Application of BL-918 in preparation of medicine for treating fibula amyotrophy 2K

Abstract

The invention belongs to the technical field of medicine and molecular biology, and particularly relates to application of BL-918 in preparation of a medicine for treating fibula amyotrophy 2K. Researches show that the BL-918 can recover mitochondrial autophagy damage caused by GDAP1 defects and improve the motion phenotype of a fibula amyotrophy zebra fish model related to GDAP1. Meanwhile, the BL-918 can be used for remarkably improving the mitochondrial functions of zebrafish with Gdap1 defects, skin fibroblasts from patients suffering from fibula amyotrophy 2K and SH-SY5Y cells knocked down by GDAP1. The invention provides a new intervention method for the treatment of the fibula amyotrophy 2K, and has important clinical application potential.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and molecular biology technology, and specifically relates to the application of BL-918 in the preparation of a drug for treating Charcot-Marie-Tooth Disease 2K. Background Art

[0002] Mutations in the GDAP1 gene cause various forms of Charcot-Marie-Tooth disease (CMT), including autosomal recessive demyelinating CMT4A, autosomal recessive axonal AR-CMT2K, and intermediate RI-CMT, in which both myelin and axons are affected. In addition, a small number of cases present with autosomal dominant AD-CMT2K. The typical clinical phenotype of CMT is length-dependent motor and sensory peripheral neuropathy, characterized by distal muscle weakness and atrophy of the limbs, abnormal gait, crane legs, and foot deformities, some with sensory impairment. Recessive GDAP1 mutations cause severe early-onset peripheral neuropathy, with complete loss of walking ability in the twenties and thirties, and living on a wheelchair. In the late stages of the disease, most patients also experience vocal cord paralysis and diaphragmatic weakness. The phenotype caused by dominant GDAP1 mutations is usually characterized by slow progression and adult onset, but there are also AD-CMT2K patients who present with rapidly progressive respiratory failure and acute vocal cord dysfunction.

[0003] GDAP1 protein is localized in the outer membrane of mitochondria and is mainly expressed in neurons and Schwann cells. GDAP1 gene mutations are associated with multiple mitochondrial dysfunctions, including impaired mitochondrial axonal transport, imbalanced mitochondrial calcium homeostasis, mitochondrial oxidative stress, and mitochondrial-lysosomal membrane contact defects. However, the key pathophysiological mechanisms of GDAP1-related Charcot-Marie-Tooth disease remain unclear, which has greatly delayed the development of targeted therapeutics.

[0004] Mitochondria, as the ultimate clearance mechanism of mitochondrial dysfunction, is an important part of maintaining mitochondrial homeostasis. In order to meet the bioenergetic needs of neuronal axons, neurons transport healthy mitochondria to axons through complex mechanisms and clear damaged mitochondria in distal areas, and this process depends on redundant mitophagy pathways. In mammals, the PINK1 / Parkin-mediated mitophagy pathway has been widely studied. In addition, the AMPK / ULK1 axis is involved in the regulation of mitophagy by regulating the PINK1 / Parkin signaling pathway. BL-918 is a ULK1-specific activator that has been shown to exhibit good effects in the treatment of Parkinson's disease and subarachnoid hemorrhage by activating the ULK1 / PINK1 / Parkin signaling pathway. Summary of the invention

[0005] The purpose of the present invention is to provide the use of BL-918 in the preparation of a drug for treating Charcot-Marie-Tooth Disease 2K, thereby overcoming the deficiencies of the prior art, BL-918 can restore the impaired mitochondrial autophagy caused by GDAP1 deficiency, and improve the motor phenotype of the GDAP1-related Charcot-Marie-Tooth Disease zebrafish model. In addition, BL-918 can significantly improve the mitochondrial function of Gdap1-deficient zebrafish, skin fibroblasts from Charcot-Marie-Tooth Disease 2K patients, and GDAP1 knockdown SH-SY5Y cells.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] The present invention provides application of BL-918 in preparing a drug for treating Charcot-Marie-Tooth Disease 2K.

[0008] In some embodiments, the medicament is a composition.

[0009] In some embodiments, the composition includes an active ingredient of a drug and a pharmaceutically acceptable carrier and medium thereof.

[0010] In some embodiments, the active ingredient of the drug is BL-918 or one of its pharmaceutically acceptable salts, esters, isomers, prodrugs, polymorphs and solvates, and the chemical formula of BL-918 is C 23 H 15 F8N3OS, CASNo.: 2101517-69-3.

[0011] In some embodiments, the dosage form of the composition is selected from one of tablets, capsules, liquid preparations, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely dispersed powders or aerosols for inhalation, sprays, sterile aqueous or oily solutions for extra-gastrointestinal administration, suspensions, and sterile emulsions.

[0012] In some embodiments, the solvent used in the liquid preparation is one of sterile water, water-propylene glycol solution and ethylene glycol aqueous solution.

[0013] In some embodiments, the liquid preparation includes an active ingredient, a colorant, a flavoring agent, a stabilizer, and a thickener.

[0014] In some embodiments, the drug is administered orally.

[0015] Aqueous solutions for oral administration can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavoring agents, stabilizers and thickeners as required. Aqueous suspensions for oral use can be prepared by dispersing the finely divided active ingredient in water together with a viscous material, which can be a natural or synthetic gum, resin, methylcellulose, carboxymethylcellulose and other suspending agents known in the pharmaceutical art.

[0016] The pharmaceutical composition may be in unit dosage form. In these forms, the composition is divided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form may be a packaged preparation containing discrete quantities of the preparation, such as boxed tablets, capsules, and powders in vials or ampoules. The unit dosage form may also be a capsule, cachet, or tablet, or may be in the form of an active powder.

[0017] In some embodiments, the Charcot-Marie-Tooth Disease 2K is an inherited motor sensory neuropathy caused by a mutation in the GDAP1 gene.

[0018] In some embodiments, the drug has one or more of the following effects:

[0019] (1) Restoring the impaired mitophagy caused by GDAP1 deficiency;

[0020] (2) improve the motor phenotype of the GDAP1-related Charcot-Marie-Tooth disease zebrafish model;

[0021] (3) Improved mitochondrial function in Gdap1-deficient zebrafish, skin fibroblasts from Charcot-Marie-Tooth Disease 2K patients, and GDAP1 knockdown SH-SY5Y cells.

[0022] Beneficial effects of the present invention:

[0023] The present invention reports for the first time the use of the ULK1 agonist BL-918 in the preparation of a drug for treating Charcot-Marie-Tooth Disease 2K. The present invention found that BL-918 can restore the impaired mitochondrial autophagy caused by GDAP1 deficiency and improve the motor phenotype of the GDAP1-related Charcot-Marie-Tooth Disease zebrafish model. At the same time, BL-918 can significantly improve the mitochondrial function of Gdap1-deficient zebrafish, skin fibroblasts from Charcot-Marie-Tooth Disease 2K patients, and GDAP1 knockdown SH-SY5Y cells. The present invention provides a new intervention method for the treatment of Charcot-Marie-Tooth Disease 2K, which has important clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 The ubiquitin level of phosphorylated Ser65 in the control and GDAP1 knockdown SH-SY5Y cells after CCCP treatment or CCCP and BL-918 combined treatment for 6 hours in the examples of the present invention;

[0026] Figure 2 The mitochondrial autophagy flux was observed by live cell confocal imaging in the control cells expressing mt-Keima and the SH-SY5Y cells knocked down for GDAP1 after CCCP treatment or CCCP and BL-918 combined treatment in the embodiments of the present invention;

[0027] Figure 3 The movement trajectory diagram and heat map of the zebrafish larvae with gdap1 knocked down after BL-918 treatment in the embodiment of the present invention;

[0028] Figure 4 The cumulative swimming duration and total movement distance of the gdap1 knocked-down zebrafish larvae after BL-918 treatment in the embodiment of the present invention;

[0029] Figure 5 ATP levels in Gdap1-deficient zebrafish, Charcot-Marie-Tooth Disease 2K patient-derived skin fibroblasts, and GDAP1 knockdown SH-SY5Y cells after BL-918 treatment in the embodiments of the present invention;

[0030] Figure 6 In the present invention, the Seahorse extracellular flux analysis method was used to measure the cellular oxygen consumption rate (OCR) in GDAP1-deficient Charcot-Marie-Tooth Disease 2K patient-derived skin fibroblasts and GDAP1 knockdown SH-SY5Y cells after BL-918 treatment. DETAILED DESCRIPTION

[0031] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Specific conditions are not specified in the examples, and the conventional conditions or conditions recommended by the manufacturer are used. The components used without indicating the manufacturer are all conventional products available on the market.

[0032] According to the present invention, the concept of "treatment" refers to any measure suitable for treating Charcot-Marie-Tooth Disease 2K and related diseases. The term "treatment" should be understood in the broadest sense and does not necessarily imply receiving treatment until full recovery. Treatment includes alleviating the symptoms of a specific disease or reducing the risk of a specific disease, or pre-intervention to prevent or reduce the occurrence of such diseases or symptoms.

[0033] The subject refers to an object that has been treated, observed or experimented, including humans and non-human animals, such as zebrafish. The "therapeutically effective amount" refers to the amount of active compounds or agents, including compounds of the present invention, which can cause a biological or medical response of the target tissue system, animal or human, and the effect observed by researchers, veterinarians, doctors or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the nature of the present invention and the form, route and site of administration, as well as the optimal dosage and dosing interval of the active ingredient treated are determined by the specific animal and external conditions, and this optimal dosage can be determined by conventional techniques. At the same time, it must also be recognized that the daily dose and the optimal course of treatment can be determined by methods known in the art to ensure the effective effect of the compound within a specified time.

[0034] Example 1

[0035] Evaluation of the effect of BL-918 on the ubiquitin level of phosphorylated Ser65 in GDAP1 knockdown SH-SY5Y cells

[0036] Mitochondrial damage, such as loss of membrane potential, causes PINK1 kinase to accumulate in the mitochondrial outer membrane, which then phosphorylates the 65th serine residue of ubiquitin and the ubiquitin-like domain of Parkin. Activated Parkin ubiquitinates mitochondrial outer membrane proteins, recruits a series of autophagy receptor proteins, and initiates the formation of autophagosomes.

[0037] Cell culture medium: DMEM medium (Maichen, Cat#CM15019), in addition to 1x penicillin-streptomycin mixture (Solabo, Cat#P1400) and 10% fetal bovine serum (Gibco, Cat#A3161002C).

[0038] Cell culture method: SH-SY5Y cells were cultured in a 37°C, 5% CO2 incubator using the above cell culture medium. Fresh culture medium was replaced every 3-4 days, and the cells were subcultured at a ratio of 1:2 to 1:3. The cells were trypsinized at room temperature for 1-2 minutes, and the subculture cycle was usually 4-5 days.

[0039] Construction of the human neuroblastoma cell line SH-SY5Y with stable knockdown of GDAP: A lentiviral silencing vector targeting human GDAP1 (pCLenti-U6-shGDAP1-CMV-Puro-WPRE) and a non-targeting control shRNA (shCTRL) were purchased from Heyuan Biotechnology. The sequence of shRNA is as follows: the nucleotide sequence of the DNA corresponding to shGDAP1 is GGTTGAAACTGAATTGCAA (as shown in SEQ ID NO.1); the nucleotide sequence of the DNA corresponding to shCTRL is TTCTCCGAACGTGTCACGT (as shown in SEQ ID NO.2). The lentiviral-coated shRNA vector was transfected into SH-SY5Y cells and selected with puromycin to establish a stable cell line with GDAP1 silencing.

[0040] Prepare 100 mM CCCP stock solution: Dissolve 100 mg CCCP powder in 4.887 ml DMSO.

[0041] Prepare 15 mM BL-918 stock solution: Dissolve 1 mg BL-918 powder in 125 μl DMSO.

[0042] Culture medium containing 10 μM CCCP and containing 10 μM CCCP and 5 μM BL-918: Culture medium with a final concentration of 10 μM CCCP (diluted from 100 mM CCCP stock solution and cell culture medium) and 5 μM BL-918 (diluted from 15 mM BL-918 stock solution and cell culture medium) was additionally added to the cell culture medium.

[0043] Prepare vehicle solution: dilute 100% DMSO to 0.01% using cell culture medium as vehicle control solution.

[0044] Experimental groups and experimental process:

[0045] For SH-SY5Y cells in the shCTRL group, the treatment methods included vehicle solution alone, 10 μM CCCP alone, and 10 μM CCCP combined with 5 μM BL-918.

[0046] The treatment method of SH-SY5Y cells in the shGDAP1 group was the same as that of SH-SY5Y cells in the shCTRL group.

[0047] Cell samples (SH-SY5Y cells in the shCTRL group and SH-SY5Y cells in the shGDAP1 group) were collected, and total protein was extracted using RIPA lysis buffer (Solebio, Cat#R0020). Protein quantification was performed using the BCA method (Thermo Fisher Scientific, Cat#A55865), followed by immunoblotting. β-actin was used as an internal reference protein to observe the level of phosphorylated Ser65 ubiquitin. The results are shown in Figure 1 shown.

[0048] from Figure 1 It can be seen that under CCCP-induced mitophagy, the level of phosphorylated Ser65 ubiquitin in the GDAP1 knockdown group was reduced compared with the control group, indicating that mitophagy function was impaired. BL-918 treatment significantly restored the level of phosphorylated Ser65 ubiquitin in GDAP1 knockdown SH-SY5Y cells.

[0049] Example 2

[0050] Evaluation of the effect of BL-918 on mitochondrial autophagy flux in GDAP1 knockdown SH-SY5Y cells

[0051] The mitochondrial-targeted fluorescent protein mito-Keima is known for its pH sensitivity and dual-wavelength excitation properties. At the physiological pH of mitochondria, mito-Keima is excited at 405nm; as mitophagy proceeds, mitochondria are sent to acidic lysosomes, and its excitation wavelength shifts to 561nm.

[0052] The preparation methods of the cell culture medium containing 10 μM CCCP and containing 10 μM CCCP and 5 μM BL-918 are the same as those in Example 1.

[0053] Experimental groups and experimental process:

[0054] SH-SY5Y cells in the shCTRL group that were transfected with mito-Keima were treated with 10 μM CCCP alone for 6 hours.

[0055] SH-SY5Y cells in the shGDAP1 group transfected with mito-Keima were treated with 10 μM CCCP or 10 μM CCCP combined with 5 μM BL-918 for 6 h.

[0056] like Figure 2 As shown, live cell imaging suggested that after CCCP induced mitophagy, the mitophagy rate in the GDAP1 knockdown group was decreased compared with the control group, and BL-918 treatment significantly increased the mitophagy flow.

[0057] Example 3

[0058] Evaluation of swimming trajectory of gdap1 knockdown zebrafish by BL-918

[0059] The preparation method of 15mM BL-918 stock solution is the same as that in Example 1.

[0060] Zebrafish embryo medium containing 1 μM BL-918: Zebrafish embryo medium is supplemented with additional BL-918 to a final concentration of 1 μM.

[0061] Experimental groups and experimental process:

[0062] ctrl-MO and gdap1-MO were microinjected into 1-2 cell stage AB zebrafish embryos, and the embryos were raised in zebrafish embryo medium at a constant temperature of 28°C. Three days after fertilization, the gdap1-MO group larvae were treated with 1 μM BL-918 embryo medium, and the medium was replaced every 24 hours until the movement trajectory was recorded 5 days after fertilization. The swimming behavior of zebrafish larvae was recorded in the dark for 15 minutes using the DanioVision system (EthoVision XT 14.1324, Noldus).

[0063] like Figure 3 As shown in the figure, the swimming path of the larvae in the control group was clear, while the swimming path of the larvae with gdap1 knockdown was irregular. The heat map of the swimming trajectory showed that the larvae in the control group tended to swim along the edge of the culture dish, while the larvae with gdap1 knockdown stayed in a fixed area for a longer time. BL-918 significantly improved the movement defects of the larvae with gdap1 knockdown.

[0064] Example 4

[0065] Evaluation of swimming time and distance of gdap1 knockdown zebrafish by BL-918

[0066] The zebrafish embryo culture medium containing 1 μM BL-918 is the same as that in Example 3.

[0067] The experimental grouping and experimental process are the same as in Example 3.

[0068] like Figure 4 As shown, the cumulative movement time and total swimming distance of gdap1 knockdown larvae were shorter than those of the control group, while BL-918 significantly increased the total swimming distance and cumulative movement time of gdap1 knockdown larvae.

[0069] Example 5

[0070] Evaluation of the effect of BL-918 on ATP levels in GDAP1 deficiency models in vivo and in vitro

[0071] Culture medium containing 5 μM BL-918: a culture medium obtained by adding BL-918 at a final concentration of 5 μM to the cell culture medium; the zebrafish embryo culture medium containing 1 μM BL-918 is the same as that in Example 3.

[0072] Cell culture method: The skin fibroblasts were cultured in a 37°C, 5% CO2 incubator using the cell culture medium in Example 1. Fresh culture medium was replaced every 3-4 days, and the cells were passaged at a ratio of 1:2 to 1:3. The cells were trypsinized at room temperature for 1-2 minutes, and the passage cycle was usually 4-5 days.

[0073] Experimental groups and experimental process:

[0074] Skin fibroblasts were divided into: a group of skin fibroblasts derived from healthy controls treated with cell culture medium containing corresponding DMSO concentrations for 6 hours; a group of skin fibroblasts derived from Charcot-Marie-Tooth Disease 2K patients carrying GDAP1 gene mutations treated with cell culture medium containing corresponding DMSO concentrations for 6 hours; and a group of skin fibroblasts derived from Charcot-Marie-Tooth Disease 2K patients carrying GDAP1 gene mutations treated with 5 μM BL-918 for 6 hours.

[0075] SH-SY5Y cells were divided into: shCTRL group SH-SY5Y cells treated with cell culture medium containing corresponding DMSO concentrations for 6 hours; shGDAP1 group SH-SY5Y cells treated with cell culture medium containing corresponding DMSO concentrations for 6 hours; shGDAP1 group SH-SY5Y cells treated with 5 μM BL-918 for 6 hours.

[0076] The zebrafish were divided into the following groups: ctrl-MO group treated with embryo medium containing corresponding DMSO concentrations 3 days after fertilization; gdap1-MO group treated with embryo medium containing corresponding DMSO concentrations 3 days after fertilization; gdap1-MO group treated with BL-918, specifically, gdap1-MO group fry were treated with embryo medium containing 1 μM BL-918 3 days after fertilization. The zebrafish embryo culture medium was changed every 24 hours until ATP levels were detected 5 days after fertilization.

[0077] ATP levels were detected by an enhanced ATP assay kit (Biyuntian, Cat#S0027). Cells and zebrafish samples were collected and lysed using an ATP assay lysate. In particular, zebrafish were homogenized using a glass tissue grinder. The samples were then centrifuged to obtain supernatant for ATP determination. Sample protein concentrations were quantified using a BCA protein assay kit (Thermo Fisher Scientific, Cat#A55865), and ATP concentrations were standardized according to protein levels.

[0078] like Figure 5As shown, the relative ATP levels in Gdap1-deficient zebrafish, Charcot-Marie-Tooth Disease 2K patient-derived skin fibroblasts, and SH-SY5Y cells were lower than those in the control group, while BL-918 treatment significantly increased the reduced ATP levels.

[0079] Example 6

[0080] Evaluation of the effect of BL-918 on mitochondrial bioenergetics in an in vitro GDAP1-deficient model

[0081] The culture medium containing 5 μM BL-918 was the same as that in Example 5.

[0082] Experimental groups and experimental process:

[0083] The grouping of skin fibroblasts and SH-SY5Y cells was the same as that in Example 5.

[0084] Oxygen consumption rate (OCR) was assessed using an Agilent Seahorse XFe24 analyzer and the Seahorse XF Cell Mitochondrial Stress Test. Skin fibroblasts and SH-SY5Y cells were seeded into XF24V7 cell culture microplates. In the BL-918-treated group, 5 hours after seeding, when cells were well attached, medium containing BL-918 was added to a final concentration of 5 μM. Mitochondrial stress tests were performed 24 hours after treatment. After the cells were plated, OCR was measured by sequentially injecting the following compounds: 1 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone / antimycin A. Sample protein concentration was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Cat# A55865) and used for data normalization. OCR parameters were calculated using the Agilent / Seahorse XF Report Generator Software.

[0085] like Figure 6 As shown, basal respiration and ATP production-related respiration were reduced in skin fibroblasts from Charcot-Marie-Tooth Disease 2K patients and GDAP1 knockdown SH-SY5Y cells compared with controls, although spare respiratory capacity was not affected. BL-918 treatment significantly enhanced the impaired mitochondrial bioenergetics caused by GDAP1 deficiency, as manifested by a significant increase in the reduced basal respiration and ATP production-related respiration rates.

[0086] Based on the above research, we know that:

[0087] (1) The present invention demonstrates for the first time that BL-918 can restore the impairment of mitophagy caused by GDAP1 deficiency;

[0088] (2) This invention demonstrates for the first time that BL-918 can significantly improve the motor defects of the GDAP1-related Charcot-Marie-Tooth disease zebrafish model;

[0089] (3) The present invention demonstrates for the first time that BL-918 can significantly improve the mitochondrial function of Gdap1-deficient zebrafish, skin fibroblasts from Charcot-Marie-Tooth Disease 2K patients, and SH-SY5Y cells.

[0090] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments or to replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of BL-918 in the preparation of drugs for the treatment of Charcot-Marie-Tooth disease 2K.

2. The use according to claim 1, characterized in that The medicine is a composition.

3. The use according to claim 2, characterized in that The composition comprises active ingredients of the drug and pharmaceutically acceptable carriers and media.

4. The use according to claim 3, characterized in that The active ingredient of the drug is BL-918 or one of its pharmaceutically acceptable salts, esters, isomers, prodrugs, polymorphs and solvates.

5. The use according to claim 3, characterized in that The dosage form of the composition is selected from one of tablets, capsules, liquid preparations, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely dispersed powders or aerosols for inhalation, sprays, sterile aqueous or oily solutions for extra-gastric administration, suspensions and sterile emulsions.

6. The use according to claim 5, characterized in that The solvent used in the liquid preparation is one of sterile water, water-propylene glycol solution and ethylene glycol aqueous solution.

7. The use according to claim 5, characterized in that The liquid preparation comprises active ingredients, colorants, flavoring agents, stabilizers and thickeners.

8. The use according to claim 1, characterized in that The drug is administered orally.

9. The use according to claim 1, characterized in that Charcot-Marie-Tooth Disease 2K is a hereditary motor and sensory neuropathy caused by mutation of the GDAP1 gene.

10. The use according to claim 1, characterized in that The drug has one or more of the following effects: (1) Restoring the impaired mitophagy caused by GDAP1 deficiency; (2) improve the motor phenotype of the GDAP1-related Charcot-Marie-Tooth disease zebrafish model; (3) Improved mitochondrial function in Gdap1-deficient zebrafish, skin fibroblasts from Charcot-Marie-Tooth Disease 2K patients, and GDAP1 knockdown SH-SY5Y cells.

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