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WO2020224364A1 - Application d'un inhibiteur de protéine mcl1 dans la préparation d'un médicament pour le traitement d'une inflammation et d'une maladie neurodégénérative - Google Patents

Application d'un inhibiteur de protéine mcl1 dans la préparation d'un médicament pour le traitement d'une inflammation et d'une maladie neurodégénérative Download PDF

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WO2020224364A1
WO2020224364A1 PCT/CN2020/083442 CN2020083442W WO2020224364A1 WO 2020224364 A1 WO2020224364 A1 WO 2020224364A1 CN 2020083442 W CN2020083442 W CN 2020083442W WO 2020224364 A1 WO2020224364 A1 WO 2020224364A1
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inflammation
disease
mcl1
umi
protein inhibitor
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Chinese (zh)
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夏宏光
岑旭峰
许正平
盛静浩
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Zhejiang University ZJU
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Zhejiang University ZJU
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention belongs to the technical field of inflammation treatment, and specifically relates to the application of UMI-77 and its derivatives as mitochondrial autophagy inducers in the preparation of drugs for treating inflammation and neurodegenerative diseases.
  • Mitophagy plays an important role in the inhibition of inflammation. Inflammation is the basic strategy of the body to fight infection, and it is also the main cause of exacerbation of diseases, such as certain neurodegenerative diseases and Alzheimer's disease. Therefore, screening new safe and effective mitochondrial autophagy inducers is a possible therapeutic strategy to inhibit inflammation and alleviate the disease.
  • the main principle of the existing mitochondrial autophagy inducers is to destroy the function of mitochondria and force cells to undergo mitochondrial autophagy. Although it can induce mitochondrial autophagy, it cannot play a role in clinical applications due to its greater toxicity.
  • MCL1 inhibitors can be used as mitochondrial autophagy inducers, and there are no drugs targeting mitochondrial autophagy inducers or MCL1 protein inhibitors to treat inflammation and Alzheimer's disease.
  • the purpose of the present invention is to provide a mitochondrial autophagy inducer and its application.
  • an MCL1 protein inhibitor for the preparation of a medicine or composition for one or more purposes selected from the following group:
  • the inflammation includes inflammation in a tissue selected from the group consisting of adipose tissue, kidney tissue, liver tissue, muscle tissue, skin tissue, pancreas tissue, lung tissue, blood vessel, heart, bone, brain, spinal cord , Retina, gastrointestinal tissue, reproductive system, or a combination thereof.
  • the inflammation includes hepatitis (such as acute hepatitis), enteritis (such as ulcerative enteritis), autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis), metabolic disease inflammation (such as diabetes caused Nephritis).
  • hepatitis such as acute hepatitis
  • enteritis such as ulcerative enteritis
  • autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis
  • metabolic disease inflammation such as diabetes caused Nephritis.
  • the neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), different types of spinocerebellar ataxia (SCA), Pick disease.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • ALS amyotrophic lateral sclerosis
  • MS multiple sclerosis
  • SCA spinocerebellar ataxia
  • the MCL1 protein inhibitor includes a compound having MCL1 protein inhibitory activity disclosed in patent document US2014/0235702A1.
  • the MCL1 protein inhibitor is a compound with the structure shown in Formula I or a pharmaceutically acceptable salt thereof:
  • R1 is X is halogen
  • R2 is R 3 is H, halogen, or hydroxyl.
  • the MCL1 protein inhibitor is compound UMI-77 or UMI-59:
  • the medicine or composition contains a safe and effective amount of MCL1 protein inhibitor and a pharmaceutically acceptable carrier.
  • the drug or composition further includes an antioxidant.
  • the drug or composition is in unit dosage form.
  • the drug or composition includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.
  • the medicine or composition includes tablets, pills, powders, granules, capsules, syrups, emulsions, suspensions, injections, patches, drops, ointments, suppositories or sprays.
  • the administration method of the drug or composition includes oral, intramuscular injection, intravenous injection, intravenous drip, spray, or intraperitoneal injection.
  • the safe and effective amount refers to 5-1000 mg/kg; preferably 50-1000 mg/kg.
  • the safe and effective amount includes 50-200 mg/kg (mouse), or 500-1000 mg/kg (human).
  • the safe and effective amount is 100-700 mg/kg, more preferably 500-700 mg/kg.
  • the second aspect of the present invention provides a method for inducing mitochondrial autophagy, which includes the steps of: administering an MCL1 protein inhibitor to a subject in need (such as cells cultured in vitro).
  • the MCL1 protein inhibitor is as described above.
  • the method is an in vitro non-therapeutic method.
  • the concentration of the MCL1 protein inhibitor is about 0.001 ⁇ M-1000 ⁇ M, preferably about 0.1 uM-100 ⁇ M.
  • the third aspect of the present invention provides a method for promoting the interaction between MCL1 protein and LC3A protein, which includes the step of applying an MCL1 protein inhibitor in a system comprising MCL1 protein and LC3A protein.
  • the MCL1 protein inhibitor is as described above.
  • the system containing MCL1 protein and LC3A protein is a cell culture medium.
  • the fourth aspect of the present invention provides a composition for inhibiting inflammation or treating inflammation-related diseases, the composition comprising an MCL1 protein inhibitor.
  • the MCL1 protein inhibitor is as described above.
  • the composition further includes a pharmaceutically acceptable carrier.
  • the composition further includes an antioxidant.
  • the fifth aspect of the present invention provides a method for preventing or treating neurodegenerative diseases, the method comprising the steps of: administering a safe and effective amount of an MCL1 protein inhibitor to a subject in need.
  • the MCL1 protein inhibitor is as described above.
  • Figure 1 shows whether the HEK293Tmtkeima system of human embryonic kidney transformed cells can indicate mitochondrial autophagy according to the present invention
  • Figure 2 shows the high-throughput screening of more than 2000 FDA-approved drugs in the present invention
  • the intensity of the color represents the contrast between the 586nm excitation fluorescence and the negative control
  • the darker the color represents the stronger the induction of mitochondrial autophagy
  • Figure 3 is the second verification of 33 positive drugs of the present invention.
  • the bar graph represents the degree of mitochondrial autophagy induction, and the higher the induction, the stronger the induction;
  • the solid line is the negative control baseline, and the dashed line is the 1.5-fold increase line;
  • FIG. 4 shows the UMI-77 of the present invention induces degradation of mitochondrial marker proteins Tom20 and Tim23;
  • Fig. 5 is the result of Caspase 3 detected by UMI-77 processing cell immune hybridization (western blot) of the present invention
  • Figure 6 is the result of mitochondrial autophagy induced by the HEK293Tmtkeima system of human embryonic kidney transformed cells of the present invention under UMI-77 treatment; the image is taken by biotek cytation3, magnification 4X; red (the upper and lower images corresponding to EM586) is mtkeima586nm excitation fluorescence, representative Mitochondria where mitochondrial autophagy occurs. Green (the two upper and lower pictures corresponding to EM469) is 488nm excitation fluorescence representing normal mitochondria;
  • Fig. 7 is a laser confocal picture of live cells after HEK293Tmtkeima cells are treated with UMI-77 to observe the co-localization of mitochondria and lysosomes;
  • Figure 8 shows the degradation of mitochondria through lysosomes caused by treatment of HEK293T and Hela cells with UMI-77 of the present invention
  • Fig. 9 is the result of mitochondria after UMI-77 treatment of HEK293T cells by transmission electron microscope of the present invention.
  • Figure 10 is the results of the determination of the activity of alanine aminotransferase and aspartate aminotransferase in the blood of the mice of the present invention.
  • UMI-77 inhibits hepatocyte damage;
  • Figure 11 shows the HE (hematoxylin-eosin) stained mouse liver section of the present invention showing that UMI-77 restores the normal morphology of liver cells;
  • Figure 12 shows the MCL1 protein required for UMI-77 to induce mitochondrial degradation of the present invention
  • Figure 13 is the present invention In Situ Red Starter Kit Mouse/Rabbit kit (purchased from Sigma-Aldrich, #DUO92101) to detect the interaction between MCL1 and LC3A;
  • Figure 14 is the immunoprecipitation experiment of the present invention showing that UMI-77 induces enhanced interaction between LC3A and MCL1;
  • Figure 15 is the immunoprecipitation of the present invention showing that the interaction between MCL1 and LC3A is achieved through LIR motif;
  • Figure 16 is a mouse inflammatory bowel disease model of the present invention and a flow chart of UMI77 treatment experiment;
  • Figure 17 is the weight statistics results of mice of the present invention.
  • UMI77 can significantly slow down the weight loss, suggesting that it can protect inflammatory bowel disease;
  • Figure 18 is the statistical result of the disease activity index of the mouse of the present invention, which is calculated based on the weight change, blood in the mouse stool and stool characteristics;
  • Fig. 19 is a representative diagram of the colorectal in the mouse of the present invention, and the statistical results of the length of the colorectal in the mouse;
  • Figure 20 is a HE staining image of the colorectal of a mouse of the present invention, and histopathological score of the colorectal of the mouse;
  • Figure 21 is the expression level of inflammatory factors in the colorectal tissue of the mouse of the present invention.
  • Figure 22 is a record of the escape latency of each group of mice in the positioning cruise experiment of the present invention.
  • Figure 23 is the space search experiment of the present invention to record the number of times the mouse has passed through the original platform
  • Figure 24 is the movement track of a representative mouse recorded in the space search experiment of the present invention.
  • MCL1 protein inhibitors can induce mitochondrial autophagy and promote the interaction between MCL1 protein and LC3A protein.
  • Experimental results show that MCL1 protein inhibitors have a significant inhibitory effect on inflammation, and can be used for the prevention or treatment of inflammation-related diseases.
  • the MCL1 protein inhibitor has a significant therapeutic effect on Alzheimer's disease, so it can be used for the prevention and treatment of neurodegenerative diseases.
  • the present invention has been completed.
  • Mcl-1 myeloid cell leukemia-1) protein is one of the important anti-apoptotic protein members in the Bcl-2 (B-cell lymphoma-2) protein family, and it is overexpressed in a variety of tumor cells.
  • the overexpression of Mcl-1 protein is not only closely related to the occurrence and development of tumors, but also causes tumor cells to develop resistance to chemotherapeutic drugs. Therefore, small molecule inhibitors targeting Mcl-1 are the focus of future anti-tumor drug development. one.
  • MCL1 inhibitors can be used as mitochondrial autophagy inducers, and there are no drugs that target mitochondrial autophagy inducers or MCL1 protein inhibitors to treat inflammation and Alzheimer's disease.
  • the MCL1 protein inhibitor includes a compound having MCL1 protein inhibitory activity disclosed in patent document US2014/0235702A1.
  • the structure of the MCL1 protein inhibitor is shown in formula I:
  • R1 is X is halogen (such as fluorine, chlorine, bromine, iodine, preferably bromine);
  • R2 is R 3 is H, halogen, or hydroxyl.
  • the MCL1 protein inhibitor is compound UMI-77 or UMI-59:
  • the MCL1 protein inhibitors that can be used in the present invention include the compound itself or its derivatives.
  • the derivatives include polymorphs of the compound or pharmaceutically acceptable salts thereof, such as hydrochloride, sulfate, and phosphate. , Sulfonate, Carbonate, Acetate, Tartrate, Formate, Acetate or Isethionate, etc.
  • the preparation of these derivatives can usually be obtained by conventional technical means, and their effects and effects can be obtained according to cell experiments or equivalence experiments, which are all within the scope that those skilled in the art can understand and expect.
  • the MCL1 protein inhibitor or derivative thereof of the present invention can be directly used in the desired object, or mixed with a pharmaceutically acceptable carrier or prepared, and then administered in the form of a composition.
  • the dosage of the MCL1 protein inhibitor or derivative thereof that can be used in the present invention is usually 5-150 mg/kg/d, for example 50-120 mg/kg/d, preferably 80-90 mg/kg/d.
  • the administered dose is within the expected range of the clinician or laboratory staff. For example, the dose can be appropriately adjusted through effectiveness and safety tests to obtain the optimal dose.
  • the present invention provides an application of UMI-77 as a mitochondrial autophagy inducer in the preparation of drugs for the treatment of inflammation and neurodegenerative diseases.
  • the present invention discloses the application of UMI-77 as a mitochondrial autophagy inducer in the preparation of drugs for treating inflammation and neurodegenerative diseases.
  • UMI-77 induces mitochondria to enter lysosomes to cause mitochondrial autophagy.
  • the inflammation is acute hepatitis or inflammatory bowel disease.
  • the neurodegenerative disease is Alzheimer's disease.
  • the present invention also discloses a human embryonic kidney transformed cell HEK293Tmtkeima stable cell line, which was deposited in the China Type Culture Collection on March 7, 2019, with the preservation number: CCTCC NO: C201940.
  • the invention also discloses a method for constructing a stable cell line HEK293Tmtkeima transformed from human embryonic kidney, which comprises the following steps:
  • Lentiviral plasmid preparation After the mtkeima sequence is synthesized, it is connected to the lentiviral plasmid pCDH to form pCDH-mtkeima by restriction enzyme digestion, and the endotoxin-free plasmid pCDH-mtkeima is extracted, and the lentiviral packaging plasmids pMD2.0G and psPAX2 are extracted;
  • HEK293T cells were seeded in a 15cm culture dish at a density of 1*10 6 cells/ml, and three plasmids pCDH-mtkeima, pMD2.0G and psPAX2 were transfected 24 hours later, each plasmid was 10 ⁇ g; At 48 hours and 72 hours after staining, collect the supernatants, combine the supernatants and filter them with a 0.45 ⁇ m filter membrane, add 7.5ml virus concentrate solution to each 30ml filtered supernatant, add an appropriate amount of concentrated solution, and mix well.
  • HEK293T cells were planted in a 10cm culture dish at 5*10 5 cells/ml. After 24 hours, the prepared lentivirus was taken out from -80°C, fully dissolved, and dropped into the cell culture. Observe the infection rate after 48 hours. If the infection rate is greater than 90%, it is considered qualified. Add 1 ⁇ g/ml of puromycin to the qualified infected cell culture to remove uninfected cells. After 2-3 days, change the culture medium and expand the culture. The cells were cryopreserved, and the stable cell line HEK293Tmtkeima transformed into human embryonic kidney was obtained.
  • drug or composition refers to a composition to be administered for a specific purpose, including a pharmaceutical composition or a health care product composition.
  • active ingredient refers to an MCL1 protein inhibitor or derivative thereof.
  • administration method of the active ingredient or the pharmaceutical composition of the present invention is not particularly limited.
  • Representative administration methods include (but are not limited to): oral, intramuscular injection, intravenous injection, intravenous drip, enema, spray, external application, or intraperitoneal injection.
  • Solid dosage forms for oral administration include tablets, pills, powders, granules, or capsules.
  • the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectant, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and g
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of active ingredients or compounds in such compositions may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active ingredient can also be formed into a microcapsule form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the dosage forms of the compound of the present invention for topical administration include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the compound of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is the pharmaceutically effective dosage considered to be administered.
  • the daily dose administered is usually about 1 to 2000 mg, preferably about 6 to 600 mg.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skill range of a skilled physician.
  • the "safe and effective amount" refers to the amount of the compound that is sufficient to significantly improve the condition without causing serious side effects.
  • the present invention provides a new clinical application of MCL1 protein inhibitors, namely for inhibiting inflammation, preventing or treating inflammation-related diseases, and preventing or treating neurodegenerative diseases.
  • MCL1 protein inhibitors can significantly inhibit inflammation and show excellent therapeutic effects on neurodegenerative diseases.
  • the active ingredients of the present invention can also be used to induce mitochondrial autophagy in cultured cells, or to promote the interaction between MCL1 protein and LC3A protein.
  • concentration of the active ingredient of the present invention added is about 0.001 ⁇ M-1000 ⁇ M, preferably 0.01-100 ⁇ M. This concentration can be further adjusted and verified according to the desired effect. All are within the range conventionally obtained by those skilled in the art.
  • modulation includes treatment, prevention or interference.
  • treatment refers to the administration of the active ingredient of the present invention to a subject in need of treatment for the purpose of curing, relieving, improving, alleviating, or affecting the disease, symptom, and predisposition of the subject.
  • subject to treatment refers to mice, humans and other mammals.
  • inflammation refers to one or more of the following symptoms. Such as pain, fever, redness, swelling, temporary or permanent loss of function. Inflammation has many manifestations, including but not limited to, acute, viscous, atrophic, catarrhal, chronic, sclerotic, diffuse, diffuse, exudative, fibrous, fibrogenic, localized, granuloma, hyperplasia Sexuality, hypertrophy, interstitial, metastatic, necrotic, occlusive, substantial, plasticity, production, proliferation, pseudomembranous, purulent, sclerosing, serous fibrin, plasma, simple , Specificity, subacute, purulent, toxic, traumatic, and/or ulcerative.
  • inflammation also includes, but is not limited to, the following vascular appearances (polyarteritis, temporal arteritis); joint appearances (arthritis-crystalline, skeletal, psoriasis, reactive, rheumatic, and dependent characteristics); stomach Intestinal appearance (colitis); skin (dermatitis); or appearance of various tissues and organs (systemic lupus erythematosus).
  • vascular appearances polyarteritis, temporal arteritis
  • joint appearances arthritis-crystalline, skeletal, psoriasis, reactive, rheumatic, and dependent characteristics
  • stomach Intestinal appearance colitis
  • skin dermatitis
  • appearance of various tissues and organs systemic lupus erythematosus
  • the inflammation includes inflammation in a tissue selected from the group consisting of adipose tissue, liver tissue, muscle tissue, skin tissue, pancreas tissue, lung tissue, blood vessels, heart, bones, brain, spinal cord, retina, gastrointestinal tissue, reproduction System, or a combination.
  • inflammation-related diseases includes: multiple sclerosis, inflammatory bowel disease, arthritis, liver fibrosis, pulmonary fibrosis and the like.
  • neurodegenerative diseases includes: Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA) ), Pick disease, etc.
  • the present invention can obtain the following technical effects:
  • the present invention establishes a high-throughput screening model for mitochondrial autophagy.
  • mitochondrial autophagy Through the characteristics of mtkeima protein, mitochondrial autophagy can be conveniently indicated; the inducer UMI-77 that can gently induce mitochondrial autophagy is selected; UMI-77 is A specific inhibitor of MCL1 protein.
  • the MCL1 inhibitor UMI-77 of the present invention can induce mitochondrial autophagy.
  • the MCL1 inhibitor UMI-77 has a new function as an inducer of mitochondrial autophagy.
  • MCL1 inhibitors can be used as mitochondrial autophagy inducers.
  • the UMI-77 of the present invention has a significant inhibitory effect on inflammation; judging from the effect on acute hepatitis and inflammatory bowel disease mouse models, UMI-77 has a significant inhibitory effect on inflammation.
  • the UMI-77 of the present invention has a significant therapeutic effect on Alzheimer's disease; judging from the effect in the APP/PS1 mouse model, UMI-77 has a significant improvement effect on Alzheimer's disease.
  • the present invention utilizes Keima fluorescent protein and mitochondrial localization sequence to be fused to form mtKeima protein, and the human embryonic kidney transformed cell HEK293Tmtkeima stable cell line is constructed by this; the construction steps:
  • HEK293T cells were seeded in a 15cm culture dish at a density of 1*10 6 cells/ml, and three plasmids pCDH-mtkeima, pMD2.0G and psPAX2 were transfected 24 hours later, each plasmid was 10 ⁇ g.
  • At 48 hours and 72 hours after transfection collect the supernatants, combine the supernatants and filter them with a 0.45 ⁇ m filter membrane, add 7.5ml virus concentrated solution per 30ml filtered supernatant, add an appropriate amount of concentrated solution, and mix well. Mix it upside down every 30 minutes for 3-5 times for a total of 4 times. Place overnight at 4°C. Centrifuge at 4000g for 20 minutes at 4°C the next day, discard the supernatant, and resuspend the virus pellet with 1ml PBS (phosphate buffered saline). Store at -80°C.
  • PBS phosphate buffered saline
  • HEK293T cells are planted in a 10cm culture dish at 5*10 5 cells/ml. After 24 hours, the prepared lentivirus is taken out of the minus 80 degree refrigerator, fully dissolved and then dropped into the cell culture , Observe the infection rate after 48 hours, and the infection rate is greater than 90% as qualified. Add 1 ⁇ g/ml puromycin to the qualified infected cell culture to remove uninfected cells. After 2-3 days, change the medium and pass the cells, expand the culture, and freeze the cells to obtain the human embryonic kidney transformed cell HEK293Tmtkeima stable cell line. The cell line was deposited in the China Type Culture Collection on March 7, 2019, with the deposit number: CCTCC NO: C201940.
  • the excitation light of keima protein is 586nm under acidic conditions and 440nm under neutral conditions is used to indicate the occurrence of mitochondrial autophagy, and the high-throughput imaging system biotek cytation3 and software are used to analyze the number of (586nm positive cells/440nm total). Cell number) is an indicator for high-throughput quantitative analysis.
  • KH buffer and PBS are positive controls, which can induce autophagy (including mitochondrial autophagy).
  • KH buffer and PBS phosphate buffer
  • the fluorescence excited by 586nm increases significantly.
  • NC untreated did not increase significantly.
  • UMI-77 induces mitochondrial autophagy (mtkeima signal).
  • the bar graph represents the degree of mitochondrial autophagy induction. The higher the induction, the stronger the induction.
  • HEK293T cells are seeded at a density of 2*10 5 cells/ml
  • Hela cells and U2OS cells are seeded in a 6-well plate at a density of 1.5*10 5 cells/ml, 2 ml per well. Each cell has 6 wells.
  • UMI-77 was added at 0 hours, 3 hours, 6 hours, and 9 hours respectively, and the final concentration was 5 ⁇ M.
  • Collect samples with 250 ⁇ l 2XSDS loading buffer at 12 hours. Heat at 100°C for 10 minutes.
  • Immunohybridization load 10 ⁇ l of each sample, electrophoresis 90V2h; transfer membrane 300mA, 1h.
  • 5% skim milk was blocked at room temperature for 1 hour, and antibody (Tom20 antibody was purchased from Cell Signaling Technology, #42406S; Tim23 antibody was purchased from Proteintech, #11123-1-AP; Tubulin antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., #M1305-2; Calnexin antibody was purchased from Cell Signaling Technology, #2433S) Incubate overnight at 4 degrees at a certain dilution ratio (1:5000 for Tubulin antibody, and 1:1000 for the rest), and wash 3 times each time with PBST (phosphate buffer saline + 0.1% Tween20) 10 minutes.
  • PBST phosphate buffer saline + 0.1% Tween20
  • Goat anti-Mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31430; Goat anti-Rabbit IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31460) diluted 1: 20000 Incubate for 1 hour than room temperature, wash 3 times with PBST for 10 minutes each time. ECL color development.
  • U2OS cells are seeded in a 6-well plate at a density of 1.5*10 5 cells/ml, 2 ml per well, 5 wells. After 24 hours, UMI-77 was added at 0 hours, 12 hours, 24 hours, and 36 hours, and the final concentration was 7.5 ⁇ M. Collect samples with 250 ⁇ l 2XSDS loading buffer at 48 hours. Add staurosporine to a final concentration of 1 ⁇ M 3 hours before the collection. Heat at 100 degrees for 10 minutes. Immunohybridization: load 10 ⁇ l of each sample, electrophoresis 90V2h; transfer membrane 300mA, 1h.
  • Goat anti-Mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31430; Goat anti-Rabbit IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31460) diluted 1: 20000 Incubate for 1 hour than room temperature, wash 3 times with PBST for 10 minutes each time. ECL color development.
  • HEK293T cells are seeded in a 6-well plate at a density of 2*10 5 cells/ml, 2 ml per well, 5 wells. Hela cells were seeded in a 6-well plate at a density of 1.5*10 5 cells/ml, 2 ml per well, 5 wells. After 24 hours, UMI-77 was added at a final concentration of 5 ⁇ M and treated for 24 hours. MG132 2 ⁇ M, E64D 2 ⁇ M and ammonium chloride 20mM, Leupeptin (leupeptin) 50 ⁇ M were added 12 hours before sampling. Collect samples with 250 ⁇ l 2XSDS loading buffer in 24 hours. Heat at 100 degrees for 10 minutes.
  • Immunohybridization Load 10 ⁇ l of each sample, electrophoresis at 90V for 2h; transfer membrane to 300mA, 1h. Blocked with 5% skimmed milk for 1h, antibody (Tom20 antibody was purchased from Cell Signaling Technology, #42406S; Tim23 antibody was purchased from Proteintech, #11123-1-AP; Tubulin antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., #M1305-2) A certain dilution ratio (1:5000 for Tubulin antibody, 1:1000 for the rest) was incubated overnight at 4 degrees, and washed with PBST 3 times for 10 minutes each time.
  • Goat anti-Mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31430; Goat anti-Rabbit IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31460) diluted 1: 20000 After incubating for 1 hour than room temperature, washing with PBST 3 times for 10 minutes each time, ECL develops color.
  • HEK293T cells are seeded in a 6-well plate at a density of 2*105 cells/ml, 2ml per well, 5 wells. After 24 hours, add UMI-77 to a final concentration of 5 ⁇ M and treat for 24 hours. Add E64D 2 ⁇ M, ammonium chloride 20mM, and Leupeptin (leupeptin) 50 ⁇ M 12 hours before sampling. After being fixed with glutaraldehyde, the samples were prepared and tested by electron microscope.
  • the screened effective mitochondrial autophagy inducer UMI-77 has the following characteristics:
  • Mitochondrial degradation passes through the lysosomal pathway.
  • adding lysosomal inhibitor E64D or ammonium chloride and Leupeptin (leupeptin) can prevent the degradation of mitochondrial marker proteins Tom20 and Tim23;
  • Mitochondria will be wrapped by autophagy lysosome membrane. If observed with electron microscope, it can be seen that multiple mitochondria are wrapped by autophagy lysosome.
  • mice with C57BL/6 genetic background were purchased from Nanjing Model Animal Center. Divided into 4 groups, each with 6 animals, and the experiment was carried out after normal feeding for 1 week. All drugs are dissolved in a solvent of 2% DMSO + 30% PEG400 + water. Weigh each mouse and inject 200 ⁇ l of the drug into the intraperitoneal cavity of UMI-77 30mg/kg and Nec1 2.5mg/kg according to the body weight. The LPS induction group was intraperitoneally injected with 200 ⁇ l solvent. The NC group does not do processing.
  • LPS/D-Gal lipopolysaccharide/D-galactosamine concentration
  • D-Gal 400 mg/kg
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • UMI-77 can significantly inhibit liver damage.
  • Nec1 is a known RIPK1 protein inhibitor, and UMI-77 is more effective than Nec1.
  • UMI-77 can significantly inhibit liver injury. UMI-77 restores the integrity of liver cells.
  • Example 4 UMI-77 induces mitochondrial autophagy by inducing the interaction between MCL1 and LC3A (autophagy marker protein)
  • HEK293T cells are seeded at a density of 2*10 5 cells/ml, and Hela cells are seeded in a 6-well plate at a density of 1.5*10 5 cells/ml, 2 ml per well. Each cell has 6 wells.
  • Transfection method pre-mix 50 ⁇ l serum-free DMEM medium and 1 ⁇ g DNA, 50 ⁇ l serum-free DMEM medium and 2 ⁇ l lip2000. Finally, the two components were mixed, allowed to stand at room temperature for 20 minutes, and dropped into the culture medium.
  • 5% skim milk was blocked at room temperature for 1 hour, and the antibody (Tom20 antibody was purchased from Cell Signaling Technology, #42406S; Tim23 antibody was purchased from Proteintech, #11123-1-AP; Tubulin antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., #M1305-2, MCL1 antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., #ET1606-14) and incubated at a certain dilution ratio (1:5000 for Tubulin antibody, and 1:1000 for the rest) at 4 degrees overnight, PBST (phosphate buffer saline + 0.1% Tween 20) ) Wash 3 times for 10 minutes each time.
  • PBST phosphate buffer saline + 0.1% Tween 20
  • Goat anti-Mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31430; Goat anti-Rabbit IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific, #31460) diluted 1: 20000 Incubate for 1 hour than room temperature, wash 3 times with PBST for 10 minutes each time. ECL color development.
  • HEK293T cells were seeded in a 12-well plate at 2*10 5 cells/ml, 1 ml per well, and plated with immunofluorescence slides in advance. After 24 hours, add UMI-77 10 ⁇ M to treat for 5 hours, remove the medium, wash twice with PBS, and add an appropriate amount of 4% paraformaldehyde for fixation for 20 minutes. Wash twice with PBS, use Blocking solution in the In Situ Red Starter Kit Mouse/Rabbit kit (purchased from Sigma-Aldrich, #DUO92101) was blocked for 60 minutes at 37 degrees.
  • the antibody diluent in the above kit to dilute the antibody (MCL1 antibody purchased from Santa cruz, #sc-69840, 1:200 dilution), LC3A antibody purchased from Abcam, #ab62720, 1:500 dilution), incubate overnight at 4 degrees . Wash the film twice with the washing reagent A in the kit, each time for 10 minutes. Incubate the secondary antibodies Anti-rabbit-PLUS and Anti-mouse-MINUS according to the instructions of the kit at 37 degrees for 60 minutes. After that, wash the film twice with washing reagent A, and ligase for 30 minutes at 37 degrees according to the instructions of the kit.
  • Experimental method HEK293T cells were seeded in a 10cm petri dish at 2*10 5 cells/ml. After 24 hours, pCMV3-MCL1-3xFlag and pCDNA3.1-HA-LC3A or pCDNA3.1-HA-LC3B plasmids were co-transfected, 4 ⁇ g each.
  • Transfection method pre-mix 500 ⁇ l serum-free DMEM medium and 8 ⁇ g DNA, 500 ⁇ l serum-free DMEM medium and 16 ⁇ l PEI (polyethyleneimine) transfection reagent. Finally, the two components were mixed, allowed to stand at room temperature for 20 minutes, and dropped into the culture medium.
  • PEI polyethyleneimine
  • HEK293T cells were seeded in a 10cm petri dish at 2*10 5 cells/ml. After 24 hours, pCMV3-MCL1-3xFlag and mutants and pCDNA3.1-HA-LC3A plasmids were co-transfected, 4 ⁇ g each.
  • Transfection method pre-mix 500 ⁇ l serum-free DMEM medium and 8 ⁇ g DNA, 500 ⁇ l serum-free DMEM medium and 16 ⁇ l PEI (polyethyleneimine) transfection reagent. Finally, the two components were mixed, allowed to stand at room temperature for 20 minutes, and dropped into the culture medium. After 24 hours of transfection, UMI-77 10 ⁇ M was added for 5 hours, and the cells were washed twice with PBS.
  • the remaining prepared cell lysis supernatant was mixed with the pretreated agarose gel coupled with Flag antibody, and mixed overnight at 4 degrees. The next day, the agarose gel was collected by centrifugation at 2000 rpm for 3 minutes, and washed with RIPA 3 times, and centrifuged at 2000 rpm for 3 minutes each time. Finally, collect the agarose gel and resuspend it in 30 ⁇ l of 2X SDS loading buffer, and boil it at 100°C for 10 minutes. Immunohybridization: Load 10 ⁇ l of each sample, electrophoresis at 90V for 2h; transfer membrane to 300mA, 1h.
  • the two LIR motifs contained in MCL1 were mutated separately.
  • the first LIR motif mutation has four forms: W261A, I264A, W261A/I264A, ⁇ LIR.
  • the second LIR motif mutation has three forms: F318A, V321A, F318A/V321A.
  • the first LIR motif mutation significantly affected the interaction between MCL1 and LC3A.
  • the second LIR motif has almost no effect.
  • mice 7-8 weeks old male wild-type mice with C57BL/6 genetic background freely drink 2% DSS solution (the solution needs to be freshly prepared) for 6 days, then change to normal water until the end of the experiment.
  • UMI77 5mg/kg body weight
  • the weight change, stool shape and blood in the stool were recorded every day.
  • the histopathology score is divided into inflammatory cell infiltration score and tissue damage score refer to Immunity.2010; 32(3) :379-91, finally calculate the disease activity index.
  • UMI77 can significantly slow down weight loss, reduce disease activity index, restore the normal state of intestinal tissue and its cells, and reduce the expression of inflammatory factors, suggesting that it can protect inflammatory bowel disease.
  • Example 6 restores the spatial memory and learning ability of APP/PS1 model mice
  • APP/PS1 mice One of the commonly used mouse models of Alzheimer's disease is APP/PS1 mice.
  • APP/PS1 mice with genetic background of C57BL/6 and control mice were purchased from Nanjing Model Animal Center. Divided into three groups, the control group, the APP/PS1 model group, and the APP/PS1 model administration group, each with 5-7 animals.
  • APP/PS1 began intraperitoneal injection of UMI-77 13mg/kg at the age of 4 months. The drug was administered every other day for 4 months, after which a water maze experiment was performed.
  • the experiment is mainly divided into two parts: positioning cruise experiment and space search experiment.
  • the positioning cruise experiment is used to measure the learning and memory ability of mice in the water maze.
  • the spatial search experiment measures the ability of mice to retain memory of the platform's spatial location.
  • Positioning cruise experiment Divide the pool containing titanium dioxide water into 4 quadrants (northwest, northeast, southwest, southeast), and place the platform about 1cm underwater in the southeast quadrant. Put the mouse head towards the pool wall and put it into the water 4 times in the order of northwest, northeast, southeast and southwest. Record each time the mouse finds the underwater platform. If the time exceeds 60 seconds, guide the mouse to the platform, let it stay for 20 seconds, and record the time to find the platform as 60 seconds. Each mouse was trained 4 times a day for a total of 4 days, and the time to find the platform (escape latency) was recorded.
  • Space search experiment 24 hours after the positioning cruise experiment is over, the platform will be removed and the space search experiment will start for 60 seconds. Place the mouse into the water from the opposite side of the original platform quadrant, and record the number of times the mouse passes through the original platform position and the movement track.
  • Alzheimer's disease is a serious neurodegenerative disease, and currently there is no specific treatment. In previous studies, it was found that there is a large amount of amyloid accumulation and damaged mitochondria accumulation in the brain of Alzheimer's patients.
  • the currently known clinical drugs are mainly aimed at the clearance and degradation of amyloid.
  • UMI-77 can induce mitochondrial autophagy and help eliminate damaged mitochondria, indicating its potential for the treatment of Alzheimer's disease.

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Abstract

L'invention concerne une application d'un inhibiteur de protéine MC1 dans la préparation d'un médicament pour le traitement d'une inflammation et d'une maladie neurodégénérative. L'effet de l'inhibiteur MCL1 UMI-77 pour induire une autophagie mitochondriale est proposé. Selon ses effets sur l'hépatite aiguë, la maladie intestinale inflammatoire et les modèles de souris APP/PS1, UMI-77 a un effet inhibiteur significatif sur l'inflammation, et peut améliorer significativement la maladie d'Alzheimer.
PCT/CN2020/083442 2019-05-09 2020-04-07 Application d'un inhibiteur de protéine mcl1 dans la préparation d'un médicament pour le traitement d'une inflammation et d'une maladie neurodégénérative Ceased WO2020224364A1 (fr)

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