Application of YAP inhibitors in combination with DRD2 inhibitors in the preparation of drugs for the treatment of breast cancer and / or liver cancer

Abstract

This invention discloses the application of YAP inhibitors in combination with DRD2 inhibitors in the preparation of therapeutic drugs for breast cancer and / or liver cancer. This invention utilizes different YAP inhibitors (CA3, MGH-CP1, verteporfen) and different DRD2 inhibitors (perphenazine, trifluoperazine hydrochloride) to treat breast cancer cell lines and liver cancer cell lines, demonstrating that both YAP inhibitors and DRD2 inhibitors have a synergistic effect in killing tumor cells. Subsequent experiments confirmed that the combined use of YAP inhibitors and DRD2 inhibitors can indeed achieve synergistic treatment of breast cancer. Therefore, targeted therapy for breast cancer and / or liver cancer can be achieved through the combined use of YAP inhibitors and DRD2 inhibitors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology. More specifically, it relates to the use of YAP inhibitors in combination with DRD2 inhibitors in the preparation of therapeutic drugs for breast cancer and / or liver cancer. Background Technology

[0002] Breast cancer is a common malignant tumor with the fastest-growing and most prevalent incidence rate among women worldwide, posing a serious threat to women's health and lives. Molecular subtyping of breast cancer is based on the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and the cell proliferation marker Ki-67, classifying breast cancer into four subtypes: Luminal A, Luminal B, HER2-overexpressing, and triple-negative breast cancer (TNBC). Luminal A breast cancer is characterized by high expression of ER or PR, HER2 negativity, and low Ki-67 expression (<14%). It exhibits low tumor proliferation activity, weak invasiveness, low risk of metastasis, high degree of tumor cell differentiation, and relatively stable cell cycle regulation. Clinically, endocrine therapy is the primary treatment, with tamoxifen or aromatase inhibitors significantly delaying disease progression by blocking estrogen signaling pathways. Luminal B breast cancer is divided into two types: one type is characterized by high ER or PR expression, HER2 negativity, but high Ki-67 expression (>14%), and the other type is characterized by high ER or PR expression and HER2 positivity. Luminal B type tumors have higher proliferative activity, are prone to cell cycle dysregulation, are more invasive than Luminal A type, and are more likely to metastasize to distant sites. HER2-overexpressing breast cancer is characterized by ER and PR negativity and HER2 positivity. These tumors have unique membrane receptor signals, leading to continuous activation of the PI3K-AKT-mTOR signaling pathway, driving rapid cell proliferation. Therefore, its clinical treatment has moved from the era of simple chemotherapy to the new era of targeted therapy. Triple-negative breast cancer, that is, breast cancer that is negative for ER, PR, and HER2, often has high Ki-67 expression, is highly invasive, has a tendency for early recurrence, and a risk of brain / lung metastasis. Currently, chemotherapy is the main treatment, and due to the lack of classic therapeutic targets, it is considered a "refractory subtype".

[0003] The Hippo signaling pathway, initially discovered in cellular gene studies of Drosophila mutants, is an evolutionarily highly conserved pathway. Its core function is to limit excessive tissue growth and maintain organ homeostasis through a kinase cascade. The core molecular mechanism of this pathway is initiated by a complex composed of mammalian sterile 20-like kinase 1 / 2 (MST1 / 2) and Salvador homolog 1 (SAV1), which phosphorylates and activates the downstream kinase Large Tumor Suppressor Kinase 1 / 2 (LATS1 / 2). Activated LATS1 / 2 further phosphorylates Yes-associated protein (YAP), leading to YAP retention in the cytoplasm and binding to 14-3-3 protein, or degradation through ubiquitination. When the Hippo pathway is inactivated, the non-phosphorylated YAP / TAZ (Transcriptional co-activator with PDZ-binding motif, TAZ) translocates to the nucleus and binds to proteins of the transcriptional enhanced associate domain (TEAD) family, forming a transcriptional complex. This complex drives the expression of proliferative genes such as connective tissue growth factor (CTGF) and cysteine-rich protein 61 (CYR61), and anti-apoptotic genes such as baculoviral IAP Repeat Containing 5 (BIRC5) and BCL2L1 (B-celllymphoma 2-like 1), thereby promoting cell proliferation, inhibiting apoptosis, and enhancing stem cell characteristics. Increasing research indicates that abnormalities in the Hippo signaling pathway are associated with the development of various tumors. Wang XD et al. used immunohistochemistry to detect YAP expression in 69 breast cancer tissues and found that YAP was expressed in 75.4% of the breast cancer samples. In vivo experiments also confirmed that YAP overexpression can promote tumor formation and growth. Therefore, exploring the role of YAP in the development and progression of breast cancer is crucial for developing new targeted therapies for breast cancer.

[0004] DRD2 (dopamine D2 receptor) is a key dopamine receptor subtype in the central nervous system, belonging to the G protein-coupled receptor (GPCR) superfamily. Its molecular structure contains a typical seven-transmembrane domain and primarily functions through Gi / o protein-coupled signaling pathways, including inhibiting adenylate cyclase activity, reducing intracellular cAMP levels, and regulating downstream signaling molecules such as MAPK and β-arrestin. DRD2 is a core target for the treatment of neuropsychiatric disorders. Typical antipsychotics (such as perphenazine), as potent DRD2 antagonists, effectively control psychotic symptoms but are prone to causing motor disorders and elevated prolactin levels. Atypical drugs (such as risperidone) reduce side effects by balancing the antagonistic effects of DRD2 and 5-HT2A receptors. The innovative design of the partial agonist aripiprazole has achieved "stabilized" regulation of dopaminergic signaling, while selective DRD2 / D3 agonists (such as pramipexole) have become the mainstay of Parkinson's disease treatment. Patent CN118079005A discloses the application of YAP inhibitors in combination with EGFR inhibitors and / or TGFβ1 receptor inhibitors in the treatment of breast cancer. However, there are currently no reports on the role of YAP inhibitors in combination with DRD2 inhibitors in the treatment of breast cancer / liver cancer. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of YAP inhibitors combined with DRD2 inhibitors in the preparation of drugs for the treatment of breast cancer and / or liver cancer.

[0006] The purpose of this invention is to provide a therapeutic agent for breast cancer and / or liver cancer.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention utilizes high-throughput screening to identify drugs with synergistic effects with the YAP inhibitor CA3. The high-throughput screening results show that the DRD2 inhibitor perphenazine can synergistically kill breast cancer cells with CA3. Further cell experiments, including cell proliferation, colony formation, and apoptosis assays, revealed that the combined use of the YAP inhibitor CA3 and the DRD2 inhibitor perphenazine significantly inhibited the proliferation of breast cancer cells and promoted their apoptosis, demonstrating a strong synergistic effect. Furthermore, this invention treated different breast cancer and liver cancer cell lines with different YAP inhibitors verteporfin (VP) and MGH-CP1 (CP1), as well as the DRD2 inhibitors perphenazine and trifluoperazine hydrochloride (TFP), finding that different YAP and DRD2 inhibitors all exhibited synergistic tumor-killing effects. Finally, using nude mouse tumorigenesis models and in situ breast tumorigenesis models, this invention found that CA3 and perphenazine alone had limited inhibitory effects on breast cancer, while their combined use effectively inhibited the in vivo growth of breast cancer tumors, confirming that the combined use of YAP and DRD2 inhibitors can indeed achieve synergistic treatment for breast cancer. Therefore, targeted therapy for breast cancer and / or liver cancer can be achieved by combining YAP inhibitors with DRD2 inhibitors.

[0009] Therefore, the present invention first provides the application of YAP inhibitors in combination with DRD2 inhibitors in the preparation of therapeutic drugs for breast cancer and / or liver cancer.

[0010] Furthermore, the YAP inhibitor is selected from CA3 (CIL56) (CAS No.:300802-28-2), MGH-CP1 (CAS No.:896657-58-2) or Verteporfin (CAS No.:129497-78-5).

[0011] Preferably, the YAP inhibitor is CA3 (CIL56) (CAS No.:300802-28-2).

[0012] Furthermore, the DRD2 inhibitor is selected from perphenazine (CAS No.:58-39-9) or trifluoperazine dihydrochloride (CAS No.:440-17-5).

[0013] Preferably, the DRD2 inhibitor is selected from perphenazine (CAS No.: 58-39-9).

[0014] Furthermore, the treatment is to inhibit the proliferation of breast cancer and / or liver cancer and promote apoptosis of breast cancer and / or liver cancer cells.

[0015] The present invention also provides a therapeutic agent for breast cancer and / or liver cancer, the therapeutic agent comprising a YAP inhibitor and a DRD2 inhibitor.

[0016] Furthermore, the molar concentration ratio of the YAP inhibitor to the DRD2 inhibitor is (0.5-1):(5-20).

[0017] Preferably, the molar concentration ratio of the YAP inhibitor to the DRD2 inhibitor is 1:10.

[0018] Furthermore, the YAP inhibitor is selected from CA3 (CIL56) (CAS No.:300802-28-2), MGH-CP1 (CAS No.:896657-58-2) or verteporfen (CAS No.:129497-78-5).

[0019] Preferably, the YAP inhibitor is CA3 (CIL56) (CAS No.:300802-28-2).

[0020] Furthermore, the DRD2 inhibitor is selected from perphenazine (CAS No.:58-39-9) or trifluoperazine hydrochloride (CAS No.:440-17-5).

[0021] Preferably, the DRD2 inhibitor is selected from perphenazine (CAS No.: 58-39-9).

[0022] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides the application of YAP inhibitors in combination with DRD2 inhibitors in the preparation of therapeutic drugs for breast cancer and / or liver cancer. This invention utilizes different YAP inhibitors (CA3, MGH-CP1, verteporfen) and different DRD2 inhibitors (perphenazine, trifluoperazine hydrochloride) to treat breast cancer cell lines and liver cancer cell lines, demonstrating that both YAP inhibitors and DRD2 inhibitors have a synergistic effect in killing tumor cells. Subsequent experiments also confirmed that the combined use of YAP inhibitors and DRD2 inhibitors can indeed achieve synergistic treatment of breast cancer. Therefore, targeted therapy for breast cancer and / or liver cancer can be achieved through the combined use of YAP inhibitors and DRD2 inhibitors. Attached Figure Description

[0025] Figure 1 This is for high-throughput screening of drugs that have a synergistic effect with the YAP inhibitor CA3; among them, Figure 1In Figure A, the chemical structural formulas of YAP inhibitor CA3 and DRD2 inhibitor perphenazine are shown; in Figure B, the results of the first screening are shown; and in Figure C, the effects of YAP inhibitor CA3 and DRD2 inhibitor perphenazine on the viability of breast cancer cells MCF7 are shown.

[0026] Figure 2 The effects of the YAP inhibitor CA3 and the DRD2 inhibitor Perphenazine on the proliferation and apoptosis of breast cancer cells; among them, Figure 2 In Figure A, CA3 and Perphenazine have effects on the proliferation and cloning of MCF7 breast cancer cells; in Figure B, CA3 and Perphenazine have effects on the apoptosis of MDA-MB-231 breast cancer cells; and in Figure C, CA3 and Perphenazine have effects on the apoptosis of MCF7 breast cancer cells.

[0027] Figure 3 The effects of different YAP inhibitors and DRD2 inhibitors on the cell viability of different breast cancer cell lines and liver cancer cell lines; among them, Figure 3A shows the effect of the YAP inhibitor CA3 and the DRD2 inhibitors Perphenazine and Trifluoperazine dihydrochloride (TFP) on the viability of MDA-MB-231 breast cancer cells; B shows the effect of the YAP inhibitor CA3 and the DRD2 inhibitors Perphenazine and Trifluoperazine dihydrochloride (TFP) on the viability of Huh-7 liver cancer cells; C shows the effect of the YAP inhibitor CA3 and the DRD2 inhibitors Perphenazine and Trifluoperazine dihydrochloride (TFP) on the viability of MCF7 breast cancer cells; D shows the effect of the YAP inhibitor MGH-CP1 (CP1) and the DRD2 inhibitors Perphenazine and Trifluoperazine dihydrochloride (TFP) on the viability of MDA-MB-231 breast cancer cells; E shows the effect of the YAP inhibitor MGH-CP1 (CP1) and the DRD2 inhibitors Perphenazine and Trifluoperazine dihydrochloride (TFP) on the viability of MDA-MB-231 breast cancer cells. The effect of dihydrochloride (TFP) on the viability of MCF7 breast cancer cells; F represents the effect of the YAP inhibitor Verteporfin (VP) and the DRD2 inhibitors Perphenazine and Trifluoperazine dihydrochloride (TFP) on the viability of MDA-MB-231 breast cancer cells; G represents the effect of the YAP inhibitor Verteporfin (VP) and the DRD2 inhibitors Perphenazine and Trifluoperazine dihydrochloride (TFP) on the viability of MCF7 breast cancer cells.

[0028] Figure 4 The effects of YAP inhibitor CA3 and DRD2 inhibitor Perphenazine on breast cancer tumors in vivo; among them... Figure 4 In the figure, A represents the effect of CA3 and Perphenazine on the tumor weight of breast cancer in a nude mouse tumorigenesis model; B represents the effect of CA3 and Perphenazine on the tumor weight of breast cancer in an in situ breast cancer model; C represents the pathological section results of breast cancer tumors in both the nude mouse tumorigenesis model and the in situ breast cancer model using CA3 and Perphenazine; D and E represent the immunofluorescence staining results of pathological sections of breast cancer tumors in both the nude mouse tumorigenesis model and the in situ breast cancer model using CA3 and Perphenazine, respectively. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] Example 1: High-throughput screening of drugs with synergistic effects with YAP inhibitors

[0032] (1) MCF7 breast cancer cells were pre-seeded into 96-well plates, with 5000 cells per well. The next day, after cell adhesion, the YAP inhibitor CA3 (1 μM) was added, along with FDA-approved drug libraries (10 μM), ensuring only one FDA-approved drug was added to each well. Cell viability was assessed using a CCK8 assay 36 hours after treatment. The first results were obtained by comparing the cell viability data of the dual-drug combination (CA3 with each FDA-approved drug) with the group treated with CA3 alone. After initial screening, 136 drugs were obtained (…). Figure 1 (Medium B) These 136 drugs were then added to MCF7 cells in 96-well plates, with or without CA3. After 36 hours of drug treatment, cell viability was detected using a CCK8 assay. Further results were obtained by comparing the cell viability data of the two-drug combination (i.e., the combination of CA3 and the 136 drugs obtained from the initial screening) with the group treated with the 136 drugs alone. Ultimately, drugs that can kill breast cancer cells when used in combination with CA3 were identified.

[0033] The results are as follows Figure 1 As shown, high-throughput screening of drugs with synergistic effects with the YAP inhibitor CA3 revealed that the DRD2 inhibitor Perphenazine can synergistically kill breast cancer cells with CA3. Figure 1 The structural formulas of the YAP inhibitor CA3 and the DRD2 inhibitor Perphenazine are as follows: Figure 1 As shown in Figure A.

[0034] Example 2: Effects of YAP inhibitor CA3 and DRD2 inhibitor Perphenazine on the proliferation and apoptosis of breast cancer cells.

[0035] (1) Cell proliferation-toxicity assay to detect the synergistic inhibitory effect of YAP inhibitor CA3 and DRD2 inhibitor Perphenazine on breast cancer cells: Breast cancer cells were treated with CA3 and Perphenazine alone or in combination (the working concentration of CA3 was 1 μM and the working concentration of Perphenazine was 10 μM) for 48 h, and the viability of breast cancer cells was detected by CCK8 kit.

[0036] (2) Colony formation assay to detect the synergistic inhibitory effect of YAP inhibitors CA3 and Perphenazine on breast cancer cells: 1000 breast cancer cells were seeded in each well of a 6-well plate. The cells were treated with CA3 alone or in combination with Perphenazine (the working concentration of CA3 was 1 μM and the working concentration of Perphenazine was 10 μM). After 24 hours, the culture medium containing the drugs was discarded, and the cells were cultured in complete culture medium until the cell clones were visible to the naked eye. The cells were then fixed and stained with crystal violet to observe the cell clone formation.

[0037] (3) Detection of the synergistic killing effect of YAP inhibitor CA3 and Perphenazine on breast cancer cells: Different breast cancer cells (MDA-MB-231, MCF7) were treated with CA3 alone or in combination with Perphenazine (the working concentration of CA3 was 1 μM and the working concentration of Perphenazine was 10 μM). After 48 h, the cells were collected and the apoptosis status of the cells was detected by Annexin V-FITC / PI apoptosis kit (Vazyme, A211-01).

[0038] The results are as follows Figure 2 As shown, through cell proliferation experiments and colony formation experiments ( Figure 2 (A), apoptosis experiment ( Figure 2 A study by the Chinese Academy of Medical Sciences (CMS) found that the combined use of the YAP inhibitor CA3 and the DRD2 inhibitor Perphenazine significantly inhibited the proliferation of breast cancer cells and promoted their apoptosis, demonstrating a "1+1>2" effect and a strong synergistic effect.

[0039] Example 3: Effects of different YAP inhibitors and DRD2 inhibitors on different breast cancer cell lines and liver cancer cell lines

[0040] Tumor cells were pre-seeded into 96-well plates with 5000 cells per well. After cell adhesion, different concentrations and types of YAP inhibitors were added the next day. The tumor cells were then treated with DRD2 inhibitors Perphenazine or Trifluoperazine dihydrochloride (TFP) (DRD2 inhibitor working concentration of 5 μM) for 48 h. The viability of breast cancer cells was detected using a CCK8 assay kit, and the results were plotted.

[0041] The results are as follows Figure 3As shown, different breast cancer cell lines and liver cancer cell lines were treated with different YAP inhibitors CA3 and Verteporfin (VP), as well as MGH-CP1 (CP1) and DRD2 inhibitors Perphenazine and TFP. It was found that different YAP inhibitors and DRD2 inhibitors all had a synergistic effect in killing tumor cells. Figure 3 AG).

[0042] Example 4: Effects of YAP inhibitor CA3 and DRD2 inhibitor Perphenazine on breast cancer tumors in vivo.

[0043] (1) Nude mouse tumor model: 4-5 week old female BALB / c nude mice were housed in an SPF environment and subcutaneously inoculated with 5×10 6 CF7 cells were inoculated, and drug administration began on the fifth day after inoculation, with administration every two days. CA3 was administered intraperitoneally at a dose of 1 mg / kg; Perphenazine was administered by gavage at a dose of 20 mg / kg. Tumor volume, nude mouse weight, activity level, and diet were recorded. The experiment was terminated when the tumor volume reached 1000-1500 mm³ or when the nude mice exhibited abnormalities such as emaciation, decreased activity, or ulceration. At this point, the nude mice were euthanized by cervical dislocation, the tumor tissue was completely dissected, weighed, and photographed. Some tissue was fixed with 4% paraformaldehyde (for pathological sections), and some was flash-frozen in liquid nitrogen and stored at -80°C for subsequent molecular experiments.

[0044] (2) In situ tumorigenesis model of mammary gland: Female BALB / c mice aged 4-5 weeks were housed in an SPF environment. The hair in the fourth pair of mammary glands (groin area) was shaved, and 150,000 4T1 cells were inoculated into the middle of the mammary pad. Drug administration began on the fifth day after inoculation and was administered every two days. CA3 was administered intraperitoneally at a dose of 1 mg / kg, and Perphenazine was administered by gavage at a dose of 20 mg / kg. Tumor volume, weight of nude mice, activity status, and diet were recorded. The experiment was terminated when the tumor volume reached 1000-1500 mm³ or when the mice showed abnormalities such as emaciation, decreased activity, or ulceration. At this time, the mice were euthanized by cervical dislocation, the tumor tissue was completely dissected, weighed, and photographed. Some tissue was fixed with 4% paraformaldehyde (for pathological sections), and some was flash-frozen in liquid nitrogen and stored at -80 degrees Celsius for subsequent molecular experiments.

[0045] The results are as follows Figure 4 As shown, using nude mouse tumorigenesis models and in situ breast tumorigenesis models, it was found that CA3 and Perphenazine alone had a small inhibitory effect on breast cancer, while the combination of the two could effectively inhibit the in vivo growth of breast cancer tumors. Figure 4(AE). The results of this invention indicate that the combined use of YAP inhibitors and DRD2 inhibitors can synergistically treat breast cancer and / or liver cancer, and that targeted therapy for breast cancer and / or liver cancer can be achieved through the combined use of YAP inhibitors and DRD2 inhibitors.

Claims

1. The application of YAP inhibitors combined with DRD2 inhibitors in the preparation of breast cancer therapeutic drugs, characterized in that, The YAP inhibitor is CA3, and the DRD2 inhibitor is perphenazine.

2. A breast cancer treatment drug, characterized in that, It contains a YAP inhibitor and a DRD2 inhibitor, wherein the YAP inhibitor is CA3 and the DRD2 inhibitor is perphenazine.

3. The drug according to claim 2, characterized in that, The molar concentration ratio of the YAP inhibitor to the DRD2 inhibitor is (0.5-1):(5-20).

4. The drug according to claim 3, characterized in that, The molar ratio of the YAP inhibitor to the DRD2 inhibitor is 1:

10.

5. The drug according to any one of claims 2 to 4, characterized in that, It also includes pharmaceutically acceptable excipients.

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