Application of TLC69 as ARIH1 activator in preparation of medicine for treating triple negative breast cancer

Abstract

The invention provides application of TLC69 as an ARIH1 activator in preparation of a medicine for treating triple-negative breast cancer, and particularly provides a composition comprising TLC69 and a ferroptosis inducer, it is found for the first time that TLC69 can be combined with the ferroptosis inducer to synergistically treat ferroptosis-resistant tumors, and the anti-tumor effect of the TLC69 is better than that of a single TLC69 drug.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of TLC69 as an ARIH1 activator in the preparation of drugs for treating triple-negative breast cancer. Background Technology

[0002] Ferroprelation plays a crucial role in cancer treatment, particularly in killing tumor cells that are difficult to eliminate through traditional mechanisms such as apoptosis. However, existing ferroptosis inducers mostly act directly on glutathione metabolism or lipid peroxidation pathways, resulting in low selectivity and significant toxic side effects, which limits their clinical application. ARIH1 (Ariadne RBRE3 Ubiquitin Protein Ligase 1), as E3 (ubiquitin ligase), can affect cellular sensitivity to ferroptosis by regulating the protein level of PRDX4 (Peroxiredoxin 4), but currently, no ferroptosis agonists targeting ARIH1 have been developed for anti-tumor therapy.

[0003] Therefore, there is an urgent need in this field to develop ferroptosis-inducing antitumor drugs based on the ARIH1 target. Summary of the Invention

[0004] The purpose of this invention is to develop a ferroptosis-inducing antitumor drug based on the ARIH1 target.

[0005] Another object of the present invention is to provide new uses for TLC69.

[0006] Another object of the present invention is to provide a method for activating ARIH1 in vitro.

[0007] Another object of the present invention is to provide a method for preventing and / or treating diseases related to ARIH1 activity.

[0008] Another object of the present invention is to provide a method for enhancing the ferrodeath effect.

[0009] A first aspect of the present invention provides a composition comprising: (i) TLC69; (ii) Ferrocyte inducers; and (iii) A pharmaceutically acceptable carrier; The structure of TLC69 is shown below: (Formula I); Ferrocyte inducers are selected from the following group: Erastin, RSL3, ML210, or combinations thereof.

[0010] In another preferred embodiment, the components (i) and (ii) in the composition account for 0.01-99.99 wt% of the total weight of the composition, more preferably 0.1-90 wt%, and more preferably 1-80 wt%.

[0011] In another preferred embodiment, the weight ratio of component (i) to component (ii) is 3-8:1, more preferably 4-5:1, such as 30:7.

[0012] In another preferred embodiment, the composition contains 1%–99% TLC69, more preferably 10%–90%, and even more preferably 50%–90% of the total weight of the active ingredients.

[0013] In another preferred embodiment, the composition comprises TLC69 and an ferroptosis inducer, with a total content of 0.01-99.99 wt%, more preferably 0.1-90 wt%, and more preferably 1-80 wt%, based on the total weight of the active ingredients.

[0014] In another preferred embodiment, the TLC69 is an ARIH1 activity enhancer.

[0015] In another preferred embodiment, the TLC69 is used to enhance the sensitivity of ferroptosis-resistant tumors to ferroptosis inducers.

[0016] In another preferred embodiment, the TLC69 treats ferroptosis-resistant tumors by modulating ferroptosis-related pathways.

[0017] In another preferred embodiment, the TLC69 treats ferroptosis-resistant tumors by allosterically activating ARIH1, thereby downregulating PRDX4 expression and upregulating 4-HNE levels.

[0018] In another preferred embodiment, the TLC69 enhances the iron death effect by morphologically activating ARIH1.

[0019] In another preferred embodiment, the TLC69 treats ferroptosis-resistant tumors by enhancing the ferroptosis effect.

[0020] In another preferred embodiment, the composition inhibits tumor growth by reversing the drug-resistant phenotype of ferroptosis-resistant tumors and enhancing the sensitivity of tumors to ferroptosis inducers.

[0021] In another preferred embodiment, the composition is used to treat ferroptosis-resistant tumors.

[0022] In another preferred embodiment, the ferroptosis-resistant tumors include triple-negative breast cancer, colorectal cancer, pancreatic cancer, and glioma.

[0023] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0024] In another preferred embodiment, the dosage form of the composition includes injectable dosage forms and oral dosage forms.

[0025] In another preferred embodiment, the oral dosage form includes tablets, capsules, films, and granules.

[0026] A second aspect of the present invention provides a medicine box, comprising: (a1) A first container, and TLC69 located in the first container, or a drug containing TLC69; (b1) A second container, and a ferroptosis inducer located in the second container, or a drug containing a ferroptosis inducer; The structure of TLC69 is shown below: (Formula I); Ferrocyte inducers are selected from the following group: Erastin, RSL3, ML210, or combinations thereof.

[0027] In another preferred embodiment, the first container and the second container may be the same or different containers.

[0028] In another preferred embodiment, the drug in the first container is a single-ingredient formulation containing TLC69.

[0029] In another preferred embodiment, the drug in the second container is a single-ingredient preparation containing an iron death inducer.

[0030] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injectable dosage form.

[0031] In another preferred embodiment, the kit also includes instructions for use.

[0032] In another preferred embodiment, the specification includes the following description: (a) A method of synergistically treating ferroptosis-resistant tumors by combining TLC69 with ferroptosis inducers.

[0033] In another preferred embodiment, the weight ratio of TLC69 to the ferroptosis inducer is 3-8:1, more preferably 4-5:1.

[0034] A third aspect of the invention provides a combination of TLC69 and a ferroptosis inducer for the preparation of a pharmaceutical composition or cartridge for the treatment of ferroptosis-resistant tumors.

[0035] In another preferred embodiment, the weight ratio of TLC69 to the ferroptosis inducer is 3-8:1, more preferably 4-5:1.

[0036] In another preferred embodiment, the ferroptosis-resistant tumors include triple-negative breast cancer, colorectal cancer, pancreatic cancer, and glioma.

[0037] In another preferred embodiment, the pharmaceutical composition or cassette inhibits tumor growth by reversing the drug-resistant phenotype of ferroptosis-resistant tumors and enhancing the tumor's sensitivity to ferroptosis inducers.

[0038] In another preferred embodiment, the effective concentration of TLC69 is 0.5-100 mg / kg, more preferably 1-50 mg / kg, and even more preferably 20-40 mg / kg.

[0039] In another preferred embodiment, the effective concentration of the ferroptosis inducer is 0.1-50 mg / kg, more preferably 0.5-20 mg / kg, and even more preferably 1-10 mg / kg.

[0040] In another preferred embodiment, the pharmaceutical composition or cartridge comprises (a) TLC69; and (b) an ferroptosis inducer; and (c) a pharmaceutically acceptable carrier.

[0041] In another preferred embodiment, the (a) TLC69 and (b) ferroptosis inducer constitute 0.01-99.99 wt%, more preferably 0.1-90 wt%, and more preferably 1-80 wt% of the total weight of the pharmaceutical composition or cassette.

[0042] A fourth aspect of the present invention provides a method for treating ferroptosis-resistant tumors, comprising: TLC69 and the ferroptosis inducer, or the composition described in the first aspect of the invention, or the kit described in the second aspect of the invention, are administered to the desired subject.

[0043] In another preferred embodiment, the object includes a human or non-human mammal suffering from ferroptosis-resistant tumors.

[0044] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0045] In another preferred embodiment, the dosage of TLC69 is 0.1 to 500 mg / kg, preferably 0.5 to 100 mg / kg, more preferably 1 to 50 mg / kg, and even more preferably 2 to 30 mg / kg.

[0046] In another preferred embodiment, the dosage of the ferroptosis inducer (such as Erastin) is 0.1 to 50 mg / kg, more preferably 0.5 to 20 mg / kg, and even more preferably 1 to 10 mg / kg.

[0047] In another preferred embodiment, the TLC69 is applied twice a day, once a day, once every two days, twice every three days, or once every four days.

[0048] In another preferred embodiment, the application time of the TLC69 is from 7 days to 1 year, preferably not less than 14 days (2 weeks), and more preferably not less than 21 days (3 weeks).

[0049] In another preferred embodiment, the ferroptosis inducer is administered once a week, three times a week, five times a week, or once a day.

[0050] In another preferred embodiment, the ferroptosis inducer is applied for 1 day to 1 year, more preferably 1 to 6 months, and most preferably 2 to 12 weeks.

[0051] In another preferred embodiment, the TLC69 is administered simultaneously or sequentially with the ferroptosis inducer.

[0052] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0053] Figure 1 This is a graph showing the effect of ARIH1 overexpression combined with a ferroptosis inducer on tumor growth curves, tumor weight, and the expression level of the ferroptosis marker ACSL4 protein in a mouse 4T1 triple-negative breast cancer (TNBC) xenograft model according to embodiments of the present invention.

[0054] Figure 2 This is a statistical chart showing the effect of in vivo screening of TLC69 on enhancing ARIH1 activity according to an embodiment of the present invention.

[0055] Figure 3 This is a graph showing the effect of TLC69 on ARIH1 activity in vitro in an embodiment of the present invention.

[0056] Figure 4 This is a statistical graph in this embodiment of the invention, which uses CCK8 and flow cytometry to jointly verify that TLC69 enhances Erastin-induced ferroptosis in a way that depends on ARIH1.

[0057] Figure 5 These are tumor growth curves, tumor weight statistics, and representative tumor photographs of TLC69 in mouse 4T1 TNBC and human TNBC PDX xenograft models (patient-derived xenograft models) in embodiments of the present invention.

[0058] Figure 6This is a graph showing the effects of TLC69 on mouse body weight and major organs (liver, kidney, spleen) in mouse 4T1 TNBC and human TNBC PDX xenograft models in this invention.

[0059] Figure 7 This is a graph showing the effect of TLC69 on the expression levels of ferroptosis markers 4-HNE and PRDX4 proteins in a mouse 4T1 TNBC xenograft model according to embodiments of the present invention.

[0060] Figure 8 This is a survival curve correlation analysis diagram of ARIH1 expression level and overall survival (OS) of clinical cancer patients in an embodiment of the present invention.

[0061] Figure 9 This is a structural diagram of Arih1-mcherry.

[0062] Figure 10 It is Smbit-ARIH1 △Ariadne and Lgbit-ARIH1 Ariadne The structural diagram.

[0063] Figure 11 This is a structural diagram of Arih1-shRNA. Detailed Implementation

[0064] Through extensive and in-depth research, the inventors have discovered for the first time that compound TLC69 can act as an allosteric activator of ARIH1, effectively enhancing ARIH1 protein activity in a concentration-dependent manner, and further inducing downstream ferroptosis, downregulating PRDX4 and upregulating 4-HNE levels. Furthermore, this compound can enhance the sensitivity of ferroptosis-resistant tumors to ferroptosis inducers. Therefore, TLC69 can be used in combination with ferroptosis inducers for synergistic treatment of ferroptosis-resistant tumors, and exhibits better anti-tumor effects than TLC69 alone. Based on these findings, the inventors have completed this invention.

[0065] compound In this invention, the specific structure of compound "TLC69" is shown in Formula I below. TLC69 can be obtained by Hangzhou Tianji Jishi Biotechnology Co., Ltd.

[0066] .

[0067] Ferrocyte inducer Ferroplastin inducers are a class of drugs or compounds that can specifically trigger or enhance ferroptosis in cells. Ferroptosis is a novel form of programmed cell death driven by iron-dependent lipid peroxidation, distinct from apoptosis, necroptosis, and autophagy. Its core biochemical characteristics lie in the abnormal accumulation of intracellular ferrous ions and the collapse of the antioxidant defense system caused by the depletion of reduced glutathione (GSH), leading to the uncontrolled accumulation and diffusion of lipid reactive oxygen species in the cell membrane phospholipid bilayer. Specifically, ferroptosis inducers exert their function by precisely intervening in key nodes of the ferroptosis signaling pathway. Their mechanisms of action mainly include: directly inhibiting the activity of glutathione peroxidase 4 (GPX4), thereby eliminating its ability to reduce lipid peroxides; or blocking systemic xc - Cystine / glutamate antitransporters disrupt cystine uptake, thereby depleting key raw materials for GSH synthesis within the cell and indirectly inhibiting GPX4 function. Furthermore, they promote the expansion of unstable intracellular iron pools (e.g., by increasing iron uptake or inhibiting iron storage), accelerating the Fenton reaction and directly catalyzing the lipid peroxidation chain reaction; and they perturb lipid metabolism, increasing the proportion of peroxidation-sensitive polyunsaturated fatty acids (PUFAs) in the cell membrane. Therefore, the essence of ferroptosis inducers is to disrupt cellular redox homeostasis and iron metabolism balance, ultimately leading to irreversible loss of cell membrane integrity and cell disintegration, marked by the massive production of lipid peroxidation products.

[0068] In a preferred embodiment, the ferroptosis inducer includes Erastin. Erastin, RSL3, and ML210 are selectively lethal drugs. The chemical structures of Erastin, RSL3, and ML210 are shown in Formulas II, III, and IV, respectively. All three drugs are available through commercial channels.

[0069] The research of this invention shows that the combined use of TLC69 of this invention with ferroptosis inducers (such as Erastin, RSL3 and ML210) in the treatment of ferroptosis-resistant tumors (such as triple-negative breast cancer and colorectal cancer) can exert a synergistic effect. The combination drug of TLC69 and ferroptosis inducers (such as Erastin, RSL3 and ML210) in the treatment of ferroptosis-resistant tumors (such as triple-negative breast cancer and colorectal cancer) has excellent efficacy and good prospects for clinical application.

[0070] Composition and method of application As used herein, the term "composition" includes (a1) a first active ingredient, wherein the first active ingredient is TLC69; and (a2) a second active ingredient, wherein the second active ingredient is a ferroptosis inducer; and (b) a pharmaceutically acceptable carrier. Furthermore, the compositions include pharmaceutical compositions.

[0071] Typically, the active ingredients of this invention can be formulated into a non-toxic, inert, and pharmaceutically acceptable carrier medium. The formulated pharmaceutical compositions can be administered via conventional routes, including (but not limited to): oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or local administration.

[0072] This invention also provides a pharmaceutical composition containing a safe and effective amount of the active ingredient of this invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of this invention can be formulated as injections, for example, prepared by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets, and capsules are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram to 10 milligrams per kilogram of body weight per day. Preferably, the dosage of TLC69 can be: 0.1 to 2000 mg daily for adults, preferably 1 to 300 mg / day. The dosage of the ferroptosis inducer can be: 0.1 to 2000 mg every two weeks for adults, preferably 1 to 300 mg / two weeks. As a drug for synergistic cancer therapy, it can be formulated as oral and non-oral preparations. Oral administration can be formulated into common dosage forms such as tablets, powders, granules, and capsules. The excipients used can be one or more of starch, lactose, sucrose, mannose, and hydroxymethyl cellulose. Disintegrants can be one or more of potato starch and hydroxymethyl cellulose. Binders can be one or more of gum arabic, corn starch, gelatin, and dextrin. In addition to the above dosage forms, oral preparations can also be formulated into emulsions and syrups.

[0073] Non-oral preparations can be formulated into injectable forms, which can be prepared with water for injection, physiological saline, or glucose solution, or with the addition of certain proportions of ethanol, propanol, ethylene glycol, etc. They can also be formulated into commonly used dosage forms such as nasal drops, sprays, rectal suppositories, and rectal retention enemas.

[0074] medicine box The present invention also provides a medicine box, the medicine box comprising: (a1) A first container, and TLC69 located in the first container, or a drug containing TLC69; (b1) A second container, and a ferroptosis inducer located in the second container, or a drug containing a ferroptosis inducer.

[0075] In a preferred embodiment, the first container and the second container may be the same or different containers.

[0076] The formulation containing TLC69 can be a unit dosage form containing TLC69, and the formulation containing the ferroptosis inducer can be a unit dosage form containing the ferroptosis inducer.

[0077] As used herein, the term "unit dosage form" refers to a dosage form in which a composition is prepared for a single dose for ease of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release formulations.

[0078] In another preferred embodiment, the instruction manual describes the following method of use: (I) Simultaneously administer to the desired subject a formulation containing TLC69 and a formulation containing an inducer of ferroptosis; and optionally... (II) Repeat steps (I)-(II).

[0079] The formulation of this invention can be taken three times a day to once every twenty days, or in a sustained-release manner once every ten days. The preferred method is once daily, as this facilitates patient adherence and significantly improves patient compliance.

[0080] When taking this medication, in most cases the total daily dose should be lower than (or in a few cases equal to or slightly greater than) the usual daily dose of each individual drug. Of course, the effective dose of the active ingredient may vary depending on the administration method and the severity of the disease being treated.

[0081] The main advantages of this invention include: (1) This invention is the first to discover that the combination of TLC69 of this invention and ferroptosis inducer can effectively treat ferroptosis-resistant tumors and has a synergistic effect.

[0082] (2) This invention provides a new use for TLC69 as a drug targeting ARIH1, as an ARIH1 activity enhancer and allosteric activator, so that it can be used to prepare drugs for treating ferroptosis-resistant tumors, such as triple-negative breast cancer.

[0083] (3) The present invention provides a method for treating ferroptosis-resistant tumors by administering TLC69 to the subject to allosterically activate ARIH1 and enhance its activity, downregulate PRDX4 expression level and upregulate 4-HNE, thereby enhancing the ferroptosis effect.

[0084] (4) The present invention provides a method for inhibiting tumor growth by enhancing the ferroptosis effect, thereby inhibiting the growth of triple-negative breast cancer tumors by activating the ARIH1-mediated ferroptosis pathway through the administration of TLC69 to the subject.

[0085] (5) This invention pioneers a novel synergistic mechanism. Unlike traditional sensitization strategies based on single targets, this invention targets and activates ARIH1 through TLC69, precisely regulating its downstream PRDX4-4-HNE signaling axis, thereby weakening the antioxidant defense capabilities of tumor cells at their source and "resetting" the sensitivity of tumor cells to ferroptosis. This mechanism provides a novel and validated combination of signaling pathways and drug targets for overcoming tumor ferroptosis resistance.

[0086] (6) The combination strategy provided by this invention has potentially wide applicability. The core of the ARIH1-mediated ferroptosis sensitization pathway lies in regulating the universal redox balance and lipid peroxidation process. Therefore, the strategy of combining TLC69 with a ferroptosis inducer in this invention is not limited to triple-negative breast cancer, but is expected to be extended to other types of malignant tumors with ferroptosis resistance (such as colorectal cancer, pancreatic cancer, and glioma), providing a promising new solution for addressing a wider range of tumor treatment resistance problems.

[0087] (7) Compared with traditional single or combined use of high-dose ferroptosis inducers, the combination strategy of the present invention has potential safety advantages. TLC69, as an allosteric activator of ARIH1, works by "sensitizing" tumor cells rather than directly causing strong oxidative stress. This makes it possible to achieve the same or better anti-tumor effect with a lower dose of ferroptosis inducer in combination therapy, thereby significantly reducing the systemic toxic side effects such as normal tissue damage and iron overload that may be caused by high-dose ferroptosis inducers, and improving the overall therapeutic window and the safety of clinical use.

[0088] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0089] Unless otherwise specified, all materials and reagents described in this invention specification are commercially available products.

[0090] Example 1: TLC69 is an activity enhancer for ARIH1. The applicant's research found that high expression of ARIH1 can reverse the drug-resistant phenotype of ferroptosis-resistant tumors, significantly increasing their sensitivity to ferroptosis inducers, thereby effectively inhibiting tumor growth, such as... Figure 1 As shown in AD. The specific implementation steps are as follows: Arih1 gene overexpression was successfully achieved in mouse triple-negative breast cancer (TNBC) 4T1 cells using lentiviral infection technology (see reference: X. Liu, X. Cen, R. Wu, Z. Chen, Y. Xie, F. Wang, B. Shan, L. Zeng, J. Zhou, B. Xie, Y. Cai, J. Huang, Y. Liang, Y. Wu, C. Zhang, D. Wang, H. Xia. ARIH1 activates STING-mediated T-cell activation and sensitizes tumors to immune checkpoint blockade. Nat Commun 2023, 14(1): 4066). The plasmid structure is shown below. Figure 9 (As shown). This example uses 6-8 week old SPF-grade female BALB / c mice (purchased from Zhejiang Vital River) and is housed in a standard SPF environment (temperature 18-22℃, humidity 50±10%, 12-hour light-dark cycle).

[0091] For the 4T1 TNBC xenograft model, 1×10 5 Four T1 cells were resuspended in 100 μL of pre-chilled PBS and subcutaneously seeded into the second pair of mammary fat pads of mice. The long diameter (L) and short diameter (W) of the tumor were measured periodically, and the volume (V = L × W) was calculated. 2 / 2). When the tumor grows to 20-50 mm 3 Then, the mice were randomly divided into four groups of 10 each: (1) wild-type tumor cells + saline solution control; (2) wild-type tumor cells + Erastin treatment (7 mg / kg, intraperitoneal injection 3 times every 4 days); (3) ARIH1 overexpressing tumor cells + saline solution control; (4) ARIH1 overexpressing tumor cells + Erastin treatment (same regimen as group 2). Figure 1As shown in the results of the AD study, ARIH1 overexpression significantly inhibited tumor growth compared to wild-type tumors; combined with Erastin treatment, tumor volume further decreased, demonstrating a synergistic anti-tumor effect. Simultaneously, Western blot analysis revealed that, compared to wild-type tumors, the expression level of the ferroptosis-related marker ACSL4 protein was significantly upregulated in the tumor tissues of mice in the ARIH1 overexpression group. These data indicate that ARIH1 overexpression can significantly enhance the anti-tumor efficacy of the ferroptosis inducer Erastin, providing crucial preclinical experimental evidence for developing a combination therapy strategy based on ARIH1 and ferroptosis induction.

[0092] The reagents and animal sources used in this embodiment are shown in Table 1 below: Table 1

[0093] Based on this discovery, targeting and activating ARIH1 has become a potential synergistic strategy for ferroptosis therapy. To address the issue of limited ARIH1 activity due to the "self-inhibition" caused by the interaction between the Ariadne domain and the RING2 domain (e.g., at the C357 site), this invention designs and constructs a high-throughput drug screening model based on a luciferase complementary system, aiming to screen small molecule compounds that specifically relieve ARIH1 self-inhibition and activate its function. The screening system employs the luciferase fusion expression system Smbit-ARIH1. △Ariadne and Lgbit-ARIH1 Ariadne(Plasmid construction method reference: S. Piech, S. Brüschweiler, J. Westphalen, KM Siess, JG Murias, R. Konrat, JW Bigenzahn, G. Superti-Furga. Identification and Characterization of Novel Small-Molecule Enhancers of the CUL3LZTR1 E3 Ligase KRAS Complex. ACS ChemBiol 2024, 19(9): 1942-1952; High-throughput drug screening method reference: X. Liu, X. Cen, R. Wu, Z. Chen, Y. Xie, F. Wang, B. Shan, L. Zeng, J. Zhou, B. Xie, Y. Cai, J. Huang, Y. Liang, Y. Wu, C. Zhang, D. Wang, H. Xia. ARIH1 activates STING-mediated T-cell activation and sensitizes tumors to immune checkpointblockade. Nat Commun 2023, 14(1): 4066, plasmid structure as follows Figure 10 As shown in the diagram, this system can regulate luciferase activity through changes in the interaction between the large and small subunits of ARIH1: when the drug relieves the autoinhibition of ARIH1, subunit spatial separation leads to a decrease in fluorescence signal, thus directly reflecting the activity state of ARIH1. The specific implementation steps are as follows: HEK293T cells were co-transfected with the recombinant ARIH1 fluorescent reporter gene plasmid (plasmid construction method as described above, plasmid structure as shown above). Figure 10 (As shown). 24 h after transfection, the cells were digested, centrifuged, and resuspended. 50 μL of cell suspension (containing approximately 5000 cells) was seeded into 384-well plates and cultured for 12 h. The test compound was then added using a pipetting station to a final concentration of 5 μM. After another 24 h of culture, the substrate furimazine was added to the wells to a final concentration of 10 μM, and the fluorescence signal intensity was immediately measured using a Cytation5. Finally, positive small molecule compounds that enhanced ARIH1 activity were selected, such as... Figure 2 As shown in Figure A.

[0094] Analysis of the screened positive compounds revealed that, compared to the dimethyl sulfoxide (DMSO) control group, the mean fluorescence level of ARIH1 in TLC69-treated cells decreased by 0.5-fold. A two-tailed t-test confirmed a statistically significant difference (e.g., ...). Figure 2 (As shown in Figure A). We also used CCK8 assays to assess cell viability in TLC69 cells in combination with Erastin. The specific implementation steps are as follows: 4T1 TNBC cell suspensions were prepared and seeded in 96-well plates with 90 µL of cell suspension per well (containing approximately 8000 cells), with 8 replicates per group. After 12 h of culture, a series of gradient concentrations of the drug were added to the wells, and the plates were cultured for another 12 h. 10 µL of CCK8 reagent was then added to each well. The culture plates were incubated at 37°C in the dark for 1–3 h, and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0095] like Figure 2 As shown in Figure B, TLC69 significantly promoted Erastin-induced 4T1 cell death. Therefore, we selected TLC69 as the primary target for our subsequent research.

[0096] The reagents used in this embodiment and their sources are shown in Table 2 below: Table 2

[0097] Example 2: In vitro allosteric activation of ARIH1 by TLC69 Next, the role of TLC69 in ARIH1 ubiquitination was examined using an in vitro ubiquitination assay combined with Western blotting (e.g., Figure 3 (As shown in Figure A). The specific implementation steps are as follows: HEK293T cells were transfected with an empty vector plasmid (purchased from Newp Biotech) and an ARIH1-WT plasmid (purchased from Miaoling Biotech), respectively. Cell precipitate proteins were lysed after 36 h. After washing once with 1×Ubiquitin buffer, UBA1 (E1) (100 ng), UBCH7 (E2) (500 ng), Ub-His (10 μg), ARIH1-HA (E3), and TLC69 were selectively added according to the experimental design. Then, 4 μL of 10×ATP Regeneration Solution was added, and the final volume was brought to 40 μL with 1×Ubiquitin buffer. The mixture was incubated in a 37°C water bath for 2 h, with the bottom of the centrifuge tube gently tapped every 20 min to mix. 40 μL of 2×SDS-PAGE Loading Buffer was added to the sample, mixed, and incubated in a 95°C metal bath for 10 min. Centrifuge at 12,000 rpm for 10 min at room temperature, and collect the supernatant for Western blot analysis.

[0098] After separation by SDS-PAGE electrophoresis, proteins were transferred to a PVDF membrane using wet transfer. The membrane was then blocked with 5% skim milk at room temperature for 1 h, the blocking buffer was discarded, and ARIH1 (1:1000) primary antibody (Ariadne RBR E3 ubiquitin ligase 1 antibody, purchased from Everest Biotech) was added and incubated overnight at 4°C. After washing the membrane with washing buffer, the membrane was incubated with the corresponding HRP-labeled secondary antibody at room temperature for 1 h. Finally, the membrane was incubated with ECL chemiluminescent substrate, and images were taken using a chemiluminescence imaging system.

[0099] like Figure 3 As shown in Figure A, at a concentration of 5 μM, TLC69 effectively increased the ubiquitination level of ARIH1. Furthermore, with increasing TLC69 concentration, ARIH1 ubiquitination further increased, suggesting it may be an effective ARIH1 activator. To provide a structural basis for elucidating the dynamic process of drug molecule activation of the ARIH1 protein, this embodiment performed molecular docking simulations on the allosteric regulatory region of the ubiquitin ligase ARIH1, systematically analyzing the dynamic binding characteristics of the activator TLC69 to the self-inhibiting Ariadne domain of ARIH1, such as... Figure 3 As shown in B.

[0100] The reagents used in this embodiment and their sources are shown in Table 3 below: Table 3

[0101] Example 3: TLC69 enhances Erastin-induced ferroptosis effect in a way that depends on ARIH1 To further determine the cellular activation effect of TLC69 on ARIH1, in this embodiment, we knocked down ARIH1 in 4T1 triple-negative breast cancer cells (ARIH1-KD) (the knockdown system construction method is based on the reference: X. Liu). X.Cen R. Wu Z. Chen Y. Xie , F. Wang, B. Shan, L. Zeng, J. Zhou, B. Xie,Y. Cai, J. Huang, Y. Liang, Y. Wu, C. Zhang, D. Wang, H. Xia. ARIH1 activatesSTING-mediated T-cell activation and sensitizes tumors to immune checkpointblockade. Nat Commun 2023, 14(1): 4066, the plasmid structure is as follows Figure 11 Afterwards, the effects of TLC69 on Erastin-induced ferroptosis in 4T1 cells were detected using CCK8 and flow cytometry (as shown in the figure). Figure 4 (As shown in Figure AB). The specific steps of flow cytometry are as follows: The content of reactive oxygen species (ROS) in live cells was detected using the BODIPY 581 / 591 C11 fluorescent probe. After counting the 4T1 tumor cells to be tested, the cells were divided into groups of 2 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Cells were then treated with a specific concentration of Erastin for 16 h. The old culture medium was discarded, and each well was incubated with 2 mL of fresh RPMI-1640 complete medium (purchased from VivaCell) diluted with BODIPY 581 / 591 C11 working solution (final concentration 5 µM), and incubated for 30 min. The culture medium was discarded, and the cells were washed 1-2 times with 1× PBS. Then, trypsin containing 0.25% EDTA was added to the cells in a 37°C cell culture incubator to digest the cells. Once the cells became rounded and completely detached, complete culture medium was added to stop the digestion. The cell suspension was transferred to 1.5 mL centrifuge tubes and centrifuged at 800 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 150 µL of PBS. The fluorescence intensity of each group of cells was detected by flow cytometry.

[0102] like Figure 4As shown in Figures AB, in wild-type 4T1 TNBC tumor cells, TLC69 significantly promoted Erastin-induced 4T1 cell death and lipid ROS accumulation. However, treatment with TLC69 on top of ARIH1 knockdown did not significantly change 4T1 cell death or lipid ROS (e.g., ...). Figure 4 (As shown in AB). This suggests that TLC69's enhanced ferroptosis-inducing effect depends on the ARIH1 protein.

[0103] The reagents used in this embodiment and their sources are shown in Table 4 below: Table 4

[0104] Example 4: Antitumor effect of TLC69 in a TNBC xenograft model In in vitro experiments, the ARIH1 activator TLC69 significantly activated ARIH1 protein activity and effectively enhanced Erastin-induced ferroptosis. Therefore, we established mouse 4T1 TNBC and human TNBC PDX xenograft models to further verify the antitumor effect of the combination of TLC69 and a ferroptosis inducer. The specific implementation steps are as follows: This embodiment uses 6-8 week old SPF-grade female BALB / c mice and BALB / c nude mice (purchased from Zhejiang Vital River), which are housed in an SPF-grade barrier environment. The environmental parameters of the animal room are strictly controlled at a temperature of 18-22℃ and a relative humidity of 50±10%, and a 12-hour light / 12-hour dark day-night cycle is adopted.

[0105] For the 4T1 TNBC xenograft model, take 1×10 5 One TNBC 4T1 cell was resuspended in 100 μL of pre-chilled 1×PBS and stored on ice for later use. After mice were fully anesthetized, they were fixed with their abdomens facing upwards, and subcutaneous injections were administered into the second pair of mammary fat pads. For the human TNBC PDX xenograft model, fresh TNBC tumor tissue was harvested, necrotic areas and connective tissues such as fat were removed, and after rinsing with PBS, it was cut into 2×2×2 mm pieces with a scalpel. 3 The tissue blocks were placed in a culture dish containing tumor sample transport and preservation solution. After shaving and disinfecting the scapular region of mice, the tumor tissue blocks were inoculated subcutaneously into the scapular region using a cannula under local anesthesia with lidocaine.

[0106] After inoculation, the long diameter (L) and short diameter (W) of the tumor are measured regularly using calipers, and the result is calculated using the formula V = L × W. 2 / 2 Calculate tumor volume. When the tumor volume reaches 20-50 mm... 3When the growth uniformity is good, the mice are randomly assigned to the following four groups, with 8-10 mice in each group: (1) solvent control group (physiological saline); (2) Erastin monotherapy group (7 mg / kg, intraperitoneal injection 3 times every 4 days); (3) TLC69 monotherapy group (30 mg / kg, intraperitoneal injection daily); (4) Erastin + TLC69 combination therapy group (Erastin 7 mg / kg, TLC69 30 mg / kg). Figure 5 As shown in the results from the study, TLC69 monotherapy significantly inhibited tumor growth compared to the solvent control group (p<0.05), and the combination therapy with Erastin further reduced tumor volume, demonstrating a synergistic anti-tumor effect. Particularly in patient-derived TNBC models, the combination therapy reduced tumor weight by 80% compared to the control group. These data indicate that TLC69 can significantly enhance the anti-tumor effect of ferroptosis inducers in combination, providing experimental evidence for the development of related combination therapies.

[0107] The reagents and animal sources used in this embodiment are shown in Table 5 below: Table 5

[0108] Example 5: Effects of TLC69 on body weight and organs in xenograft mice In this embodiment, the mouse body weight was monitored every 2-4 days in the mouse xenograft model experiment. At the end of the experiment, fresh tissue samples of the mouse liver, kidney and spleen were collected, fixed with 4% paraformaldehyde, and then embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E).

[0109] like Figure 6 As shown in the AD diagram, there were no significant differences in body weight change trends and pathological morphological characteristics of major organs (liver, kidney, and spleen) between the TLC69-treated group and the solvent control group. This result indicates that within the experimental dose range, TLC69 did not induce significant toxicity or organ damage in TNBC xenograft mice. These data confirm the good in vivo safety of TLC69, providing a safety basis for its further application in tumor treatment.

[0110] Example 6: TLC69 upregulates 4-HNE and downregulates PRDX4 in the 4T1 TNBC model. In this embodiment, fresh tumor tissue was collected at the endpoint of the mouse xenograft model experiment (day 21 post-inoculation) and fixed overnight in 4% paraformaldehyde. Subsequently, immunohistochemical staining was used to detect the effect of TLC69 on the expression level of the ferroptosis-related marker 4-HNE (e.g., Figure 7 (As shown in Figure A). The specific implementation steps are as follows: Fixed tissues were embedded in paraffin, sectioned, and stained. Paraffin sections were first baked at 60°C for 2 h, followed by dewaxing and hydration with xylene and a gradient of ethanol (from anhydrous ethanol to 75% ethanol). After rinsing with distilled water, they were incubated with 3% H2O2 at room temperature for 30 min. Microwave antigen retrieval was performed using citrate buffer (preheating for 5 min, high for 4 min, and medium for 5 min). After washing three times with PBS, the sections were blocked for 30 min, followed by incubation at 4°C overnight with 4-HNE primary antibody (1:200) (4-hydroxynonenal antibody, purchased from Abcam). After primary antibody incubation, the sections were thoroughly washed with PBS, and then incubated at 37°C for 60 min with the corresponding secondary antibody (goat anti-rabbit IgG-HRP antibody, purchased from Abcam). The sections were washed again with PBS. DAB staining and hematoxylin counterstaining were then performed, with thorough rinsing with tap water after each step. Finally, the sections were dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and then air-dried naturally.

[0111] like Figure 7 As shown in Figure A, the average percentage of 4-HNE positive staining in each group was as follows: solvent control group 0.1544%, Erastin monotherapy group 0.2099%, TLC69 monotherapy group 0.4486%, and TLC69 combined with Erastin group 1.104%. The data indicate that TLC69 monotherapy significantly increased the area of ​​4-HNE immunostaining in tumor tissue; after combined treatment with Erastin, the 4-HNE positive percentage further increased. Simultaneously, Western blot results showed that, compared with the control group, the expression level of PRDX4 protein in the tumor tissue of mice treated with TLC69 was significantly downregulated (e.g., ...). Figure 7 (As shown in Figure B). The above results demonstrate that TLC69 can significantly upregulate the expression of the ferroptosis-related marker 4-HNE and downregulate the PRDX4 protein level, suggesting that it exerts its anti-tumor effect by regulating ferroptosis-related pathways.

[0112] The reagents used in this embodiment and their sources are shown in Table 6 below: Table 6

[0113] Example 7: ARIH1 expression is positively correlated with overall patient survival. This embodiment further utilized public databases to analyze the correlation between ARIH1 expression levels and clinical prognosis in cancer patients. After acquiring data from the Kaplan-Meier Plotter platform, samples were sorted according to the level of ARIH1 mRNA expression in the tumor, and the top 25% and bottom 25% of samples were designated as the "ARIH1 high expression group" and the "ARIH1 low expression group," respectively. Figure 8As shown, in triple-negative breast cancer, hematological malignancies (acute myeloid leukemia), lung adenocarcinoma, and pancreatic cancer, patients in the ARIH1 high-expression group had significantly longer overall survival (OS) than those in the low-expression group. This indicates a positive correlation between ARIH1 mRNA expression level and patient OS, meaning that higher ARIH1 expression is associated with better prognosis. These data suggest that high ARIH1 expression may be related to increased tumor sensitivity to certain therapeutic interventions (such as ferroptosis induction). Therefore, ARIH1 expression level holds promise as a potential biomarker for predicting whether patients will benefit from treatment strategies targeting ferroptosis.

[0114] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A composition, characterized in that, The composition comprises the following substances composition: TLC69; Ferrocyte inducers; and Pharmaceutically acceptable carriers; The structure of TLC69 is shown below: ； Erastin was used as the ferroptosis inducer.

2. The composition according to claim 1, characterized in that, The weight ratio of TLC69 to ferroptosis inducer is 3-8:

1.

3. The composition according to claim 1, characterized in that, The weight ratio of TLC69 to ferroptosis inducer is 4-5:

1.

4. The composition according to claim 1, characterized in that, The TLC69 is used to enhance the sensitivity of ferroptosis-resistant tumors to ferroptosis inducers.

5. The composition according to claim 1, characterized in that, The composition inhibits tumor growth by reversing the drug-resistant phenotype of ferroptosis-resistant tumors and enhancing the sensitivity of tumors to ferroptosis inducers.

6. The composition according to claim 1, characterized in that, The composition is used to treat ferroptosis-resistant tumors.

7. The composition according to claim 6, characterized in that, The ferroptosis-resistant tumors include triple-negative breast cancer, leukemia, pancreatic cancer, and lung adenocarcinoma.

8. A medicine box, characterized in that, It consists of the following components: A first container, and TLC69 located in the first container, or a drug containing TLC69; The second container, and the ferroptosis inducer located in the second container, or a drug containing the ferroptosis inducer; The structure of TLC69 is shown below: ； Erastin was used as the ferroptosis inducer.

9. An application of a combination, characterized in that, The combination comprises TLC69 and a ferroptosis inducer, used to prepare a pharmaceutical composition or kit for treating ferroptosis-resistant tumors, including triple-negative breast cancer, leukemia, pancreatic cancer, and lung adenocarcinoma.

10. The use as described in claim 9, characterized in that, The weight ratio of TLC69 to ferroptosis inducer is 3-8:

1.

11. The use as described in claim 10, characterized in that, The weight ratio of TLC69 to ferroptosis inducer is 4-5:1.

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