ACTH hormone secretion is the primary goal of ACTH adenoma treatment; currently, there are no drugs that control pituitary ACTH hormone secretion to treat ACTH adenomas.

[0003] Transcription factors are generally believed to regulate a range of genes and play a crucial role in cell differentiation, aging, and death. Regulation of transcription factors at the transcription factor level can influence cell development and normal cellular physiological functions. As a characteristic transcription factor of PitNETs in the TPIT lineage, TPIT plays a decisive role in the cell differentiation fate and hormone secretion of ACTH-secreting cells. Previous studies have found that TRIM65, as an E3 ligase for the ubiquitination of the transcription factor TPIT, promotes TPIT protein degradation, thereby inhibiting the transcription of POMC and other downstream target genes (including transcription factors), thus suppressing ACTH synthesis and secretion, and ultimately altering cell fate. Therefore, small molecules targeting the TRIM65-TPIT interaction in ACTH adenomas, by promoting TPIT degradation and thereby inhibiting ACTH hormone synthesis and abnormal secretion, are potential drugs for the treatment of ACTH adenomas. However, no small molecules developed using this technical approach have yet been marketed. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide the application of aurinofen in the preparation of drugs for treating pituitary adrenocorticotropic hormone tumors. The invention verifies at the cellular and animal levels that aurinofen can enhance TRIM65-TPIT interaction, reduce TPIT protein levels, and reduce ACTH levels in cell culture medium / serum.

[0005] This invention provides the use of aurinophene in the preparation of drugs for treating pituitary adrenocorticotropic hormone-induced tumors.

[0006] Furthermore, the aurinofen was obtained through screening using the NanoBiT high-throughput drug screening platform based on TRIM65-TPIT protein interaction and the NanoGlo high-throughput drug screening system based on protein-level quantitative detection.

[0007] Furthermore, the aurinophene enhances the TRIM65-TPIT interaction, reduces TPIT protein levels, and decreases ACTH levels in cell culture medium / serum.

[0008] Furthermore, the drug also contains pharmaceutically acceptable carriers and / or excipients.

[0009] Furthermore, the pharmaceutically acceptable carrier and / or excipient includes at least one of diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0010] The present invention also provides a pharmaceutical composition for pituitary adrenocorticotropic hormone (ACTH) tumors, the pharmaceutical composition comprising a therapeutically effective amount of aurinophene.

[0011] Furthermore, the dosage form of the pharmaceutical composition includes at least one of tablets, pills, powders, solutions, suspensions, emulsions, and granules. Beneficial effects

[0012] This invention utilizes a high-throughput drug screening system based on NanoBiT technology to monitor TRIM65-TPIT protein interaction and NanoGlo technology to detect TPIT protein levels, identifying aurinophene as a potential drug that promotes TRIM65-TPIT interaction and reduces TPIT protein levels. Experiments using CCK8 and subcutaneous tumorigenesis in nude mice showed that aurinophene had no significant cytotoxic effect on pituitary ACTH adenomas. RT-qPCR, Western blotting, and immunohistochemistry experiments revealed that aurinophene downregulates POMC levels and reduces ACTH production and secretion. Therefore, it can be inferred that aurinophene may be a novel drug for treating pituitary ACTH adenomas. Attached Figure Description

[0013] Figure 1 To construct a high-throughput drug screening system based on NanoBiT technology to monitor the interaction between TRIM65 and TPIT; (A) Schematic diagram of TRIM65-TPIT protein luminescence induced by the complementary structure of two luciferase fragments, SmBiT and LgBiT; (B) Optimization results of NanoBiT tag, with 50 ng fixed transfection. Signal curves obtained by transfecting different concentrations of SmBiT-tag-protein on the basis of LgBiT-tag-protein; NL, N-terminal labeling of LgBiT; NS, N-terminal labeling of SmBiT; CS, C-terminal labeling of SmBiT; CL, C-terminal labeling of LgBiT; (C) NanoBiT signal generated by LgBiT-tag-TRIM65 and SmBiT-tag-TPIT compared with negative control, SmBiT-Empty is negative control; (D) The number of NanoBiT stable transfected cells is proportional to the NanoBiT signal; (E) Schematic diagram of TPIT protein with HiBiT tag luminescence caused by the complementary structure of HiBiT and LgBiT luciferase fragments; (F) HiBiT / Luciferase signal is proportional to HiBiT-tag-TPIT protein level.

[0014] Figure 2Three candidate drugs that may promote TRIM65-TPIT interaction were identified for high-throughput drug screening. (A, B) Schematic diagrams of the high-throughput drug screening process for NanoBiT and NanoGlo; (C) High-throughput drug screening results for the NanoGlo HiBiT system, where the ratio of HiBiT signal to Luciferase signal is the relative HiBiT signal, the red dashed line represents the average of all relative signal values, and the red dots represent the representative drugs with the lowest and highest relative signal values; (D) Quantitative kinetic model of the TRIM65-TPIT interaction and TRIM65-mediated TPIT ubiquitination and degradation: S(t) = (e) Venn diagram of the intersection of high-throughput screening results of NanoBiT HTS and NanoGlo HTS; (f) NanoBiT signal in cells at different time points for the three potentially effective candidate drugs obtained from the screening.

[0015] Figure 3 To screen for drugs, aurnofen was found to enhance the TRIM65-TPIT interaction and promote TPIT degradation. Specifically, (A) the reliability of the drug screening was verified in a 96-well plate system, and NanoBiT signal values ​​were obtained at different time points; (B) the effect of the candidate drug on the exogenous TPIT protein level was analyzed by Western blot; (C) the TPIT protein level was quantified using ImageJ software; (D) the effect of the candidate drug on the TRIM65 and TPIT protein levels was detected by Co-IP experiment; and (E) the TPIT protein level decreased after aurnofen treatment at different time points.

[0016] Figure 4The results show that aurinophene promotes the ubiquitination and degradation of TPIT; among them, (A) the cell ubiquitination experiment shows that aurinophene promotes TRIM65-mediated ubiquitination of TPIT; (B) the cell ubiquitination experiment shows that TRIM65 mainly promotes the K27 ubiquitin chain modification of TPIT; (C) the TPIT degradation effect of aurinophene can be reversed by the proteasome inhibitor MG132 but not by CQ; (D) schematic diagram of the chemical structure of aurinophene and the gold-containing compound disodium gold thiobutane; (E) the results of the immunoprecipitation experiment show that aurinophene can promote the protein phase of TRIM65 and TPIT. (F) Western blot results showed that auronoxine promoted a decrease in TPIT protein levels at 6 h, while auronoxine had no significant effect on TPIT protein levels; (G) Immunoprecipitation experiments showed no protein interaction between USP14 and TPIT; (H) Immunoprecipitation experiments showed a weak protein interaction between UCHL5 and TPIT; (I) Cell ubiquitination experiments showed that UCHL5 had no significant effect on TPIT ubiquitination levels; (J) Western blot verification of TRIM65 knockdown effect; (K) Cell ubiquitination experiments showed that after TRIM65 knockdown, TPIT ubiquitination levels decreased, and the effect of auronoxine promoting TPIT ubiquitination was reversed after TRIM65 knockdown.

[0017] Figure 5 The study showed that aurinophene promoted the degradation of endogenous TPIT and inhibited ACTH secretion; (A) Western blot analysis of TPIT and POMC protein levels in AtT20 cells after aurinophene time-gradient treatment; (B) Western blot analysis of whether the reduction in TPIT by aurinophene was reversed by MG132 or CQ; (C) Overexpression of TRIM65 promoted the reduction of TPIT and POMC protein by aurinophene in AtT20 cells, Western blot analysis. (D) blot detection of TPIT and POMC expression levels in each group; (E) qRT-PCR detection of TPIT and POMC mRNA levels in AtT20 cells after auron-methyl treatment; (F) ELISA detection of ACTH protein content secreted by AtT20 cells after auron-methyl treatment; (G) Tumor-bearing images of AtT-20 mice in the control group and after auron-methyl treatment; (H) Tumor volume of AtT-20 mice in the control group and after auron-methyl treatment; (I) Representative immunohistochemical images showing the levels of TPIT, POMC and ACTH proteins in tumor-bearing tissues of mice in the control group and after auron-methyl treatment; (J) Auron-methyl inhibits ACTH secretion in mouse blood. Detailed Implementation

[0018] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. Example

[0019] I. Experimental Methods (1) Enhancers of TRIM65-TPIT protein-protein interaction in high-throughput drug screening: This study employed high-throughput drug screening based on NanoBit technology. NanoBit is a reporter gene system based on Promega's latest patented luciferase NanoLuc® luciferase, used to detect protein-protein interactions in living cells. The NanoBit system consists of two subunits: a large subunit, Large BiT (LgBiT; 18 kDa), and a small subunit, Small BiT (SmBiT; 11 amino acids), which are fused to two target proteins, respectively. When the fused target proteins carrying these two subunits interact, the two subunits bind together to form an active enzyme, producing a bright luminescent signal. The operational procedure is as follows: 1) The Smibt-TPIT and Lgbit-TRIM65 plasmids were transfected into HEK-293T cells, and 25 μL of medium containing 5000 cells was transferred to CulturPlate-384 (PerkinElmer, 6007680).

[0020] 2) Subsequently, an equal volume of culture medium containing the luminescent substrate furimazine (10 μM) was added to each well of the 384-well plate. The luminescent signal emitted by the Lgbit-Smbit was quantified using the Explorer high-throughput screening platform (PerkinElmer).

[0021] 3) 1913 compounds from the FDA-approved drug library were introduced at a final concentration of 1 μM. Luminescence intensity was measured at 5, 15, 30, 60, 120, 240, and 360 minutes. This screening was conducted at the Shanghai National Translational Medicine Center.

[0022] 4) Select the best-performing candidate drugs from the above screening for validation.

[0023] (2) High-throughput drug screening for inhibitors that suppress TPIT protein levels: This study employed the Nano-Glo HiBiT dual luciferase reporter gene assay system for high-throughput screening. The Nano-Glo HiBiT dual luciferase reporter gene assay system allows for multiplex detection of HiBiT-tagged proteins and firefly luciferase (Fluc) in the same well. This system minimizes or eliminates experimental variability caused by factors such as transfection efficiency, cell number, cell viability, temperature, or assay time, thereby improving experimental reliability and data quality. When detecting changes in the level of HiBiT-tagged target proteins, this method can distinguish non-specific effects caused by overall changes in protein expression levels. The procedure is as follows: 1) Cell preparation: The plasmid plvx-Hibit-TPIT-IRES-luciferase-puro was constructed and then packaged into a lentivirus to infect HEK293T cells. The cells were then treated with puromycin (2 μg / ml) for 3 days to establish a stable cell line. Preliminary experiments were performed in 384-well plates to select appropriate cell quantities and reaction systems.

[0024] 2) Plating and Drug Addition: Cells were counted using a cell counter, and the cells were diluted to 2,000 cells per 10 μL of culture medium. The cells were then seeded into CulturPlate-384 culture plates using a Multidrop Combi dispenser. Drugs from an approved drug screening library containing 1,913 compounds were added to the 384-well plates at a final concentration of 1 μM.

[0025] 3) High-throughput signal detection: After 24 h of cell culture, 10 μL of ONE-Glo™ EX / Lgbit reagent from the Nano-Glo HiBiT Dual Fluorescent Reporter System Kit (Promega, CS1956A09) was added to the dispensing instrument, and the cells were incubated on a shaker at 300 rpm for 10 min. The luciferin luminescence signal was then detected using the explorer HT platform. Subsequently, 10 μL of NanoDLRTMStop&Glo buffer was added, and the cells were incubated on a shaker for 10 min. The Hibit-TPIT luminescence signal was then detected.

[0026] 4) Data processing: Calculate the ratio of Hibit-TPIT to fluorescein, and perform statistical analysis after correction using DMSO as the standard.

[0027] (3) Detection of protein interactions by co-immunoprecipitation (Co-IP): A common method for studying protein-protein interactions involves using "antibodies" immobilized on magnetic or agarose beads to capture the "target protein" and further capture proteins that interact with the target protein.

[0028] 1) Cell preparation: Overexpress Flag-TPIT, Myc-TRIM65 and Hb-Ub in HEK293T cells.

[0029] 2) Cell lysis and binding: Collect cells into EP tubes and centrifuge at 1,000 rpm for 3 min. Remove the supernatant, centrifuge again at 1,000 rpm for 1 min, remove the supernatant, add 1 mL Co-IP buffer and mix by pipetting. Lyse on ice for 5-10 min, then sonicate for 3-5 min. Pre-chill the centrifuge, place the tubes in the centrifuge, and centrifuge at 12,000-15,000 rpm for 10-15 min at 4 ℃. Take 60 μL of cell lysis buffer as input, add 30 μL of 3× SDS protein loading buffer, and add the sample at 95-100 ℃ for 5-10 min. Take 200 μL of the Flag beads to be used into an EP tube, wash the beads with 1 mL Co-IP buffer, centrifuge at 2000 rpm for 5 min, remove the supernatant, and repeat once. Add the remaining cell lysis buffer to the washed beads, and bind overnight at 4 ℃ with slow rotation in a mixer.

[0030] 3) Beads washing: Remove the overnight bound beads, centrifuge at 2000 rpm for 3 min, discard the supernatant, add 1 mL Co-IP buffer to wash, and then rapidly rotate on a 4 ℃ mixer for 10 min. Repeat this process 4-5 times to remove non-specific bound proteins as much as possible.

[0031] 4) Protein electrophoresis: After washing, centrifuge at 2000 rpm for 3 min, discard the supernatant, centrifuge again to remove the liquid from the centrifuge tube wall, aspirate the remaining Co-IP buffer at the bottom of the centrifuge tube with a 1 mL syringe, add 100 μL of 2× SDS protein loading buffer, heat the sample at 95-100 ℃ for 5-10 min. Centrifuge again, and collect the supernatant for electrophoresis.

[0032] (4) Western blot analysis of TPIT, POMC, and ACTH protein levels: Proteins contained in cell or tissue samples are transferred to polyvinylidene fluoride (PVDF) membranes or nitrocellulose membranes (NC membranes) using methods such as polyacrylamide gel electrophoresis. The expression levels of these proteins are then detected using specific antibodies. The specific operational steps are as follows: 1) Preparation of protein samples: Refer to the methods for total cell protein extraction and protein quantification.

[0033] 2) Polyacrylamide gel electrophoresis preparation: This involves an upper stacking gel and a lower separating gel (different concentrations of separating gel are prepared according to the molecular weight of the target protein, generally between 6% and 15%. The larger the molecular weight of the protein, the lower the concentration of the separating gel; the smaller the molecular weight of the protein, the higher the concentration of the separating gel). ① Separating gel preparation: First, wash and dry the 1.5 mm thick glass plate and the corresponding thin glass plate, fix them in the gel preparation plate holder, prepare the application concentration of separating gel (6%, 8%, 10%, 12%) according to the dosage of 7-7.5 mL per plate, and pour it between the gel plates. Add 0.5-1 mL of anhydrous ethanol and press the separating gel flat. Let it stand at room temperature for 30-45 min to allow the separating gel to solidify. Pour off the upper layer of anhydrous ethanol and let it stand for 5 min to allow the anhydrous ethanol to evaporate. ② Preparation of stacking gel: Prepare stacking gel according to the amount of 2.5-3 mL per plate. Immediately fill the remaining space of the plate with a 5 mL pipette. Immediately insert the 15-well comb that comes with the 1.5 mm glass plate into the stacking gel and let it solidify at room temperature for 15-20 minutes.

[0034] 3) Electrophoresis: Fix the prepared protein electrophoresis gel in the electrophoresis tank and fill the tank with 1× protein electrophoresis buffer. Add the prepared protein sample to the gel wells, and simultaneously add a protein marker to each well to indicate the protein molecular weight. Perform electrophoresis at a constant voltage of 80 V. Once the protein sample has migrated from the stacking gel to the separating gel and the protein markers have clearly separated, increase the voltage to 120-150 V. Electrophoresis can be terminated when the target protein molecules are clearly separated according to their size.

[0035] 4) Transfer: Cut a PVDF membrane with a pore size of 0.22 μm (6.5 cm × 9 cm) and soak it in methanol for 2-3 min to activate the PVDF membrane (methanol activation is not required if using an NC membrane). Prepare a transfer dish and pour in pre-cooled transfer buffer (1×). Place the black clamp at the bottom of the transfer clamp, then place the white sponge, 3 sheets of filter paper (Beyotime), gel, PVDF membrane, 3 sheets of filter paper (Beyotime), white sponge, white clamp, and clamp onto the black and white clamp for fixation. (Note that air bubbles between the gel and PVDF membrane will affect the transfer results; be sure to remove air bubbles during the operation). Place the clamp in the transfer tank (black clamp against black transfer tank) and perform transfer at a constant current of 250 mA or a constant voltage of 100 V for 100-120 min (the transfer time can be extended if the target protein has a large molecular weight).

[0036] 5) Blocking and Antibody Incubation: After transfer, remove the PVDF membrane and wash it rapidly with 1× TBST on a shaker for 5 min. Remove the 1× TBST and block with 5-10% skim milk at room temperature for 50-60 min. Wash the PVDF membrane three times with TBST, 5-8 min each time (until the TBST is clear). Cut the PVDF membrane according to the target band size, then place it in an antibody incubation box and add the corresponding primary antibody dilution buffer and antibody (generally diluted 1:2000). Incubate slowly overnight at 4 ℃ on a shaker. The next day, recover the antibody and wash the PVDF membrane three times with TBST on a shaker for 5-8 min each time. Prepare secondary antibody with TBST (mainly from mice, rabbits, and donkeys), with a dilution ratio of 1:3000-1:5000. When adding the secondary antibody, the liquid should completely submerge the PVDF membrane. Incubate slowly on a shaker at room temperature for 50 min. Discard the secondary antibody and wash the PVDF membrane three times with TBST for 5-8 min each time.

[0037] 6) Development and mapping: Prepare ECL developer (a mixture of solution A and solution B) according to the quantity and size of the PVDF film, and develop it using a chemiluminescence analyzer to obtain bands. Use Photoshop to crop and create the image, and then use Adobe Illustrator for layout.

[0038] (5) Immunohistochemical detection of TPIT, POMC, and ACTH protein levels: A method for detecting the expression of a specific protein in tissues or cells utilizes the specific binding of an antibody to an antigen (target protein). The labeled antibody is then visualized through staining, revealing the location and expression level of the target protein within the tissue or cell. The procedure is as follows: 1) Sampling, fixation and dehydration: Tissue samples and xenograft specimens were rinsed with DPBS to remove blood stains; tissue fixative was used for fixation and rinsed with water, and then dehydrated stepwise with 50%, 75%, 85%, 95%, 95%, 100% and 100% ethanol for 1 hour each time.

[0039] 2) Transparency and wax impregnation: Treat with anhydrous ethanol / xylene (1:1) mixture for 15 min, then soak in xylene twice for 20 min each time. Then soak in soft wax at 50-52 ℃ (in two batches, the first for 30 min and the second for 90 min); then soak in hard wax at 56-58 ℃ (in two batches, the first for 60 min and the second for 30 min).

[0040] 3) Embedding and sectioning: The tissue and tumor specimens were placed in molten paraffin. After the paraffin cooled and solidified, the tissue and tumor were embedded in the paraffin. The paraffin around the tissue and tumor was removed, and the specimens were sectioned using a paraffin microtome. The tissue sections were then laid flat on glass slides and baked in a slide warmer at 50 °C for 2 h.

[0041] 4) Dewaxing and rehydration: Dewax the dried paraffin sections in xylene (twice, 15-20 min each time), then dehydrate them sequentially with 100%, 90%, 90%, and 75% ethanol, 5-10 min each time. Rinse with tap water to remove the ethanol, then wash twice with PBST (1L DPBS + 0.5 mL Tween 20), 5-10 min each time.

[0042] 5) Antigen retrieval and blocking: Dilute sodium citrate antigen retrieval solution (50×) 50 times with ddH2O to prepare antigen retrieval solution (1×). Immerse the sections in the antigen retrieval solution and heat in a water bath at 95-100 ℃ for 15-20 min. After cooling to room temperature, wash twice with PBST for 3-5 min each time. Then, use an immunohistochemical pen to mark the tissue extent, add 5% goat serum (50 μL goat serum + 1 mL DPBS) to immerse the sections, and block at room temperature for 45-60 min.

[0043] 6) Antibody incubation: Discard the blocking serum, add the primary antibody diluted with DPBS (generally 1:200, the dilution ratio can be adjusted according to different antigen abundance and antibody titer), and incubate overnight at 4 ℃ in a humidified chamber. The next day, remove the primary antibody and wash three times with PBST, 5-8 min each time. Add the corresponding diluted secondary antibody (generally 1:200, the dilution ratio can be adjusted according to antibody titer), and incubate at 37 ℃ for 30-60 min. Discard the secondary antibody, wash three times with PBST, 5-8 min each time.

[0044] 7) DAB staining: Place the slide in DPBS and wash three times in a decolorizing shaker for 5-8 min each time. Add freshly prepared DAB staining solution (DAB staining solution A + DAB staining solution B), and observe the degree of staining under a microscope (3-10 min). Immediately after completion, immerse the slide in PBST buffer to terminate the DAB reaction. Wash twice with PBST for 3-5 min each time. Then, place the slide in hematoxylin for 3 min, rinse with water for 5 min, and then place it in the separation solution (2 mL concentrated hydrochloric acid + 400 mL 95% ethanol) 1-2 times. Immediately rinse gently with tap water for 10 min.

[0045] 8) Counterstaining: Wash 3 times with DPBS, 3 min each time. Then wash with ddH2O for 5 min, add a large drop of hematoxylin staining solution, stain nuclear proteins for a few seconds, stain cytoplasmic or cell membrane proteins for 20 s, rinse with tap water, wash with ddH2O for 5 min, use ammonia to turn blue, and rinse thoroughly with running water.

[0046] 9) Dehydration and mounting: Place the slides in 75% ethanol, 85% ethanol, anhydrous ethanol I, anhydrous ethanol II, and xylene I in sequence for dehydration and clearing, 5 min for each reagent. Remove the slides from the xylene and let them air dry slightly before mounting with neutral resin.

[0047] 10) Microscopic examination, image acquisition and analysis.

[0048] (6) Enzyme-linked immunosorbent assay (ELISA) to detect ACTH hormone levels: ELISA is a commonly used experimental technique used to detect the concentration of specific proteins or other molecules in a sample.

[0049] 1) Cell plating: Add 1 mL of culture medium containing 40,000 cells to a 12-well plate.

[0050] 2) Supernatant collection: Collect the supernatant after 24 hours of cell culture. Collect 100 μL of blood from the orbital venous plexus of mice and store at 4°C.

[0051] 3) Centrifugation of supernatant: Centrifuge the collected cell supernatant or mouse serum (4℃, 2000 g, 20 min) to remove cell residues and other impurities.

[0052] 4) ACTH assay: ACTH secretion levels were measured using an ELISA kit (Immunoway, KE1520).

[0053] II. Experimental Results 1. Construct a high-throughput drug screening system for monitoring TRIM65-TPIT interaction and TPIT protein levels. A high-throughput drug screening model was constructed targeting a library of FDA-approved small molecule compounds. The model was primarily based on two systems: one using NanoBiT technology (capable of detecting protein-protein interactions in living cells) to monitor the interaction between TRIM65 and TPIT; and the other using NanoGlo technology (capable of quantitatively detecting HiBiT-tagged proteins in cells) to detect TPIT protein levels. Figure 1A, E). First, eight possible combinations of the NanoBiT tags LgBiT and SmBiT were optimized. The combination of LgBiT-TRIM65 and SmBiT-TPIT was found to have the strongest signal. Therefore, the fusion protein combination of N-terminal SmBiT-TPIT and N-terminal LgBiT-TRIM65 was used to construct a stable HEK293T transgenic strain for subsequent high-throughput drug screening. Figure 1 BD). For the NanoGlo HiBiT screening system, a stable HEK293T transfectant expressing LgBiT protein was first constructed. Based on this, the HiBiT-TPIT-Luciferase overexpression plasmid was transfected using a concentration gradient. Simultaneously, HiBiT and Luciferase signals were detected, and the relative signal values ​​were obtained by comparing the two signals. It was found that the relative signal was directly proportional to the TPIT protein expression level. Figure 1 E and F) demonstrate that the stable transgenic strain and the detection system can reflect the intracellular TPIT protein content and can be used for subsequent high-throughput drug screening.

[0054] 2. High-throughput drug screening identified the candidate drug aurinophene. The two stable cell lines described above were used to screen candidate drugs from a library of 1913 FDA-approved small molecule drugs on a high-throughput drug screening platform. The constructed stable cell lines were plated in 384-well microplates, and drug, substrate furimazine buffer, and luciferase buffer were added according to different screening procedures to detect luminescence signals. Figure 2 A, B). Analyze the relative HiBiT signal values ​​of the drug screening results and plot them as a scatter plot, using the average relative signal value as a control (A, B). Figure 2 C). Simultaneously, the luminescence signals of cells in the drug-treated group and the luminescence signals of cells in the DMSO-treated group were standardized based on the NanoGlo screening results, and a quantitative kinetic model was established to characterize the kinetic interaction between TRIM65-TPIT interaction and TPIT degradation. Figure 2 D), and finally, the drugs with signal values ​​lower than the control in the NanoGlo screening results were intersected with the drugs with signal values ​​increased in the NanoBiT screening results, and 27 candidate drugs were screened out and cluster analysis was performed. Figure 2E, F (Table 1). These drugs were found to be categorized into three groups: 1) NanoBiT signals continuously increased within 0-6 hours, indicating that the drugs continuously promoted the interaction between TRIM65 and TPIT within 6 hours, including bortezomib, flurbiprofen, and nimodipine; 2) NanoBiT signals continuously increased after drug treatment, showing a signal peak at different detection time points within 0-6 hours, followed by a gradual decline, indicating that drug treatment promoted the interaction between TRIM65 and TPIT at different times within 0-6 hours. After reaching its peak, TPIT degraded, and the NanoBiT signal weakened, including auronoxine, niclosamide, and onalespib; 3) NanoBiT signals continuously decreased within 0-6 hours, indicating that the drugs took effect within 5 minutes after treatment, promoting the interaction between TRIM65 and TPIT. Subsequently, TPIT began to degrade, and the NanoBiT signal began to weaken, including drugs such as avapritinib, escin, and homoharringtonine.

[0055] Table 1. Comparison of Candidate Drug Numbers and Drug Names

[0056] 3. Aurinophene enhances the interaction between TRIM65 and TPIT. To further screen for effective drugs, the above candidate drugs were validated in a NanoBiT small-system in 96-well plates. The results showed that the trend of cell luminescence signals after different drug treatments was consistent with that in the high-throughput drug screening results, further validating the reliability of the high-throughput drug screening results. Figure 3 A). Western blot analysis was used to examine whether 27 candidate drugs could reduce TPIT protein levels. Only 5 drugs significantly reduced TPIT levels, including Onalespib, AZD3759 (Zorifertinib), Gramicidin, aurinol, and Homoharringtonine (HHT), with inhibition levels of 13% for AZD3759, 39% for Onalespib, 49% for HHT, 62% for aurinol, and 69% for Gramicidin. Figure 3 B, C). Further Co-IP experiments showed that only aurinophene significantly promoted the interaction between exogenous TRIM65 and TPIT proteins and TPIT degradation. Figure 3 D, E).

[0057] 5. Aurinophene promotes TRIM65-mediated TPIT ubiquitination and degradation. To determine how auronoflavone regulates TPIT expression, ubiquitination experiments were conducted. The results showed that auronoflavone promotes TRIM65-mediated TPIT ubiquitination and reduces TPIT protein levels. Simultaneously, the proteasome inhibitor MG132 reversed this effect, indicating that auronoflavone promotes TRIM65-mediated TPIT ubiquitination and reduces TPIT levels via the proteasome pathway. Figure 4 AC). Meanwhile, the ubiquitination assay ruled out the effects of other gold compounds and deubiquitinating enzymes (USP14 and UCHL5). Figure 4 DI). Knockdown of TRIM65 did not alter TPIT ubiquitination levels following auronoxine treatment. Figure 4 J, K).

[0058] 6. Auranofin inhibits POMC expression and ACTH secretion. To investigate the effect of auronoxine on POMC protein levels, Western blotting was performed, revealing that auronoxine reduced TPIT and POMC levels, but the proteasome inhibitor MG132 could reverse this effect. Figure 5 AC). To further investigate the effect of auronoxine on POMC transcription, quantitative polymerase chain reaction (qPCR) was performed and it was found that auronoxine inhibited POMC transcription in AtT-20 cells, but did not affect TPIT transcription. Figure 5 D). Similarly, in the nude mouse tumor-bearing experiment, Western blotting and immunohistochemical experiments were performed on tumor tissues, and it was found that after auronoxine treatment, the levels of TPIT, POMC, and ACTH proteins in tumor cells decreased ( Figure 5 FI). Enzyme-linked immunosorbent assay (ELISA) was used to detect the effect of aurinol on ACTH secretion. The results showed that the drug inhibited ACTH secretion in AtT-20 cells and mouse blood. Figure 5 E, J).

Claims

1. Application of aurinophene in the preparation of drugs for treating pituitary adrenocorticotropic hormone-induced tumors.

2. The application according to claim 1, characterized in that: The aurinofen was obtained through screening using the NanoBiT high-throughput drug screening platform based on TRIM65-TPIT protein interaction and the NanoGlo high-throughput drug screening system based on protein-level quantitative detection.

3. The application according to claim 1, characterized in that: The aurinophene enhances the TRIM65-TPIT interaction, reduces TPIT protein levels, and decreases ACTH levels in cell culture medium / serum.

4. The application according to claim 1, characterized in that: The drug also contains pharmaceutically acceptable carriers and / or excipients.

5. The application according to claim 1, characterized in that: The pharmaceutically acceptable carriers and / or excipients include at least one of diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

6. A pharmaceutical composition for pituitary adrenocorticotropic hormone (ACTH) tumors, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of aurinophen as described in claim 1.

7. The pharmaceutical composition according to claim 6, characterized in that: The dosage form of the pharmaceutical composition includes at least one of tablets, pills, powders, solutions, suspensions, emulsions, and granules.

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