WO2020211819A1 - Two-step method for selecting drugs against mitochondrial diseases - Google Patents

Abstract

A two-step method for selecting drugs against mitochondrial diseases, relating to the related technical field of mitochondria. Specifically, the method comprises selecting 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside as drugs capable of treating mitochondrial diseases. Provided is new use of these compounds. A new possibility for treating the mitochondrial diseases is provided. Also provided is a method for treating mitochondrial diseases.

Description

Two-step method for screening drugs for mitochondrial diseases

Cross references to related applications

This application claims the priority of a Chinese patent application filed with the State Intellectual Property Office of China with application number 201910321293.6 and titled "Two-step screening of mitochondrial disease drugs" on April 19, 2019, the entire content of which is incorporated into this application by reference in.

Technical field

The present disclosure relates to the technical field related to mitochondria, in particular, to a two-step method for screening mitochondrial disease drugs.

Background technique

Mitochondrial dysfunction, especially oxidative phosphorylation defects, causes a series of clinical phenotypic diseases that are collectively referred to as mitochondrial diseases, and its incidence is conservatively estimated to be about 1/6500. Mitochondrial diseases in a narrow sense specifically refer to genetic diseases caused by mtDNA mutations, which mainly affect tissues and organs with high energy demands such as the heart, brain, and skeletal muscle. Mitochondrial diseases usually present genetic heterogeneity and clinical heterogeneity, and there is still a lack of specific and effective cures.

Mitochondria are not only an important place for cell energy metabolism, but also closely related to cell apoptosis, reactive oxygen species (ROS) production and Ca ²⁺ homeostasis. Mitochondrial functions are regulated by nuclear genes (nDNA) and mitochondrial genes (mtDNA). Of the approximately 1,500 mitochondrial proteins, most of them are encoded by nuclear genes. mtDNA only encodes 13 mitochondrial electron transport chain complex subunits, 22 tRNAs and 2 types of rRNA. Since Wallace et al. reported in 1988 that the m.11778G>A mutation is an important pathogenic factor in Lerber’s hereditary optic neuropathy (LHON), hundreds of disease-related mtDNA mutations have been discovered so far. These mitochondrial diseases mainly include: mitochondrial myopathy, mitochondrial encephalopathy, mitochondrial cardiomyopathy, deafness, optic neuropathy, mitochondrial encephalomyopathy with lactateemia and stroke seizure syndrome (MELAS), myoclonic epilepsy and broken erythrofibrosis ( MERRF) and many other clinical phenotypes, but also related to some metabolic diseases (such as essential hypertension, diabetes, hypercholesterolemia, etc.), neurodegenerative diseases (such as Parkinson’s disease, Alzheimer’s disease, etc.) and The susceptibility of tumors (such as prostate cancer, breast cancer, etc.) is related.

The genetic heterogeneity and clinical heterogeneity of mitochondrial diseases are mainly manifested in that the same mtDNA mutation can trigger different disease phenotypes, and the same disease phenotype can be induced by multiple different mtDNA mutations. Therefore, the diagnosis and treatment of mitochondrial diseases are difficult.

Drug screening refers to the preliminary pharmacological activity detection and testing of substances that may have medicinal value. Traditional target-centric drug discovery has always been the mainstream of the pharmaceutical industry. However, it is not easy to analyze disease-related drug targets. Due to the existence of compensation mechanisms and feedback pathways, those targets that are effective in non-cellular systems may be greatly reduced in the complex cellular environment. This target-based drug discovery process is usually lengthy and expensive. In recent years, phenotypic drug screening based on disease models has re-entered people’s field of vision and has received more and more attention. Since each mammalian cell contains hundreds or even thousands of mitochondria with a double-layer membrane structure, and each mitochondria contains multiple copies of mtDNA molecules, there is no specific and efficient genetic operating system that can perform site-directed mutation or modification of mtDNA. . It is still difficult to construct cell models and animal models of mitochondrial diseases. Currently, drug screening for known mitochondrial diseases is a single model strategy.

Summary of the invention

The purpose of the present disclosure includes providing an application of a compound in the preparation of a medicament for the treatment of mitochondrial diseases. The compound is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside. The present disclosure The new use of the compound is disclosed, and it also provides a new possibility for the treatment of mitochondrial diseases.

The purpose of the present disclosure includes providing a medicine for the treatment of mitochondrial diseases, providing a broader approach for the treatment of mitochondrial diseases, so as to optimize the existing treatment methods.

The purpose of the present disclosure also includes providing a method for screening drugs for the treatment of mitochondrial diseases. The screening method is based on mitochondrial function screening and supplemented by cell phenotype screening, which can effectively and quickly screen out drugs with significant drug effects against mitochondrial diseases. It has the advantages of high efficiency and low cost.

The present disclosure provides the application of the compound in the preparation of a medicine for treating mitochondrial diseases. Specifically, the compound is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, its structural formula is shown in formula 1:

The 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside is derived from Polygonum multiflorum. At present, the known uses of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside are the treatment of atherosclerosis, lipid metabolism, vascular and cardiac remodeling, and vascular fibrosis. , Cardiac and cerebral ischemia, learning and memory disorders, neuroinflammation, Alzheimer’s disease and Parkinson’s disease, diabetes complications, hair growth, etc. In this application, it is used to treat mitochondrial diseases. Preferably, it helps treat maternal hereditary hypertrophic cardiomyopathy.

The present disclosure also provides a medicine for treating mitochondrial diseases, which includes 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

Preferably, the mitochondrial disease refers to maternal hereditary hypertrophic cardiomyopathy associated with the m.2336T>C mutation.

In addition, the present disclosure also provides a method for screening drugs for the treatment of mitochondrial diseases, which includes adopting a patient-specific mitochondrial model for high-throughput screening of candidate drugs, and then subjecting the candidate drugs to a patient-specific iPSCs directed differentiation cell model. Two-step screening. In the embodiment of the present disclosure, the candidate drug includes 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

Optionally, when performing high-throughput screening of candidate drugs, the mitochondrial function index related to the m.2336T>C mutation is selected as a parameter. More preferably, the mitochondrial function index related to the m.2336T>C mutation includes one of the following or Multiple indicators: mitochondrial membrane potential, ATP content, ROS level and/or oxygen consumption rate;

Preferably, when the second step of screening is performed, the physiological cell phenotypic indicators of cases related to mitochondrial diseases are used as parameters for drug screening; preferably, the pathophysiological cell table related to the m.2336T>C mutation The type index is a parameter, and further preferably, the pathophysiological cell phenotype index related to the m.2336T>C mutation includes one or more of the following indexes: cell survival rate and/or action potential of cardiomyocytes.

Preferably, in the screening method, the mitochondrial disease is specifically related to the m.2336T>C mutation, and more preferably hypertrophic cardiomyopathy (HCM).

Specifically, the construction of the mitochondrial model includes immortal lymphocyte lines and mitochondrial cell lines, and transferring mitochondria cells refers to the cytoplasm of immortalized lymphocytes or fibroblasts from patients with the nucleus removed. , Fuse and screen with p ^o cells lacking mtDNA to construct nuclear and cytoplasmic heterozygous cells with consistent nuclear gene background. It excludes the interference of nuclear genes and can directly reflect the impact of mtDNA mutations on mitochondrial function.

In the prior art, the construction, expansion, and targeted differentiation of iPSCs not only require well-trained scientific researchers, but also very expensive reagents and consumables. For example, the cost of iPSCs culture medium is more than 60 times that of ordinary culture medium. Therefore, high-throughput screening based on iPSCs and their targeted differentiated cells is a huge burden in terms of manpower and expense.

The screening method provided in the present disclosure adopts a patient-specific mitochondrial model for high-throughput screening of candidate drugs, and then performs a second-step screening of the candidate drugs through the patient-specific iPSCs directed differentiation cell model, which effectively reduces the cost of drug discovery. Moreover, iPSCs are perfectly combined with high-throughput drug screening to increase the efficiency and results of drug screening.

The present disclosure successfully screened the m.2336T>C mutation-related maternal hereditary hypertrophic cardiomyopathy (HCM) specific drugs through this screening method.

Hypertrophic cardiomyopathy (HCM) is a primary heart disease characterized by asymmetrical hypertrophy of the left ventricle and ventricular septum. It is one of the most common causes of sudden cardiac death in teenagers and athletes. Familial HCM is mainly inherited as an autosomal dominant inheritance caused by mutations in the myocardial sarcomere protein gene. Some HCM cases have maternal inheritance characteristics, which are related to mitochondrial gene (mtDNA) mutations.

In 1991, Zeviani et al. reported the first case of mitochondrial tRNA gene m.3206A>G mutation related to maternal hereditary cardiomyopathy. So far, multiple HCM-related mtDNA pathogenic mutation sites have been found, mainly located in the complex of mitochondrial tRNA gene and electron transport chain. Subunits, and mitochondrial rRNA gene-related mutations are rarely reported. HCM-related mitochondrial tRNA pathogenic genes mainly include MT-TH (OMIM590040), MT-TI (OMIM590095), MT-TK (OMIM 590060), MT-TL1 (OMIM 590050) and MT-TG (OMIM 590035), etc.; HCM Related mitochondrial structural protein pathogenic genes mainly include MT-ATP6 (OMIM 516060), MT-ATP8 (OMIM 516070) and MT-CYB (OMIM516020).

The inventors identified a maternally inherited HCM-related m.2336T>C mutation. The m.2336T>C site is highly conserved. The m.2336T>C mutation is only found in HCM patients. This mutation disrupts the normal 2336U-A2438 base pairing of mitochondrial 16S rRNA, reduces the steady-state expression level of 16S rRNA, and causes mitochondrial oxygen. Reduced consumption rate and damage to ultrastructure. A model of m.2336T>C mutation to mitochondrial cells was constructed. Functional studies have confirmed that the m.2336T>C mutation can cause mitochondrial dysfunction such as decreased mitochondrial membrane potential, decreased ATP synthesis ability, and increased reactive oxygen species (ROS) levels.

Further construct HCM patient-specific iPSCs and their directional differentiated cardiomyocytes (HCM-iPSC-CMs) models, and found that they have increased size, delayed action potential depolarization, and action potential duration prolongation, etc., similar to HCM cardiomyocytes. feature. Using the above-mentioned cell model, candidate drugs that can significantly rescue mitochondrial function were screened from the mitochondrial small molecule compound library and natural product library.

The candidate drug that significantly rescues mitochondrial function is: 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

In addition, the present disclosure also provides a method of treating mitochondrial diseases, the method comprising administering an effective therapeutic dose of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D to patients with mitochondrial diseases -Glucoside.

Optionally, the treatment method includes increasing the mitochondrial membrane potential of the cells of the mitochondrial disease patient.

Optionally, the treatment method includes increasing the ATP synthesis ability of the cells of the mitochondrial disease patient.

Optionally, the treatment method includes reducing ROS levels in cells of patients with mitochondrial diseases.

Optionally, the mitochondrial disease is a mitochondrial disease caused by mtDNA mutations.

Optionally, the mitochondrial disease is a mitochondrial disease caused by the m.2336T>C mutation.

Optionally, the mitochondrial disease is maternal hereditary hypertrophic cardiomyopathy associated with the m.2336T>C mutation.

The present disclosure has the following beneficial effects:

The embodiment of the present disclosure provides the application of a compound in the preparation of a medicament for the treatment of mitochondrial diseases. The compound is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside. The present disclosure The new application of the compound is provided, and a new possibility is provided for the treatment of mitochondrial diseases.

The embodiments of the present disclosure also provide a medicine for treating mitochondrial diseases, which provides a wider way to treat mitochondrial diseases, so as to optimize the existing treatment methods.

In addition, the embodiments of the present disclosure also provide a method for screening drugs for the treatment of mitochondrial diseases. The screening method is based on mitochondrial function screening and supplemented by cell phenotype screening, which can effectively and quickly screen out drugs that target mitochondrial diseases and have significant drug effects. Medicine has the advantages of high efficiency and low cost.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present disclosure more clearly, the following will briefly introduce the drawings that need to be used in the embodiments. It should be understood that the following drawings only show certain embodiments of the present disclosure, and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative work.

Figure 1 is a family diagram of maternal hereditary hypertrophic cardiomyopathy provided in Example 1 of the disclosure;

Figure 2 shows the identification of the m.2336T>C mutation of 16S rRNA in Example 1 of the disclosure; Figure 2A is a partial sequencing diagram of the 16S rRNA gene PCR products of the proband (III-3) and normal control individuals; Figure 2B is The m.2336T>C mutation disrupts the normal 2336U-A2438 base pairing of 16S rRNA; Figure 2C shows that the patient’s peripheral blood cells, hair follicle cells, and oral mucosal epithelial cells all showed m.2336T>C homogeneous mutations;

Figure 3 is the construction and identification of the transgenic mitochondrial cell line in Example 1 of the disclosure; Figure 3A is the growth status in the selective medium; Figure 3B is the detection of mtDNA copy number; Figure 3C is the detection of cell doubling time;

Figure 4 shows the mitochondrial function analysis of the mitochondrial cell line in Example 1 of the disclosure; Figure 4A is the total cell ATP; Figure 4B is the mitochondrial ATP level; Figure 4C-4D is the mitochondrial membrane potential; Figure 4E is the detection of reactive oxygen species; Figure 4F is the active oxygen quantification;

Figure 5 shows the construction and identification of iPSCs in Example 1 of the present disclosure; among them, Figure 5A is ordinary optical microscope observation, alkaline phosphatase staining, and immunofluorescence identification; Figure 5B is the expression of three germ layer marker genes of iPSCs differentiated embryoid bodies Fluorescence quantitative PCR detection; Figure 5C is the immunohistochemical analysis of iPSCs teratoma;

Fig. 6 shows the directionally differentiated cardiomyocytes of iPSCs in Example 1 of the disclosure; Fig. 6A is a schematic diagram of a cardiomyocyte differentiation scheme; Fig. 6B is an immunofluorescence identification of a cardiomyocyte marker protein;

Fig. 7 is an identification diagram of the increased volume of HCM-iPSCs differentiated cardiomyocytes in Example 1 of the disclosure;

Figure 8 is the iPSC-cardiomyocyte action potential detection in Example 1 of the disclosure; Figure 8A is the whole-cell patch clamp detection of action potentials; Figure 8B is a typical single action potential diagram;

FIG. 9 is a partial result diagram of preliminary screening of mitochondrial drugs using the membrane potential as an indicator of the mitochondrial cell model in Example 1 of the disclosure;

FIG. 10 is a partial result diagram of preliminary screening of mitochondrial drugs using the ATP production as an indicator of the mitochondrial cell model in the disclosed embodiment 1;

Figure 11 is a graph showing the results of the growth of iPSC-cardiomyocytes in galactose medium in Example 1 disclosed.

detailed description

The following combines experiments and examples to further describe the claims of the present disclosure in detail, but it does not constitute any limitation to the present disclosure. Any limited modification made within the protection scope of the claims of the present disclosure is still in the claims of the present disclosure. Within the scope of protection. If the specific conditions are not specified in the implementation, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.

The application of the compounds of the embodiments of the present disclosure in the preparation of mitochondrial disease drugs will be specifically described below.

The application of a compound provided in the embodiment of the present disclosure in the preparation of a medicament for the treatment of mitochondrial diseases, said compound is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

Optionally, in this application, the above-mentioned compounds can all increase the mitochondrial membrane potential of cells from patients with mitochondrial diseases.

It should be noted that increasing the mitochondrial membrane potential of the cells of patients with mitochondrial diseases refers to increasing their mitochondrial membrane potential for patients with mitochondrial diseases, especially those whose mitochondrial diseases are mitochondrial diseases caused by mtDNA mutations; preferably Patients with mitochondrial diseases caused by m.2336T>C mutations; more preferably, they refer to patients with maternally inherited hypertrophic cardiomyopathy associated with m.2336T>C mutations. The above compounds can be used to increase the mitochondrial membrane potential of patients close to or return to the mitochondrial membrane potential level of normal people.

Optionally, in this application, the above-mentioned compound can increase the ATP synthesis ability of the cells of patients with mitochondrial diseases. Specifically, the patients with mitochondrial diseases here are the same as the patients mentioned above for improving the mitochondrial phantom of mitochondrial patients, and will not be repeated here. The above-mentioned compounds are used to increase the ATP synthesis ability of patients with mitochondrial diseases to approach or restore the ATP synthesis ability of normal people. Level.

Optionally, in this application, the above-mentioned compound can be applied to reduce the ROS level of patients with mitochondrial diseases. The mitochondrial patients here are the same as above and will not be repeated. The above compounds are used to reduce the ROS level of mitochondrial patients to close to or restore the ROS level of normal people.

It should be noted that, in some embodiments of the present disclosure, mitochondrial diseases refer to mitochondrial diseases caused by mtDNA mutations; preferably, mitochondrial diseases are mitochondrial diseases caused by m.2336T>C mutations; further preferably, the mitochondrial disease The disease is maternal hereditary hypertrophic cardiomyopathy associated with m.2336T>C mutation.

The embodiment of the present disclosure also provides a medicine for treating mitochondrial diseases, the medicine includes 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

It should be noted that drugs composed of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside or drugs containing the above-mentioned compounds and used to treat mitochondrial diseases belong to Within the scope of protection of this application.

Optionally, treating mitochondrial diseases refers to increasing the mitochondrial membrane potential of patients with mitochondrial diseases;

And/or the treatment of mitochondrial diseases refers to improving the ATP synthesis ability of patients with mitochondrial diseases;

And/or the treatment of mitochondrial diseases refers to reducing the level of ROS in patients with mitochondrial diseases.

Preferably, the mitochondrial disease is maternal hereditary hypertrophic cardiomyopathy associated with the m.2336T>C mutation.

In addition, the embodiments of the present disclosure also provide a method for screening drugs for the treatment of mitochondrial diseases. The screening method is a two-step method for screening drugs for mitochondrial diseases. Specifically, the high-throughput screening of candidate drugs using a patient-specific mitochondrial model is then passed through the patient Specific iPSCs directed differentiation cell model for the second step screening of the candidate drugs;

Among them, candidate drugs include 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

Optionally, when performing high-throughput screening of candidate drugs, select mitochondrial function indicators related to mitochondrial diseases as parameters; preferably, select mitochondrial function indicators associated with m.2336T>C mutations as parameters, preferably, and m. Mitochondrial function indicators related to the 2336T>C mutation include one or more of the following indicators: mitochondrial membrane potential, ATP content, ROS level and/or oxygen consumption rate;

Preferably, when the second step of screening is performed, the physiological cell phenotypic indicators of cases related to mitochondrial diseases are used as parameters for drug screening; preferably, the pathophysiological cell table related to the m.2336T>C mutation The type index is a parameter. Further preferably, the pathophysiological cell phenotype index related to the m.2336T>C mutation includes one or more of the following indexes: cell survival rate and action potential of cardiomyocytes.

In addition, the present disclosure also provides a method of treating mitochondrial diseases, the method comprising administering an effective therapeutic dose of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D to patients with mitochondrial diseases -Glucoside.

Optionally, the treatment method includes increasing the mitochondrial membrane potential of the cells of the mitochondrial disease patient.

Optionally, the treatment method includes increasing the ATP synthesis ability of the cells of the mitochondrial disease patient.

Optionally, the treatment method includes reducing ROS levels in cells of patients with mitochondrial diseases.

Optionally, the mitochondrial disease is a mitochondrial disease caused by mtDNA mutations.

Optionally, the mitochondrial disease is a mitochondrial disease caused by the m.2336T>C mutation.

Optionally, the mitochondrial disease is maternal hereditary hypertrophic cardiomyopathy associated with the m.2336T>C mutation.

The features and performance of the present disclosure will be further described in detail below in conjunction with embodiments.

Example 1

This embodiment provides a method for screening drugs for treating mitochondrial diseases, which includes the following steps:

1. Construction of a model of patient-specific mitochondrial cell transfer.

1. Construction of immortalized lymphocyte line

The proband of the core family of maternal hereditary HCM is a male, who was diagnosed with non-obstructive hypertrophic cardiomyopathy at the age of 23. There are four generations in the family, and the maternal members include the proband, the proband’s mother, uncle, and brother, all of whom have hypertrophic cardiomyopathy. Please refer to Figure 1, where the arrow points to the proband III-3 .

Perform full-sequence PCR-sequencing on the peripheral blood mtDNA of all maternal lineage members, and found that all maternal lineage members have a new unreported 16S rRNA m.2336T>C mutation, please refer to Figure 2A. Molecular structure analysis showed that 2336T is located in domain III (Stem 24) of mitochondrial ribosomal 16S rRNA. This mutation disrupts the normal 2336U-A2438 base pairing of 16S rRNA. Please refer to Figure 2B. Pyrosequencing showed that the patient’s peripheral blood cells, hair follicle cells, and oral mucosal epithelial cells all showed m.2336T>C homogenous mutations. Please refer to Figure 2C.

Collect approximately 6 mL of peripheral blood from patients (HCM patients) and normal control individuals in a heparin anticoagulation tube, mix them upside down, and place them in a foam box with ice packs. Lymphocytes were separated within 24 hours after blood sample collection and cultured in a 5% CO ₂ incubator. Cells could be seen to grow in clusters around 24 hours. The pH of the liquid changed in 4 days (3 to 5 days), which was orange-yellow. After 6 days (5-7 days), the culture medium (RPMI 1640 + 15% FBS + 1% double antibody + 2 μg/mL CsA) was changed in half after transformation. The morphology of the transformed cells changed in about a week, with irregular burr-like protrusions on the outer cell wall. White cell clusters visible to the naked eye appeared about 2 weeks. The cells can be passaged in about 1 month, and the immortalized lymphocyte lines obtained from the mutant group and the control group are transferred to a liquid nitrogen tank for cryopreservation.

2. Construction of Transmitting Mitochondrial Cell Line

Add different concentrations of Ficoll to the nitrocellulose centrifuge tube to form a gradient gel. Ficoll was dissolved in DMEM medium containing cytochalasin B and 5% fetal bovine serum, the final concentration was 12.5%.

Cells were taken from the immortalized lymphocyte lines of the mutant group and the control group obtained in step 1, respectively, and resuspended in 5mL Ficoll solution after centrifugation. The two groups of cells were added to the corresponding centrifuge tubes for centrifugation. It can be seen that the cytoplasm of the donor cells is located at the 14% and 16% Ficoll interface, while the nucleus is located at the 17% and 25% Ficoll interface. The cytoplasmic layer was collected and suspended in cytoplasmic fluid (DMEM+20%FBS+50μg/mL uracil) by centrifugation, resuspended with 2mL cytoplasmic fluid, and the cytoplasmic suspension was placed in a 37℃ incubator for 30 Minutes to restore the cytoplasm to a spherical shape.

Ρ ⁰ 206 cells lacking mtDNA are derived from bromodeoxyuridine (BrdU)-resistant osteosarcoma 143B.TK- cells, cultured in (DMEM+5% FBS+100μg/mL BrdU+50μg/mL uracil) culture medium .

The cytoplasmic suspension and ρ ⁰ 206 cells were gently mixed and centrifuged, resuspended in 40% polyethylene glycol (PEG) solution, and then added (DMEM+10% FBS+50μg/mL uracil) culture medium after 1 minute at room temperature Dilution and incubation; (DMEM+5% dialyzed FBS+50μg/mL BrdU) culture medium is cultured and screened to isolate a stable monoclonal fusion cell line, that is, a mitochondrial cell line.

Please refer to Figure 3A for the growth status of the cells in the selection medium; please refer to Figure 3B for the results of the mtDNA copy number of the cells; refer to Figure 3C for the results of the cell doubling time.

Analysis of the mitochondrial function of the transgenic cell line. The m.2336T>C mutation can lead to mitochondrial dysfunction such as reduced mitochondrial membrane potential, weakened ATP synthesis capacity, and increased reactive oxygen species (ROS) levels. Please refer to Figure 4A for total cell ATP, mitochondrial ATP Please refer to Figure 4B for the level; refer to Figure 4C and Figure 4D for the mitochondrial membrane potential; refer to Figure 4E for the detection results of active oxygen; refer to Figure 4F for the quantification of active oxygen.

2. Construction of patient-specific iPSCs and their directional differentiation cardiomyocyte model

1. Construction of patient's primary cell line

Collect the mid-section urine of the control group and the patients (HCM patients). After centrifugation and purification, the urine cells are inoculated into a gelatin-coated 6-well plate and allowed to stand for more than 3 days. When clones appear, change the urine cell culture medium , Change the fluid every other day. Passage after cell clones grow densely or overgrown the well plate, culture in a 37°C, 5% CO ₂ incubator, and replace the culture medium every 2 days.

2. Construction of patient-specific iPSCs model

(1) Establishment of iPSCs: Transfect the pMX retroviral vector plasmid carrying Oct-4, Sox-2, C-myc and Klf4 and the packaging plasmid pCL-ECO into 293T cells, and collect the viral supernatant 48 hours after transfection Fluid infects urine cells. On 4-5 days after virus infection, the deformed primary cells are transferred to feeder cells for culture, and the human embryonic stem cell culture medium is replaced. On the 25th day after the cells are infected with the virus, embryonic stem cell-like (ES cell)-like clones can be observed, which are picked out and cultured in a 12-well plate. After culturing in a 12-well plate for 2-3 generations, transfer to a 6-well plate for cultivation. Collect cells (iPSCs) for biological identification of iPSCs.

(2) Identification of iPSCs

Perform the following identification steps on the iPSCs obtained in step (1):

Alkaline phosphatase staining (AP staining): Shows alkaline phosphatase activity. Cells are fixed with paraformaldehyde, and AP coloring solution is protected from light for coloring. Observe under a microscope. Please refer to Figure 5A for the results of ordinary optical microscope observation, alkaline phosphatase staining, and immunofluorescence identification, in which H1 embryonic stem cells are the positive control.

Immunofluorescence detection: analysis of ES cell-specific cell surface markers SSEA3, SSEA4, tumor-associated antigens TRA-1-60, TRA-1-81, and nuclear protein NANOG, etc. Please refer to Figure 5A, where H1 embryonic stem cells are the positive control, Con-iPSCs are the iPSCs of the normal control group, and HCM-iPSCs are the iPSCs of HCM patients.

Fluorescence quantitative PCR detection: analyze the expression of endogenous pluripotency genes OCT4, SOX2, NANOG and REX1, and silence the exogenous reprogramming factors OCT4, SOX2, KLF4 and C-MYC.

Methylation detection of pluripotency gene promoter: extract genomic DNA, EcoRⅤ restriction enzyme digestion, sodium bisulfite treatment, nested PCR amplification of Nanog and Oct4 gene promoter regions, transform into E. coli, select positive clones for sequencing, find Out the methylation site.

Karyotype analysis: The cells in the exponential growth phase were treated with 200ng/mL colchicine, then hypotonic treatment and fixed, Giemsa staining, and observation and photographing under a microscope.

Differentiation of embryoid bodies: culture iPSCs to 80% in feeder layer, suspension culture after digestion, and change medium every two days. Adherent culture on the 8th day after suspension culture, and RNA was collected and extracted after the cells became full. qPCR detection of endoderm genes AFP, GATA4, SOX17, mesoderm gene TBX1, ectoderm genes PAX6, SOX1, and endogenous pluripotent genes Oct4, Nanog, using H1 embryonic stem cells as a positive control, iPSCs differentiated into the three germ layers of embryoid bodies Please refer to Figure 5B for the results of fluorescent quantitative PCR detection of marker gene expression.

In vivo differentiation of teratomas: iPSCs were cultured without feeder layer, cells were harvested when they were overgrown, and injected into immunodeficient mice, respectively, with 200 μL of back/armpit subcutaneous injection and 100 μL of leg muscle injection. After 8-10 weeks, the teratoma was taken, and the three germ layers were observed by HE staining. Please refer to Figure 5C for the immunohistochemical analysis results of iPSCs teratoma.

The test results show that the patient-specific induced pluripotent stem cells (HCM-iPSCs) of this HCM family have multi-directional differentiation potential and can differentiate into different tissue cells of three germ layers. In vitro embryoid body formation experiment and fluorescence quantitative PCR detection revealed that the expression of marker genes of the three germ layers was significantly increased, while the pluripotency genes were almost not expressed. In vivo differentiated teratoma experiment and HE staining observed a variety of tissue cells in the three germ layers.

3. Construction of iPSCs directed differentiation of cardiomyocyte model

(1) Oriented differentiation of cardiomyocytes: Digest the iPSCs obtained in the model construction procedure of patient-specific iPSCs with Accutase, resuspend in DMEM/F12 centrifugation, and spread 45×10 ⁴ cells on a 12-well plate coated with Matrigel. 1mL mTeSR1+5μmol/L Y-27632 was cultured for 24h; on day 2-4, mTeSR was changed every day; on day 5, 1640+B27-insulin+12μmol/L CHIR; on day 6-7, 1640+B27-insulin; Change to 1640+B27-insulin+5μmol/L IWP2 on days 8-9; change to 1640+B27-insulin on days 10-11; change to 1640+B27 after day 12. Incubate at 37℃, 5% CO _{2 for} 2 weeks. Cardiomyocytes are beating and mature after 6-8 weeks.

(2) Cardiomyocyte identification

Observation and recording of spontaneous contraction of cardiomyocytes: After 2 weeks of directional differentiation, the cardiomyocytes can be seen beating. Observe and record video with an inverted microscope.

Cardiomyocyte specific gene expression and identification: qPCR method and immunofluorescence detection are used, mainly including TNNI3, α-Actinin, MLC2a and MLC2v, etc. For the immunofluorescence identification results of cardiomyocyte marker proteins, please refer to Figure 6B. HCM-iPSCs-differentiated cardiomyocytes express troponin (TNNI3), α-actinin (α-Actin), ventricular myosin-2 (MLC2v) and atrial myosin-2 (MLC2a) and other cardiomyocyte markers protein.

Electrophysiological detection of cardiomyocytes: 30×10 ⁴ cells were plated on a 24-well plate and cultured for 2 days. Place the slide in the groove of the inverted microscope in the 37°C constant temperature perfusion tank, and the extracellular fluid is continuously perfused. Whole cell patch clamp records spontaneous action potentials of cardiomyocytes. pClamp 10.2 and Lab Chart 8.0 software for data recording, analysis and graphing.

The results show that HCM-iPSCs-cardiomyocytes have increased volume (Figure 7), electrophysiological abnormalities, and delayed action potential depolarization (Figure 8, where HCM-iPSC cardiomyocytes have delayed depolarization (DAD). ), Figure 8A is the whole-cell patch clamp detection of action potentials), action potential duration extension (Figure 8B is a typical single action potential diagram, in which HCM-iPSC cardiomyocyte action potential duration is prolonged), etc., L-type Ca ²⁺ A cardiomyocyte phenotype similar to hypertrophic cardiomyopathy, such as significant reduction in current (I _caL ), and irregular rhythmic beating.

Three, two-step method to screen mitochondrial drugs

High-throughput screening of candidate drugs using patient-specific mitochondrial model

1. Drug screening based on membrane potential (preliminary screening based on membrane potential)

Transmit mitochondrial cells obtained in the construction step of the patient-specific mitochondrial cell model were seeded at 8×10 ³ cells/well into a 96-well or 384-well plate with a black bottom, and 143B cells were used as a control. Add the drugs in the mitochondrial drug library (TOPSCIENCE, MedChemExpress, BioChemPartner, etc.) with a final concentration of 30μmol/L to both normal and mutant mitochondrial cells, and set 3 multiple wells for each drug as the experimental group, and set a blank control and a negative control at the same time , Positive control, placed in 37 ℃, 5% CO ₂ incubator for 48 hours. Remove the medium, add 50 μL of DMEM medium to the experimental group, blank control and negative control, and add 50 μL of CCCP solution (decoupling agent) to the positive control well. Place in 37°C, 5% CO ₂ incubator for 30-60 min. Add 25 μL of the prepared JC-10 solution (for the detection of mitochondrial membrane potential) to each well, and incubate in a 37°C, 5% CO ₂ incubator for 30-60 minutes. The fluorescence value of Ex/Em=490nm/525nm and 540nm/590nm was detected by the microplate reader. After subtracting the corresponding blank control group from each well, compare PL590/PL525.

Using the m.2336T>C mutant mitochondrial cell model, using membrane potential as an indicator, preliminary screening of mitochondrial small molecule compound library to obtain 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (drug A).

Please refer to Figure 9. Figure 9 shows other candidate drugs such as Tetramethylpyrazine, Vitamin E, Asiatic acid, etc., from which we can see that 2,3,5,4'-tetrahydroxy The effect of stilbene-2-O-β-D-glucoside (drug A) on the increase of mitochondrial membrane potential has the potential to restore mitochondrial function.

2. ATP-based drug re-screening (preliminary screening based on ATP output)

ATP bioluminescence assay kit HS Ⅱ was used to detect cellular ATP synthesis in Synergy ^TM H1 detector. By adding glucose, or glucose and oligomycin A to the detection buffer, the total cell ATP production, glycolytic ATP production, etc. are measured respectively.

The mitochondrial cells were inoculated in a 6cm ² culture dish, and the final concentration of the drug was 30μmol/L. The cells were placed in a 37°C, 5% CO ₂ incubator for 48 hours. After the cells grow to occupy 80-90% of the area of the bottom of the dish, add 0.5 mL of 0.25% trypsin-EDTA for digestion, and collect the cells. Each cell is divided into two tubes, A and B. 3×10 ⁵ cells are sucked separately, and the detection buffer is used to make up to 1 mL, and 10 μL of 1M D-glucose (final concentration 10 mmol/L) is added to tube A and tube B is added. 10 μL 2-deoxy-D glucose (final concentration 0.5 mmol/L) + 5 μL sodium pyruvate (final concentration 5 mmol/L), treated in a 37° C., 5% CO ₂ incubator for 2 hours, and collected the cell suspension. Add 50μL/well of cell suspension to 96-well plate, add 50μL/well of ATP standard curve gradient dilution solution and mix well, then add 50μL/well detection solution, and mix for 10min in the dark. Leave it at room temperature for 2 minutes, and put it in a fluorescence microplate reader for detection. Through protein quantification, calculate the total cell ATP level and mitochondrial oxidative phosphorylation ATP level (μmol/g protein).

Please refer to Figure 10 for the results. It can be seen that 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (drug A) has the potential to restore mitochondrial function due to the effect of increasing ATP production.

Patient-specific iPSCs directed differentiation of cardiomyocyte model for the second step screening of the candidate drugs

3. The effect of candidate drugs on cell growth (screening based on cell survival rate)

Inoculate 4×10 ⁴ cells/well in a 12-well plate and incubate in a galactose medium at 37° C. and 5% CO _{2 in an} incubator for at least 16 hours to allow the cells to attach to the culture dish. The cells of the experimental group (cardiomyocytes constructed by the patient-specific iPSCs directional differentiation cardiomyocyte model) were replaced with galactose medium containing 30μmol/L candidate drug, and the cells of the control group were replaced with galactose medium containing 30μmol/L DMSO, each group Set up 3 multiple holes for processing. Collect the cells in each well every 24h, and count the cells 3 times in a row.

Taking advantage of the characteristic that galactose can only produce ATP through mitochondrial oxidative phosphorylation, the mutant cardiomyocytes died in a large number of cultures in galactose medium for 3 days. Candidate mitochondrial drugs (2,3,5,4'- Tetrahydroxystilbene-2-O-β-D-glucoside), the survival rate of mutant cardiomyocytes is significantly improved, suggesting that candidate mitochondrial drugs can improve the state or activity of mutant cardiomyocytes, please refer to Figure 11.

Therefore, it is known that drug A (2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside) can significantly improve the survival rate of HCM-iPSC cardiomyocytes in galactose medium.

4. The effect of candidate drugs on cell electrophysiology

Spread cell slides on a 24-well plate, add 200μL of Matrigel and incubate for 1h. Cardiomyocytes (cardiomyocytes constructed by patient-specific iPSCs directional differentiation cardiomyocyte model) were digested with Collagenase B and incubated at 37°C for 30 min. Add twice the volume of 1640 medium to resuspend the cells, centrifuge at 1000 rpm for 5 min; discard the supernatant, and add 20% FBS+1640 medium containing 30 μmol/L drug candidate to resuspend the cells. Take 30×10 ⁴ cells and spread them on a 24-well plate, and incubate them at 37°C and 5% CO _{2 for} 2 days. The cell slide was taken out and placed in a groove of an inverted microscope with a constant temperature perfusion tank at 37°C, and continuously perfused with extracellular fluid. Whole-cell patch clamp technique was used to record spontaneous action potentials of cardiomyocytes. The patch clamp amplifier adopts 700B amplifier. The glass microelectrode is filled with the electrode liquid, and the electrode resistance used when recording the action potential is 3-5MΩ, forming a high resistance (1-5GΩ) seal between the electrode and the cell membrane. Use negative pressure to break the cell membrane, adjust the capacitance and series resistance compensation, the sampling frequency is l0k Hz, the low-pass filter frequency is 2k Hz, let the electrode fluid and the cell fluid balance for 5 minutes and then record the spontaneous beating cardiomyocyte action potential or L Type Ca ²⁺ current (I _caL ). The data were analyzed with pClamp 10.2 and Lab Chart 8.0 software. All experimental data were expressed as mean±standard error. One-way analysis of variance and t test were used. When P<0.05, the difference was considered statistically significant.

In summary, the embodiments of the present disclosure provide an application of a compound in the preparation of a medicine for the treatment of mitochondrial diseases. The compound is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D- Glucoside, the present disclosure provides new uses of the compound and provides new possibilities for the treatment of mitochondrial diseases.

The embodiments of the present disclosure also provide a medicine for treating mitochondrial diseases, which provides a broader approach for the treatment of mitochondrial diseases, so as to optimize the existing treatment methods.

In addition, the embodiments of the present disclosure also provide a method for screening drugs for the treatment of mitochondrial diseases. The screening method is based on mitochondrial function screening and supplemented by cell phenotype screening, which can effectively and quickly screen out drugs that target mitochondrial diseases and have significant drug effects. Medicine has the advantages of high efficiency and low cost.

In addition, the present disclosure also provides a method for treating mitochondrial diseases, which provides an effective treatment method for mitochondrial diseases.

The foregoing descriptions are only preferred embodiments of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Industrial applicability

The present disclosure provides an application of a compound in the preparation of a medicament for treating mitochondrial diseases. The compound is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside. The present disclosure provides The new use of the compound provides new possibilities for the treatment of mitochondrial diseases. The present disclosure also provides a medicine for treating mitochondrial diseases, which provides a broader approach for the treatment of mitochondrial diseases, so as to optimize the existing treatment methods. The present disclosure also provides a method for screening drugs for the treatment of mitochondrial diseases. The screening method is based on mitochondrial function screening and supplemented by cell phenotype screening. It can effectively and quickly screen out drugs with significant pharmacological effects against mitochondrial diseases. The advantages of efficiency and low cost. In addition, the present disclosure also provides a method for treating mitochondrial diseases, which provides an effective treatment method for mitochondrial diseases.

Claims (17)

1. An application of a compound in the preparation of a medicine for treating mitochondrial diseases, characterized in that the compound is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.
2. The application according to claim 1, wherein the treatment of mitochondrial diseases is to increase the mitochondrial membrane potential of cells of patients with mitochondrial diseases.
3. The use according to claim 1, wherein the treatment of mitochondrial diseases is to increase the ATP synthesis ability of cells of patients with mitochondrial diseases.
4. The application according to claim 1, wherein the treatment of mitochondrial diseases is to reduce the level of ROS in cells of patients with mitochondrial diseases.
5. The use according to any one of claims 1 to 4, wherein the mitochondrial disease is a mitochondrial disease caused by mtDNA mutation; preferably, the mitochondrial disease is a mitochondrial disease caused by m.2336T>C mutation .
6. The application according to claim 5, wherein the mitochondrial disease is maternal hereditary hypertrophic cardiomyopathy associated with the m.2336T>C mutation.
7. A medicine for treating mitochondrial diseases, which is characterized in that the medicine includes 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.
8. The medicament according to claim 7, wherein the treatment of mitochondrial diseases is to increase the mitochondrial membrane potential of cells of patients with mitochondrial diseases;

   And/or the treatment of mitochondrial diseases is to increase the ATP synthesis ability of cells of patients with mitochondrial diseases;

   And/or the treatment of mitochondrial diseases is to reduce the level of ROS in cells of patients with mitochondrial diseases;

   Preferably, the mitochondrial disease is hypertrophic cardiomyopathy.
9. A method for screening drugs for the treatment of mitochondrial diseases, characterized in that it comprises adopting a high-throughput screening of candidate drugs using a mitochondrial disease patient-specific trans mitochondrial cell model, and then using the mitochondrial disease patient-specific induction of pluripotent stem cells (iPSCs) to differentiate cell models Screening the candidate drugs in the second step;

   The candidate drugs include 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.
10. The screening method according to claim 9, wherein when performing high-throughput screening of candidate drugs, mitochondrial function indicators related to mitochondrial diseases are selected as parameters;

    Preferably, the mitochondrial function index related to the m.2336T>C mutation is selected as a parameter. Preferably, the mitochondrial function index related to the m.2336T>C mutation includes one or more of the following indicators: mitochondrial membrane potential, ATP content, ROS level And/or oxygen consumption rate;

    Preferably, when the second step of screening is performed, the physiological cell phenotypic indicators of cases related to mitochondrial diseases are used as parameters for drug screening; preferably, the pathophysiological cell table related to the m.2336T>C mutation The type index is a parameter. Further preferably, the pathophysiological cell phenotype index related to the m.2336T>C mutation includes one or more of the following indexes: cell survival rate and action potential of cardiomyocytes.
11. A method for treating mitochondrial diseases, characterized in that the method comprises administering an effective therapeutic dose of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside to patients with mitochondrial diseases .
12. 11. The method of claim 11, wherein the method comprises increasing the mitochondrial membrane potential of the cells of the patient with mitochondrial disease.
13. The method of claim 11, wherein the method comprises increasing the ATP synthesis ability of the cells of the patient with mitochondrial disease.
14. The method according to claim 11, wherein the method comprises reducing the level of ROS in cells of patients with mitochondrial diseases.
15. The method according to claim 11, wherein the mitochondrial disease is a mitochondrial disease caused by mtDNA mutations.
16. The method according to any one of claims 11-15, wherein the mitochondrial disease is a mitochondrial disease caused by the m.2336T>C mutation.
17. The method of claim 16, wherein the mitochondrial disease is a maternal hereditary hypertrophic cardiomyopathy associated with the m.2336T>C mutation.

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