Use of imperatorin in preparation of drugs for inhibiting hedgehog signal pathway

Abstract

The present application relates to the application of imperatorin in the preparation of a drug for inhibiting the Hedgehog signal pathway, and discloses that imperatorin can inhibit the Hedgehog signal pathway activated by Shh, inhibit the Hedgehog signal pathway activity activated by SMO overexpression, and inhibit the Hedgehog signal pathway activity activated by SMO drug-resistant mutation, and can obviously inhibit the growth of tumors in vivo caused by abnormal activation of the Hedgehog, and in particular, it is disclosed that imperatorin can inhibit the Hedgehog signal pathway activity activated by SMO drug-resistant mutation, which provides a new strategy for treating tumors caused by abnormal activation of the Hedgehog, and expands the application field of imperatorin.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and in particular to the use of imperatorin in the preparation of drugs that inhibit the Hedgehog signaling pathway. Background Technology

[0002] The Hedgehog (Hh) signaling pathway is a highly conserved pathway that plays a crucial role in embryonic development, organ repair, and homeostasis. Key signaling molecules in the classic Hh signaling pathway include HH ligands (Shh, Ihh, Dhh), patched receptor (PTCH), smoothed (SMO) receptor, SUFU protein, and transcription factors GLI (GLI1, GLI2, GLI3). In the absence of HH ligands, PTCH inhibits SMO protein activity, ultimately suppressing the transcription of downstream target genes such as GLI1 and PTCH1. When HH ligands bind to PTCH, they relieve the inhibitory effect of PTCH on SMO, and the activated SMO promotes the entry of transcription factor GLI into the nucleus, initiating the transcription of target genes. Of the three homologous proteins of transcription factor GLI, GLI2 primarily activates transcription, GLI3 represses transcription, while GLI1, being a target gene of the Hh signaling pathway itself, is often used to indicate the activity level of the Hh signaling pathway.

[0003] Studies have shown that sustained activation of the Hh signaling pathway is closely related to the occurrence and development of various tumors, such as medulloblastoma, rhabdomyosarcoma, and colorectal cancer. Blocking abnormally activated Hh signaling in tumors can effectively inhibit tumor growth, providing an effective strategy for cancer treatment. Developing anti-tumor drugs targeting key molecules in the Hh signaling pathway has become a focus of research for major pharmaceutical companies and research institutions. To date, three anti-tumor drugs targeting the Hh signaling pathway, specifically SMO, have been marketed. In recent years, China has also made some progress in the research and development of drugs targeting the Hh signaling pathway, but currently only four drugs are in early clinical trials, and no related drugs have been marketed.

[0004] On the other hand, clinical use has revealed that existing SMO inhibitors are prone to resistance after use, and the toxic side effects limit the long-term efficacy of these drugs. Studies show that mechanisms leading to SMO inhibitor resistance include SMO point mutations, SUFU loss-of-function mutations, GLI2 amplification, and non-classical pathways such as SRF-MKL1-induced GLI activation. Improving the efficacy of cancer treatment and expanding the patient population that benefits from it has become a key objective for the industry. However, there are currently no new drugs on the market that can overcome SMO inhibitor resistance; therefore, developing novel Hh signaling pathway inhibitors that can overcome SMO inhibitor resistance has potential clinical value. Summary of the Invention

[0005] The purpose of this invention is to provide the application of imperatorin in the preparation of drugs that inhibit the Hedgehog signaling pathway, especially in the preparation of drugs resistant to existing SMO inhibitors.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The present invention provides the application of imperatorin in the preparation of drugs and / or drug combinations that inhibit the Hedgehog signaling pathway. The drugs are, for example, Hedgehog signaling pathway inhibitors.

[0008] In some specific embodiments, the drug and / or drug combination is a drug and / or drug combination for inhibiting the activity of the Shh-activated Hedgehog signaling pathway.

[0009] In some specific embodiments, the drug and / or drug combination is a drug and / or drug combination for inhibiting the activity of the Hedgehog signaling pathway activated by SMO overexpression.

[0010] In some specific embodiments, the drug and / or drug combination is a drug and / or drug combination for inhibiting the activity of the Hedgehog signaling pathway activated by SMO resistance mutations.

[0011] In some specific embodiments, the drug and / or drug combination is a drug and / or drug combination for inhibiting Gli1 expression in the SMO-activated Hedgehog signaling pathway.

[0012] In some specific embodiments, the drug and / or drug combination is a drug and / or drug combination for inhibiting Ptch1 expression in the Hedgehog signaling pathway activated by SMO.

[0013] In some specific embodiments, the drug and / or drug combination is an antitumor drug and / or antitumor drug combination for inhibiting the activation of tumors by the Hedgehog signaling pathway.

[0014] In some specific embodiments, the tumors treated with the antitumor drugs and / or combinations of antitumor drugs include glioma, basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, and colorectal cancer.

[0015] In some specific embodiments, the dosage forms of the drug and / or drug combination include tablets, granules, capsules, oral solutions, injections, and liposomes.

[0016] In some specific embodiments, the drug combination also includes pharmaceutically permissible excipients.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention reveals that imperatorin can effectively inhibit the Hedgehog signaling pathway (inhibit the Hedgehog signaling pathway activated by Shh, inhibit the Hedgehog signaling pathway activity activated by SMO overexpression, and inhibit the Hedgehog signaling pathway activity activated by SMO drug resistance mutation), and can significantly inhibit the abnormal activation of Hedgehog signaling to promote tumor growth in vivo. In particular, imperatorin can inhibit the Hedgehog signaling pathway activity activated by SMO drug resistance mutation.

[0019] (2) This invention reveals a novel application of imperatorin in the preparation of drugs against tumors abnormally activated by the Hedgehog signaling pathway, particularly in the preparation of drugs to inhibit tumors resistant to existing SMO inhibitors caused by SMO mutations. By revealing that imperatorin can be prepared or modified into new antitumor drugs, this invention provides a new strategy for treating tumors with abnormal Hedgehog activation, provides a scientific basis for the clinical use of imperatorin, and expands the application field of imperatorin. Attached Figure Description

[0020] Figure 1 The dose-response curves of GLI-luciferase activity after 36 hours of treatment with different concentrations of imperatorin in Light 2 cells under the activation of the Hh signaling pathway ligand Shh are shown.

[0021] Figure 2 The dose-response curves of GLI-luciferase activity after 36 h of treatment with different concentrations of imperatorin in Light 2 cells under SMO plasmid overexpression stimulation are shown.

[0022] Figure 3 DAOY cells, which are medulloblastoma cells, were treated with imperatorin (20 μM) and GDC-0449 (10 μM) for 72 h, and the CCK8 bar graph was analyzed.

[0023] Figure 4 The effect of imperatorin on the growth of Shh-medulloblastoma xenografts in nude mice.

[0024] Figure 5 This is a Western blot image showing the expression of GLI1 protein in nude mouse tumor tissue bearing Shh-medulloblastoma.

[0025] Figure 6 The bar chart shows the expression of Gli1 mRNA and Ptch1 mRNA in Light 2 cells after 24 h of treatment with imperatorin (20 μM) and GDC-0449 (10 μM) under SMO D473H plasmid overexpression stimulation.

[0026] Figure 7 The bar chart shows the expression of Gli1 mRNA and Ptch1 mRNA in Light 2 cells after 24 h of treatment with imperatorin (20 μM) and GDC-0449 (10 μM) under SMO W535L plasmid overexpression stimulation. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0028] Unless otherwise specified, the raw materials or processing techniques used in the following embodiments and comparative examples are all conventional commercially available raw materials or conventional processing techniques in the art.

[0029] Example 1: Imperatorin inhibits Shh and suppresses the activity of the Hh signaling pathway activated by SMO overexpression.

[0030] The dual-luciferase reporter system was used for detection. Light 2 cells were NIH3T3 cells that were modified to stably express GLI1-bindingsite-luciferase (GLI-luciferase) and the TK-Renilla reporter gene (Light 2 cell reference: PMID:10984056.). Hh signaling pathway inhibitors effectively inhibited the reporter gene activity of this cell. Light 2 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v / v) fetal bovine serum, 0.4 mg / mL G418, 0.15 mg / mL Zeocin, and 1× penicillin / streptomycin (Hyclone) at 37°C in a 5% CO2 incubator.

[0031] 1) Imperatorin inhibits the activity of the Shh-activated Hh signaling pathway:

[0032] According to 8×10 per hole 3Light 2 cells were seeded in 96-well plates. After 24 hours, the medium was replaced with serum-free medium, and Shh supernatant (Shh supernatant collection reference: PMID: 32855528) and different concentrations of imperatorin (Aladdin) were added. Cells were treated for 36 hours, and the treatment was terminated. Detection was performed according to the instructions of the Dual-Luciferase Reporter Gene Detection Kit (Promega, E1960). Each concentration was used in triplicate. The Luciferase values ​​of each group were normalized to Renilla Luciferase to obtain the relative fluorescence intensity. This relative intensity was then divided by the corresponding fluorescence intensity of the control group (the untreated group, i.e., the group treated with Shh supernatant but not imperatorin) to obtain the relative transcriptional activity of the reporter gene in each group.

[0033] See results Figure 1 The results showed that imperatorin significantly inhibited Shh-stimulated GLI-luciferase activity in a dose-dependent manner, with a corresponding IC50 value of [missing information]. 50 The value is 2.07 μM.

[0034] 2) Imperatorin inhibits the activity of the Hh signaling pathway activated by SMO overexpression:

[0035] According to 1×10 per hole 4 Light 2 cells were seeded in 96-well plates. After 24 hours, the medium was replaced with serum-free medium. 25 ng of Lipo2000 overexpression SMO plasmid (Origene, RC203451) was administered per well. A control group was transfected with an empty vector plasmid. After 36 hours, different concentrations of imperatorin (Aladdin) were added to the cells for another 36 hours before treatment. Detection was performed according to the instructions of the Dual-Luciferase Reporter Gene Detection Kit (Promega, E1960). Each concentration was tested in triplicate. The relative fluorescence intensity of each group of firefly luciferase was normalized to that of Renilla luciferin and divided by the corresponding fluorescence intensity of the control group (the untreated group, i.e., the group using Lipo2000 overexpression SMO plasmid without imperatorin) to obtain the relative transcriptional activity of the reporter gene in each group.

[0036] See results Figure 2 The results showed that imperatorin significantly inhibited the activity of GLI-luciferase stimulated by SMO overexpression in a significant dose-dependent manner, with a corresponding IC50 value of [missing information]. 50 It is 6.77 μM.

[0037] Example 2: Imperatorin inhibits Hh-dependent medulloblastoma cell proliferation

[0038] CCK8 assay: Shh subtype human medulloblastoma cells from DAOY (Chinese Academy of Sciences Cell Bank) were cultured in MEM containing 10% (v / v) fetal bovine serum and 1× penicillin / streptomycin (Hyclone) at 37°C in a 5% CO2 incubator. 1×10⁶ cells were seeded. 4 DAOY cells were placed in 96-well plates. After 24 hours, imperatorin (20 μM) or GDC-0449 (10 μM) was added and the cells were treated for 72 hours. Detection was then performed according to the CCK8 manual. Each group was set up with three replicates, and the experiment was repeated at least three times.

[0039] See results Figure 3 Experimental results showed that imperatorin and GDC-0449 could inhibit the proliferation of DAOY cells.

[0040] Example 3: Imperatorin inhibits the in vivo growth of medulloblastoma by inhibiting Hh signaling activity.

[0041] 1) Imperatorin inhibits the in vivo growth of medulloblastoma:

[0042] Ptch+ / - genotype mice were crossed with P53- / - genotype mice (Jackson Laboratory) to obtain Ptch+ / -; P53- / - genotype mice. Spontaneously generated medulloblastomas from the cerebellum of these mice were harvested and inoculated into the axilla of immunodeficient 6-week-old nude mice (BALB / C nude). The tumors were allowed to grow to 100 mm. 3 At that time, patients were randomly assigned to groups for drug administration. Tumor volume was measured every three days. Imperatorin was administered by gavage at a dose of 50 mg / kg once daily; the control group received an equal volume of PBS. Tumor volume (V) was calculated using the formula: V = (a × b) / (a ​​× b) 2 ) / 2, where a represents the measured length of the tumor and b represents the measured width of the tumor.

[0043] See results Figure 4 Experimental results showed that imperatorin could significantly inhibit the in vivo growth of Hh-dependent medulloblastoma.

[0044] 2) Imperatorin inhibits Hh signaling activity in medulloblastoma:

[0045] After the in vivo experiment (day 16) was completed, tumor tissue was removed, proteins were extracted, and Western blot analysis was performed.

[0046] See results Figure 5 Experimental results showed that imperatorin could inhibit the expression of GLI1 protein in medulloblastoma tissue.

[0047] Example 4: Imperatorin inhibits the activity of the Hh signaling pathway activated by SMO mutation.

[0048] NIH3T3 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v / v) fetal bovine serum in an incubator at 37°C and 5% CO2.

[0049] 3×10 5 NIH3T3 cells were placed in 6-well plates. After 24 hours, the medium was replaced with serum-free medium. SMO D473H or SMO W535L plasmids were overexpressed using Lipo2000 at a dose of 100 ng per well. After 36 hours, cells were treated with imperatorin (20 μM), DMSO, and GDC-0449 (10 μM), respectively, for 24 hours before termination. Samples were extracted and analyzed by RT-qPCR as follows. Both SMO D473H and SMO W535L plasmids were constructed using the QuickChange Site-Directed Mutagenesis kit from Agilent (Santa Clara, CA), detailed in reference PMID:32855528.

[0050] Total RNA was extracted according to the Trizol (Takara) kit instructions, using endogenous Gusb as a reference, and reverse transcription was performed using a kit (…). RT Master Mix (Takara) reverse transcription. Using... Premix Ex Taq TM (Tli RNaseH Plus)(Takara) was used for quantitative PCR according to the manufacturer's instructions. Gli1 mRNA and Ptch1 mRNA expression were detected using an iCycler iQ system (Bio-Rad) real-time quantitative PCR instrument. Each group was set up with three replicates, and the experiments were repeated at least three times. The relative expression levels of the genes were calculated. The calculation formula is as follows:

[0051] Target gene expression level = 2 -ΔΔCt

[0052] Where ΔΔC t = (Ct target gene - Ct internal reference) 处理组 -(Ct target gene-Ct internal reference) 未处理组

[0053] Primer sequences are shown in Table 1:

[0054] Table 1 Primer sequences

[0055]

[0056] See results Figure 6 , 7 Experimental results showed that imperatorin significantly inhibited the expression of Hh target genes Gli1 and Ptch1 activated by overexpression of SMO D473H and SMO W535L, while GDC-0449 had no effect. This indicates that imperatorin has the potential to overcome resistance to existing SMO inhibitors.

[0057] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of imperatorin in the preparation of drugs that inhibit abnormal activation of tumors by the Hedgehog signaling pathway. The Hedgehog signaling pathway abnormality is Shh-activated Hedgehog signaling pathway activity; And / or, the Hedgehog signaling pathway aberration is SMO overexpression activating the Hedgehog signaling pathway; And / or, the Hedgehog signaling pathway aberration is Hedgehog signaling pathway activity activated by SMO resistance mutation; And / or, the Hedgehog signaling pathway anomaly is an SMO-activated Hedgehog signaling pathway. Gli1 Express; And / or, the Hedgehog signaling pathway anomaly is an SMO-activated Hedgehog signaling pathway. Ptch1 Express; The tumor is a medulloblastoma.

2. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, granules, capsules, oral liquids, injections, and liposomes.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically permissible excipients.

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