Application of mGluR5 agonist in preparation of antitumor drugs

Abstract

The invention relates to an application of an mGluR5 agonist in preparation of an anti-tumor drug. The mGluR5 agonist is a compound I, and the chemical structural formula of the compound I is as follows: # imgabs0. The small molecule compound provided by the invention can effectively inhibit the growth of tumors and the like, and has good tumor treatment potential.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of an mGluR5 agonist in the preparation of an anti-tumor drug and the use of a small molecule compound in the preparation of a drug of the mGluR5 agonist. Background Art

[0002] Tumors are complex and systemic diseases caused by the combined effects of multiple factors, posing a major challenge to global health. When the body is affected by physical, chemical, viral, bacterial and other carcinogens in the environment, or due to the effects of intrinsic factors such as its own genetics, mental emotions, endocrine, age, and immune status, it drives the activation of proto-oncogenes, the inactivation mutation of inhibitory genes and other genetic abnormalities, leading to uncontrolled excessive cell proliferation and eventually forming tumors. Tumor characteristics are characterized by persistent proliferation signals, escape from growth inhibition, resistance to cell death, unlimited replication, genomic instability and mutation, induction of angiogenesis, high invasiveness and easy distal metastasis, reprogramming of cell energy metabolism, immune escape, and promotion of tumor-related inflammation (Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011; 144(5): 646-674; Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discov. 2022; 12(1): 31-46.). With the revelation of cancer characteristics, the development of targeted therapy and immunotherapy has been promoted, which has significantly improved the treatment effect of clinical patients (Passaro A, Al Bakir M, Hamilton EG, et al. Cancer biomarkers: Emerging trends and clinical implications for personalized treatment. Cell. 2024; 187 (7): 1617-1635.). However, the current treatment strategies still have many problems such as primary or secondary drug resistance, limited response population, and low response efficiency (Xia Y, Sun M, Huang H, Jin WL. Drug repurposing for cancer therapy. Signal Transduct Target Ther. 2024; 9 (1): 92.). Therefore, there is still an unmet clinical need in tumor treatment. Finding new tumor treatment targets and discovering drugs with new modes of action are of great significance for treating tumors.

[0003] Metabotropic glutamate receptor 5 (mGluR5) is a type of excitatory G protein-coupled receptor (G Protein-Coupled Receptors, GPCRs), which is widely distributed in the central nervous system and is involved in regulating synaptic plasticity, synaptic transmission, neuronal differentiation and other physiological processes (Abd-Elrahman KS, Ferguson SSG. Noncanonical Metabotropic Glutamate Receptor 5 Signaling in Alzheimer's Disease. Annu Rev Pharmacol Toxicol. 2022 Jan 6; 62: 235-254.). mGluR5 is a typical class C GPCR, which functions in the form of a dimer and is coupled to phospholipase C (PLC) through Gαq / 11. Activated PLC catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) on the cell membrane into second messenger molecules 1,4,5-triphosphate (inositol-1,4,5-triphosphate, IP3) and diacylglycerol (1,2-diacylglycerol, DAG). IP3 mobilizes the endoplasmic reticulum to release calcium ions (Ca 2+ ) into the cytoplasm and bind to calmodulin, and then participate in a series of reactions. 2+ In collaboration with mGluR5, protein kinase C (PKC) is activated, triggering a PKC-mediated cascade reaction. In addition, mGluR5 is also involved in PI3K / AKT / mTOR signaling pathway transduction, ERK1 / 2 signal activation, etc., ultimately achieving the regulation of cell activities such as membrane receptor activity, gene transcription, and protein synthesis (Mango D, Ledonne A. Updates on the Physiopathology of Group I Metabotropic Glutamate Receptors (mGluRI)-Dependent Long-Term Depression. Cells. 2023; 12 (12): 1588.).

[0004] Abnormal mGluR5 signals play a key role in the pathophysiological mechanisms of a variety of nervous system diseases, including Alzheimer's disease, Parkinson's syndrome, stroke, depression, anxiety, schizophrenia, pain syndrome, epilepsy, etc. mGluR5 activity modulators have also been widely studied in central nervous system diseases. For example, disturbances in physiological mGluR5 signal transduction lead to pathological features such as β-amyloid deposition, Tau protein hyperphosphorylation, and synaptic loss, thereby promoting the progression of Alzheimer's disease (Kumar A, Dhull DK, Mishra PS. Therapeutic potential of mGluR5 targeting in Alzheimer's disease. Front Neurosci. 2015 Jun 9; 9: 215.). Treatment with a silencing allosteric modulator of mGluR5 restores synaptic density and reduces hyperphosphorylated Tau levels in a mouse model of Alzheimer's disease (Purrier J, Nicholson L, Fang XT, Stoner AJ, Toyonaga T, Holden D, Siegert TR, Laird W, Allnutt MA, Chiasseu M, Brody AH, Takahashi H, Nies SH, Pérez- ásA, Sadasivam P, Lee S, Li S, Zhang L, Huang YH, Carson RE, Cai Z, Strittmatter SM. Reversal of synapse loss in Alzheimer mouse models by targeting mGluR5 to prevent synaptic tagging by C1Q. Sci Transl Med. 2022Jun; 14(647):eabi8593.). In addition, mGluR5 and its downstream G protein-coupled signals are inactivated during the abnormal activation of microglia stimulated by α-synuclein, leading to the activation of transcription factors NF-κB p65 and TNFα and enhancing the release of inflammatory cytokines, inducing neurodegeneration, which is an important pathogenesis of Parkinson's syndrome (Azam S, Jakaria M, Kim J, Ahn J, Kim IS, Choi DK. Group ImGluRs in Therapy and Diagnosis of Parkinson's Disease: Focus on mGluR5Subtype. Biomedicines. 2022Apr 7; 10(4): 864.). mGluR5 is also involved in regulating anxiety-like behavior. Under chronic restraint stress, the activity of mGluR5 in hippocampal pyramidal cells decreases, triggering a decrease in the discharge of pyramidal cells, leading to anxiety-like behavior. After chronic restraint, the mGluR5 positive allosteric modulator CDPPB can reverse the damaged inhibitory synaptic input and anxiety-like behavior of mice (Li X, Du ZJ, Xu JN, Liang ZM, Lin S, Chen H, Li SJ, Li XW, Yang JM, Gao TM. mGluR5 in hippocampal CA1 pyramidal neurons mediates stress-induced anxiety-like behavior. Neuropsychopharmacology. 2023 Jul; 48 (8): 1164-1174.). Currently, a number of mGluR5 antagonists and negative allosteric modulators have entered the clinical trial stage. The mGluR5 antagonist Mavoglurant has entered the Phase II clinical trial stage for the treatment of patients with alcohol use disorder (clinical trial number: NCT06136195).In addition, two mGluR5 negative allosteric modulators, Basimglurant and Dipraglurant, are in Phase II clinical studies for patients with trigeminal neuralgia and Parkinson's disease, respectively (clinical trial numbers: NCT05217628, NCT05116813). HTL0014242 is a potent and selective mGlu5 small molecule negative allosteric modulator, currently undergoing Phase I clinical trials for the treatment of cocaine addiction and substance abuse problems (clinical trial number: NCT06025396). The mGluR5 silencing allosteric modulator BMS-984923 effectively inhibits the binding of mGluR5 and cellular prion proteins without changing the glutamate signal at physiological concentrations, thereby blocking synaptic loss in the course of Alzheimer's disease. BMS-984923 is undergoing Phase I clinical trials for Alzheimer's disease (clinical trial numbers: NCT05817643, NCT05804383). However, research on mGluR5 agonists and positive allosteric modulators is currently in the preclinical stage and mainly focuses on neurological disease models such as schizophrenia. Therefore, further promoting the development of mGluR5 activity modulators is of great significance for clarifying the mechanism of action of neurological diseases and developing treatment strategies.

[0005] In addition to participating in the regulation of the progression of nervous system-related diseases, recent studies have shown that mGluR5 is abnormally altered in various types of tumors and is closely related to cancer progression and patient prognosis (references: Pereira MSL, Klamt F, Thomé CC, Worm PV, de Oliveira DL. Metabotropic glutamate receptors as a new therapeutic target for malignant gliomas. Oncotarget. 2017; 8(13): 22279-22298. Yu LJ, Wall BA, Wangari-Talbot J, Chen S. Metabotropic glutamate receptors incancer. Neuropharmacology. 2017; 115: 193-202.). Studies have found that mGluR5 plays an important role in the occurrence, development and metastasis of oral squamous cell carcinoma. The mGluR5 agonist DHPG can promote the migration and invasion of oral squamous cell carcinoma cells, and this effect is reversed by the mGluR5 antagonist MPEP. In addition, high expression of mGluR5 significantly reduces the overall survival rate of clinical patients with oral squamous cell carcinoma, revealing that mGluR5 may serve as a potential prognostic marker for oral cancer (reference: Park SY, Lee SA, Han IH, et al. Clinical significance of metabotropic glutamate receptor 5 expression in oral squamous cell carcinoma. Oncol Rep. 2007; 17 (1): 81-87.). Overexpression of mGluR5 has also been shown to induce the progression of melanoma in transgenic mice, and mGluR5 mediates the growth of mouse melanoma by activating ERK phosphorylation (reference: Choi KY, Chang K, Pickel JM, Badger JD 2nd, Roche KW. Expression of the metabotropic glutamate receptor 5 (mGluR5) induces melanoma in transgenic mice. Proc Natl Acad Sci US A. 2011; 108 (37): 15219-15224.). mGluR5 is highly expressed in multiple myeloma cells and tissues, and the mGluR5 agonist DHPG promotes the proliferation of multiple myeloma cells and resists apoptosis. Conversely, the mGluR5 antagonists MPEP and MTEP significantly inhibit tumor cell proliferation.Mechanistic studies have found that mGluR5 promotes the growth of multiple myeloma cells by activating the Ras-MAPK signaling pathway, suggesting that mGluR5 may be a potential molecular target for the treatment of multiple myeloma (reference: Kou W, Huang H, Dai S, et al. mGluR5 promotes the progression of multiple myeloma in vitro via Ras-MAPKsignaling pathway. Adv Clin Exp Med. 2022; 31(8): 881-888.). More information on the mechanism of action of mGluR5 in tumors remains to be further elucidated. Summary of the invention

[0006] In order to solve the above problems in the prior art, the present invention provides a use of a mGluR5 agonist in the preparation of an anti-tumor drug.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides a use of a mGluR5 agonist in the preparation of an anti-tumor drug.

[0008] Preferably, the mGluR5 agonist is Aflavinine (referred to as Compound I), and the chemical structure of Compound I is as follows:

[0009]

[0010] The chemical formula of compound I is C 28 H 39 NO, molecular weight is 405.6154, CAS number is 74328-59-9. Compound I (Aflavinine) is a natural product derived from fungi such as Aspergillus, belonging to the indole alkaloid class. It has a complex tricyclic skeleton and exhibits a highly unique chemical structure. The structure of compound I (Aflavinine) is based on the indole ring as the core, combined with the fused structure of pyrrole and six-membered ring to form a tricyclic system.

[0011] Preferably, the drug contains a pharmaceutically acceptable carrier or excipient, and the pharmaceutically acceptable carrier or excipient includes but is not limited to excipients, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, etc. The dosage form of the drug can be a pharmaceutically acceptable dosage form, including but not limited to injection, powder injection, tablet, pill, capsule, dispersion, spray, etc. The route of administration of the drug can be a pharmaceutically acceptable route, including but not limited to oral, subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous, and transdermal, intranasal or oral inhalation, etc.

[0012] The present invention finds the anti-tumor mGluR5 target through compound activity screening, and the provided small molecule compound can effectively inhibit the growth of tumors, especially cervical cancer, and has good tumor treatment potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a diagram showing the cytotoxicity screening results of compound I in Example 1.

[0014] Figure 2 This is a graph showing the inhibitory activity of compound I in Example 2 on the proliferation of cervical cancer HeLa cells.

[0015] Figure 3 and Figure 4 This is a diagram showing the mechanism of apoptosis of HeLa cells induced by compound I in Example 3.

[0016] Figure 5 A and Figure 5 B is the result diagram of the decrease in mitochondrial membrane potential induced by compound I in Example 4.

[0017] Figure 6 This is a graph showing the results of compound I inducing calcium ion accumulation in mitochondria in Example 5.

[0018] Figure 7 A and Figure 7 B is the result of knocking out mGluR5 in Example 6 to reverse the apoptosis effect of compound I inducing HeLa cells. DETAILED DESCRIPTION

[0019] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0020] Example 1 Cytotoxicity screening of compound I

[0021] The CCK8 method was used to screen the cell line toxicity of compound I (nine tumor cell lines HeLa, SMMC-7721, PC12, PC3, U251, U87, MDA-MB-231, DU-145, and SH-SY5Y). The specific cytotoxicity detection steps are as follows:

[0022] 1) Select cells in the logarithmic growth phase, remove the culture medium, add 3 mL PBS to rinse the cells, then add trypsin to digest for 1-2 minutes, add 5 mL of fresh culture medium to terminate the digestion, and slowly pipette the cells to completely detach them from the culture dish.

[0023] 2) Count the cells so that there are 100,000 cells per milliliter. The steps for cell counting are as follows:

[0024] Spray the counting plate and glass slide with 75% ethanol to sterilize, then wipe the counting plate and glass slide dry for later use. Take half the volume of cells in the sample loading groove, then add the remaining amount of fresh culture medium required for plating into the sample loading groove, and mix the cells by pipetting. Take 10μL of cells and culture medium from the sample loading groove and add along the gap between the counting plate and the glass slide. Observe with a 20× or 40× microscope and count the number of cells. The counting principle is "take the top but not the bottom, take the left but not the right". Count the total number of cells in 4 16-square grids. The number of cells obtained by dividing the total number of cells by 4 is the number of tens of thousands of cells per milliliter. The specific number of cells should be selected according to the actual experimental arrangement. If there are not enough cells, they can be drawn from the mother solution. If the amount of cells is too large, fresh culture medium needs to be added to dilute to the required number of cells.

[0025] 3) Use a 100 μL pipette to pipette cells into a 96-well plate, and add 100 μL PBS to the edge of the 96-well plate.

[0026] 4) After the cells adhered for about 12 hours, 1 μL of compound I working solution of different concentrations was added to the 96-well plate, and 1 μL of DMSO was added to the control group. Three replicate wells were made for each concentration and the plates were placed in a cell culture incubator for 24 hours.

[0027] 5) During the incubation process, it is necessary to observe and record the cell morphology and death status at different time periods after drug treatment.

[0028] 6) After 24 hours, add 10 μL of CCK8 solution to each well and place in a cell culture incubator in the dark for 1 to 4 hours (depending on the cells).

[0029] 7) Place the 96-well plate in an ELISA reader for testing. Set the parameters of the ELISA reader to oscillate for 30 seconds and read and record the OD value at a wavelength of 450 nm.

[0030] 8) Calculate and plot using Graphpad Prism 7.0 software, and calculate cell viability according to the following formula: Cell viability = (OD compound I / OD DMSO )×100%

[0031] like Figure 1 As shown, compound I is highly toxic to cervical cancer HeLa cells in a concentration-dependent manner, but has little toxicity to other cell lines.

[0032] Example 2 Proliferation inhibition activity of compound I on cervical cancer HeLa cells

[0033] Based on the results of Example 1, HeLa cells were subjected to IC 50 Detection.

[0034] IC was performed using the same CCK8 method as in Example 1 50 Detection. HeLa cells were digested and treated according to the steps in Example 1, and the cells were counted and inoculated into 96-well plates. After the HeLa cells adhered to the wall, gradient dilutions of compound I (0, 1, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 30, 40 μM) were added for 24 hours, and an equal volume of DMSO was added to the control group. After 24 hours, 10 μL of CCK8 solution was added to each well and placed in a cell culture incubator for incubation in the dark for 2 hours. Finally, the absorbance value at a wavelength of 450 nm was detected using an enzyme reader. The proliferation inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1-(OD compound I / OD DMSO ))×100%. GraphPad Prism software was used to calculate and fit the IC of compound I on HeLa cells 50 value.

[0035] The results are as follows Figure 2 As shown, HeLa cells were treated with different concentrations of compound I (0, 1, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 30, 40 μM) for 24 h, and the IC 50 It is 13.93μM.

[0036] Example 3 Mechanism of Compound I inducing apoptosis in HeLa cells

[0037] This example further studies the way in which compound I causes HeLa cell death, and uses the Annexin V / PI double staining method to detect whether compound I induces cell apoptosis, wherein Annexin V-FITC marks early apoptotic cells (located in Q3), PI marks late apoptotic cells (located in Q2), normal cells are located in Q4, and necrotic cells are located in Q1. The expression of apoptosis-related proteins was further detected using Western Blot.

[0038] (1) The specific steps of Annexin V / PI double staining flow cytometry detection of cell apoptosis are as follows:

[0039] 1) HeLa cells in the logarithmic growth phase were selected, and the cells were digested and counted according to the steps of Example 1, so that each milliliter of culture medium contained 150,000 cells.

[0040] 2) Add 2 mL of mixed cell culture medium (3.0×10 5After the cells adhered to the wall for 8-12 h, different concentrations of compound I (0, 5, 10, 15, 20, 30 μM) were added to each well for 24 h.

[0041] 3) Collect the supernatant medium into a centrifuge tube, rinse the cells with PBS, add EGTA-free trypsin to digest the cells for 1-2 minutes, add 4°C pre-cooled PBS and slowly pipette the cells to completely detach the cells from the culture dish.

[0042] 4) Transfer the detached cells to the above centrifuge tube and centrifuge at 1000 rpm for 3 minutes at room temperature.

[0043] 5) Remove the supernatant and add 100 μL 1× Binding Buffer to each centrifuge tube to resuspend the cells.

[0044] 6) Add 5 μL FITC and 5 μL PI reagent to each tube in a dark environment, mix thoroughly, and incubate at room temperature for 15 minutes.

[0045] 7) Before testing, add 100-200 μL 1×Binding Buffer and store on ice away from light. Mix the sample before testing and the test must be completed within 1 hour. Use flow cytometer to detect at 480nm and 561nm channels.

[0046] (2) The specific steps of Western blotting are as follows:

[0047] 1) Preparation of protein samples

[0048] After compound I acted on HeLa cells for 24 hours, the culture medium was removed and the cells were rinsed once with pre-cooled PBS. After washing, an equal amount of cell lysis solution was added to each well, and the cells were immediately lysed on ice for 5-10 minutes. Using a sterilized scraper, the cell lysate was scraped off separately and transferred to a centrifuge tube. The centrifuge tube was placed in a 4°C centrifuge and centrifuged at 13000rpm for 15 minutes. After the high-speed centrifugation was completed, the same volume of lysate was drawn from the centrifuge tube into a new centrifuge tube (the cell pellet could not be taken, and sufficient volume for protein quantification must be left in the centrifuge tube).

[0049] Add 4× Loading Buffer and mix with the cell lysis buffer. Cook the sample in a metal bath for 5-10 minutes, and centrifuge the cooked sample in a handheld centrifuge for 20-60 seconds. Finally, store the sample at -20℃ for later use.

[0050] 2) Protein quantification

[0051] Take out 2 μL of lysate from the old centrifuge tube and add it to a 96-well plate, and set up 3 replicate wells. Add 200 μL of BCA reagent to each well (prepare BCA reagent according to A solution: B solution = 50:1). Incubate at 37℃ in the dark for 30 minutes. Measure the OD value at a wavelength of 562nm with an enzyme reader, and calculate the loading amount based on the measured protein concentration.

[0052] 3) SDS-PAGE electrophoresis

[0053] Take the prepared sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) out of the 4°C refrigerator, remove the gel comb and put it into the gel running rack, install it into the electrophoresis tank, and pour in 1× newly prepared electrophoresis solution. Use a 10μL pipette to load the sample according to the pre-set total amount of protein.

[0054] Cover the electrophoresis cover and run the concentrated protein sample at a constant voltage of 80V for 25 minutes. After all samples have entered the separation gel and are on the same horizontal line, increase the voltage to 100V until the samples run to the bottom of the electrophoresis tank, the small molecular weight marker protein is clearly separated, and the samples have not flowed out of the bottom of the gel. Stop running the gel.

[0055] 4) Transfer

[0056] Prepare 1× transfer solution in advance (preparation of transfer solution: 100mL 10× transfer solution, add 200mL methanol, add 700mL deionized water, mix well and place in a 4℃ refrigerator for use). At the same time, cut out a PVDF membrane that is the same size as the gel or slightly larger (if the target protein is a small molecule protein below 20kDa, use 0.22μm PVDF membrane, and if it is larger than 20kDa, use 0.45μm), soak it in methanol for 3 minutes and then put it into the transfer solution.

[0057] Take a clean tray and pour the pre-cooled transfer solution into the tray. Place the "sandwich" clips for transfer in the order: clips-sponge-filter paper, and soak the filter paper completely. Then take out the gel after electrophoresis and rinse it with ultrapure water. Use a prying board to gently pry open the gel along one side and cut off the required part. Put the protein gel on the filter paper close to the black side of the "sandwich" clip, and then cover the gel with the pre-activated PVDF membrane to squeeze out the bubbles. Then cover the white side of the filter paper and sponge to form a sandwich structure of filter paper-gel-membrane-filter paper. After clamping the "sandwich" clip, put it in the transfer tank, with the black side clip facing the black tank and the white side clip facing the red tank. Add a magnetic stirrer, put in an ice pack (to reduce the heat generated during the electrotransfer process), and fill the transfer tank with transfer solution. Turn on the magnetic stirrer, connect the power supply, and transfer the membrane at a constant current of 230mA (determine the transfer time according to the molecular weight of the target protein). If the electroporation time is too long (>1.5h), it is necessary to pause in the middle, replace the ice pack and then continue the transfer until the end.

[0058] 5) Closure

[0059] Take out the electroporated PVDF membrane and place it in a sealing box containing 5% milk or 5% BSA for blocking at room temperature for 2 hours, or at 4 degrees overnight. (5% milk preparation: weigh 2.5g milk powder, add 1×TBST buffer to 50mL, mix well, and store at 4℃. 5% BSA preparation: weigh 2.5g BSA (fetal bovine serum albumin), add 1×TBST buffer to 50mL, mix well, and store at 4℃.) Cut the blocked PVDF membrane according to the molecular weight of the target protein.

[0060] 6) Incubation with antibodies

[0061] Preparation of primary antibody diluent and secondary antibody diluent: transfer 1.5 mL of 5% milk into a 1.5 mL EP tube, dilute the antibody according to the proportion specified in the antibody manual, and store at -20°C for later use.

[0062] Add the prepared primary antibody diluent to a small rectangular antibody incubation box, and place the cut membrane into the primary antibody diluent, trying to cover the membrane with the primary antibody diluent. Gently shake and incubate on a shaker at room temperature for 2-3 hours, or incubate overnight at 4°C.

[0063] Take out the membrane incubated with primary antibody, recover the primary antibody dilution and store it at -20℃ for next use. Add appropriate amount of 1×TBST to wash the membrane, each time for 10 minutes, repeat 3 times.

[0064] After washing, put the membrane into the antibody incubation box, add the secondary antibody diluent, and incubate at room temperature for 2-3 hours.

[0065] Similarly, the secondary antibody was recovered and the membrane was washed three times with TBST, each time for 10 min.

[0066] 7) Development: Prepare an appropriate amount of ECL developer in proportion. Exposure is to first place the PVDF membrane under white light, take an image of the protein marker, and then soak the PVDF membrane in the developer for an appropriate length of time. Be careful not to overexposure so as not to affect observation. The image of the protein marker under white light is combined with the image after exposure by chemiluminescence, and the location of the target protein is confirmed by band comparison.

[0067] like Figure 3 As shown in the figure, after HeLa cells were treated with compound I for 24 hours, the proportion of early apoptotic cells and late apoptotic cells (Q2+Q3) increased with the increase of compound I concentration (0, 5, 10, 15, 20, 30 μM), indicating that compound I can induce apoptosis of HeLa cells in a concentration-dependent manner. Figure 4The results of Western Blot experiments showed that after 24 hours of treatment of HeLa cells with compound I, as the concentration of compound I increased (0, 5, 10, 15, 20 μM), apoptosis-related proteins Cleaved-Caspase3 and Cleaved-RARP accumulated continuously, accompanied by a decrease in the background proteins Caspase3, Caspase9 and PARP. At the same time, as the concentration of the compound increased, the pro-apoptotic protein Bax in the mitochondrial Bcl-2 family accumulated significantly, while the anti-apoptotic protein Bcl-2 was gradually downregulated, indicating that compound I-induced HeLa cell apoptosis may be closely related to mitochondria (changes in Bcl-2 / Bax affect the transformation of the permeability transition pore (PTP). Bcl-2 can protect the mitochondria in the cell and keep its membrane potential in a normal state, at which time cytochrome c will not be released. Bax destroys the integrity of the mitochondrial membrane potential, causing it to lose its electron transfer ability and reduce energy synthesis).

[0068] Example 4 Compound I induces a decrease in mitochondrial membrane potential

[0069] This example further studies whether the apoptosis of HeLa cells induced by compound I is related to the destruction of mitochondrial membrane potential. The specific steps of using Rh123 fluorescent indicator to detect mitochondrial membrane potential are as follows:

[0070] 1) HeLa cells in the logarithmic growth phase were selected, and cell digestion and cell counting were performed according to the steps in Example 1. 15×10 4 After the cells were attached to the wall (about 8 h), different concentrations of compound I (0, 5, 10, 15, 20 μM) were added and incubated for 24 h.

[0071] 2) Remove the supernatant and rinse twice with PBS. Use culture medium to dilute Rh123 to prepare a working solution of 100 nM, and add 500 μL to each well. Place the 12-well plate in a CO2 incubator at 37°C and incubate in the dark for 30 minutes.

[0072] 3) Remove the supernatant and wash twice with PBS to remove excess probes. Finally, add 500 μL PBS to cover the cells. Observe under a fluorescence microscope at 20× magnification and an excitation wavelength of 488 nm.

[0073] 4) Use ImageJ to process cell images. Import images into ImageJ, convert images to 8-bit (Image→Type→8-bit), adjust thresholds (Image→Adjust→Threshold), set detection parameters (Analyse→SetMeasurement), detect fluorescence intensity (Analyse→Measure), and obtain Excel tables. Finally, use Graphpadprism 7.0 software to draw graphs.

[0074] like Figure 5 A and Figure 5 As shown in Figure B, after HeLa cells were treated with compound I for 24 hours, the fluorescence intensity of Rh123 was observed to gradually decrease under the 488nm excitation light of the fluorescence microscope with the increase of compound I concentration (0, 5, 10, 15, 20 μM), especially starting to drop sharply at 15 μM. The above results indicate that compound I induces a decrease in mitochondrial membrane potential, causing mitochondrial dysfunction in HeLa cells and leading to cell apoptosis.

[0075] Example 5 Compound I induces calcium ion accumulation in mitochondria

[0076] Ca 2+ Accumulation is a key step in triggering mitochondrial dysfunction. High concentrations of Ca in the mitochondrial matrix 2+ This results in the opening of the transmembrane channel permeability transition pore (PTP) between the inner and outer membranes of the mitochondria, thereby regulating the mitochondrial membrane potential (MMP). This example further studies the effect of compound I on the calcium ion content in mitochondria. The fluorescent probe Rhod-2 / AM is used to detect the Ca 2+ content in mitochondria. 2+ , the experimental steps are as follows:

[0077] 1) Cell digestion and cell counting were performed according to the steps in Example 1, and 2×10 5 HeLa cells.

[0078] 2) After the cells adhered, 0, 5, 10, and 15 μM of compound I were added for 14 h.

[0079] 3) After 14 hours, remove the old culture medium, rinse the cells with PBS, and discard the PBS. Add 100 μL of trypsin to each well to make all the cells infiltrated with trypsin, discard the trypsin, and incubate in the incubator for 1.5 minutes. Then add 1 mL of PBS to each well, gently blow the cells to blow off all the attached cells, and collect the cell suspension in a 1.5 mL EP tube.

[0080] 4) Centrifuge at 1200 rpm for 3 min at room temperature. Remove the supernatant and add 1 mL of PBS.

[0081] 5) Repeat step 4) above and wash the cells with PBS three times.

[0082] 6) After centrifugation, discard the supernatant and add 200 μL Rhod-2 / AM working solution to resuspend the cells.

[0083] 7) Incubate in a 37°C incubator away from light for 30 min.

[0084] 8) Centrifuge at 1200 rpm for 3 min to remove the Rhod-2 / AM working solution.

[0085] 9) Resuspend the cells in PBS, wash by centrifugation 3 times, and then add 200 μL of PBS buffer to resuspend the cells.

[0086] 10) Place in a 37°C incubator for about 30 minutes to completely deesterify the AM bodies. After incubation, immediately test on the instrument.

[0087] like Figure 6 As shown, compound I induced the intramitochondrial Ca 2+ The increase was concentration-dependent, suggesting that compound I could disrupt Ca 2+ Homeostasis induces mitochondrial dysfunction in HeLa cells, thereby triggering apoptosis.

[0088] Example 6 Knockout of mGluR5 reverses the apoptosis effect of compound I inducing HeLa cells

[0089] Metabotropic glutamate receptor 5 (mGluR5) belongs to the class C G protein-coupled receptors, which bind to Gα q / 11 binding promotes intracellular Ca cascade through PLCβ signaling 2+ The release of Ca 2+It plays an important role in mitochondrial-related apoptosis events induced by mitochondria. In addition, studies have found that mGluR5 can regulate mitochondrial function (Gonzalez-Lozano MA, Wortel J, van der Loo RJ, van Weering JRT, Smit AB, Li KW. Reduced mGluR5 Activity Modulates Mitochondrial Function. Cells. 2021; 10 (6): 1375.). Therefore, this example further studies the role of mGluR5 in compound I-induced apoptosis. Stably transfected mGluR5 shRNA in HeLa cells, constructed cells with specific knockout of the mGluR5 gene (sh-mGluR5), and synchronously used HeLa cells stably transfected with Lacz labels (sh-Lacz) as controls. Sh-Lacz and sh-mGluR5 HeLa cells were inoculated in 12-well plates and compound I was added for 12 hours. The expression of apoptosis-related proteins was detected by Western Blot as described in Example 3.

[0090] The specific steps for constructing a stable cell line are as follows:

[0091] 1) Design and synthesize the shRNA sequence targeting mGluR5 and clone it into the empty vector plasmid pLV-H1-EF1α-puro (BiOSETTIA, #SORT-B19).

[0092] 2) Lentivirus packaging and production: The tool cells HEK293FT (Invitrogen) were inoculated in a 6-cm culture dish. After the cells adhered and the degree of fusion reached 90%, the above-constructed shRNA lentiviral backbone vector (3 μg) and packaging plasmids vsv-g (1 μg) and Δ8.9 (2 μg) were transfected into HEK-293T cells using Lipofectamine 2000 at a ratio of plasmid: Lipofectamine 2000 = 1:2.5. After 12 hours, fresh culture medium was replaced. After another 36-48 hours, the virus suspension was collected and filtered through a 0.45 μM filter membrane to obtain a lentiviral suspension for infecting the target cells.

[0093] 3) Construction of HeLa stable transfected cells: HeLa cells were inoculated in a 6-well plate, and after the cells adhered, 1 mL of the above-collected lentiviral suspension and 1 mL of fresh culture medium were added, and polybrene, a dyeing agent, was added at a final concentration of 8 μg / mL. Fresh culture medium was replaced 24 hours after viral transfection and culture was continued. Puromycin was added at a final concentration of 2 μg / mL 48 hours after transfection to select HeLa cells with stable knockdown of mGluR5.

[0094] like Figure 7 A and Figure 7 As shown in Figure B, with the increase of the concentration of compound I (0, 10μM, 20μM, 25μM, 30μM), the apoptotic proteins Cleaved-PARP and Cleaved-caspase-9 were significantly accumulated in the control cells (sh-Lacz), accompanied by the downregulation of the anti-apoptotic protein BCL-xL and the increase of the pro-apoptotic protein Bax. However, knocking out mGluR5 can hinder the upregulation of apoptotic proteins Cleaved-PARP and Bax induced by compound I, and does not cause changes in the anti-apoptotic protein BCL-xL, indicating that knocking out mGluR5 can antagonize the pro-apoptotic effect of compound I. The above results suggest that mGluR5 may be the key target of compound I inducing apoptosis of tumor cells.

[0095] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims

1. Application of mGluR5 agonists in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that: The mGluR5 agonist is compound I, and the chemical structure of compound I is as follows:

3. The use according to claim 1, characterized in that: The medicine comprises a pharmaceutically acceptable carrier or excipient.

4. The use according to claim 1, characterized in that: The tumor is cervical cancer.

5. Use of compound I in the preparation of mGluR5 agonist drugs.

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