Use of bl-918 in the preparation of a medicament for treating charcot-marie-tooth disease 2k

Abstract

The application belongs to the technical field of medicine and molecular biology, and particularly relates to application of BL-918 in preparation of a 2K drug for treating Charcot-Marie-Tooth disease, researches show that BL-918 can restore the impaired mitochondrial autophagy caused by GDAP1 defects and improve the motion phenotype of a GDAP1-related Charcot-Marie-Tooth disease zebrafish model. Meanwhile, BL-918 can significantly improve the mitochondrial function of Gdap1-deficient zebrafish, skin fibroblasts derived from Charcot-Marie-Tooth disease 2K patients and SH-SY5Y cells with GDAP1 knockdown. The application provides a new intervention method for treating Charcot-Marie-Tooth disease 2K and has important clinical application potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine and molecular biology, and particularly relates to application of BL-918 in preparation of a drug for treating Charcot-Marie-Tooth disease 2K. BACKGROUND

[0002] GDAP1 gene mutation leads to multiple forms of Charcot-Marie-Tooth disease (CMT), including autosomal recessive demyelinating CMT4A, autosomal recessive axonal AR-CMT2K, and intermediate RI-CMT with simultaneous involvement of myelin and axon. In addition, there are a few cases of autosomal dominant AD-CMT2K. The typical CMT clinical phenotype is length-dependent motor and sensory peripheral neuropathy, characterized by muscle weakness and atrophy in the distal extremities, gait abnormalities, crane legs, and foot deformities, some with sensory impairment. Recessive GDAP1 mutations lead to severe early-onset peripheral neuropathy, completely losing the ability to walk in their twenties, and relying on a wheelchair. In the late stage of the disease, most patients also have vocal cord paralysis and diaphragmatic weakness. Dominant GDAP1 mutations cause a phenotype that usually shows slow progression and adult-onset, and some AD-CMT2K patients show rapid progression of respiratory failure and acute vocal cord dysfunction.

[0003] GDAP1 protein is located in the outer membrane of mitochondria and is mainly expressed in neurons and Schwann cells. GDAP1 gene mutation is associated with multiple mitochondrial dysfunction links, including impaired mitochondrial axonal transport, mitochondrial calcium homeostasis imbalance, mitochondrial oxidative stress, and mitochondrial-lysosome membrane contact defects. However, the key pathophysiological mechanisms of GDAP1-related Charcot-Marie-Tooth disease are still unclear, which greatly delays the development of targeted therapeutic means.

[0004] Mitophagy, as the ultimate clearance mechanism of mitochondrial dysfunction, is an important link in maintaining mitochondrial homeostasis. To meet the bioenergy needs of neuronal axons, neurons transport healthy mitochondria to axons through complex mechanisms and remove damaged mitochondria in the distal region, and this process depends on the redundant mitophagy pathway. In mammals, the PINK1 / Parkin-mediated mitophagy pathway has been extensively studied. In addition, the AMPK / ULK1 axis participates in the regulation of mitophagy by regulating the PINK1 / Parkin signaling pathway. BL-918 is a specific activator of ULK1, which has been shown to activate the ULK1 / PINK1 / Parkin signaling pathway and exhibit good effects in the treatment of Parkinson's disease and subarachnoid hemorrhage. SUMMARY

[0005] The application aims to provide the application of BL-918 in the preparation of a drug for treating Charcot-Marie-Tooth disease 2K, so as to overcome the shortcomings of the prior art, and BL-918 can restore the impaired mitochondrial autophagy caused by GDAP1 defects and improve the motor phenotype of GDAP1-related Charcot-Marie-Tooth disease zebrafish models. In addition, BL-918 can significantly improve the mitochondrial function of Gdap1-deficient zebrafish, Charcot-Marie-Tooth disease 2K patient-derived skin fibroblasts and GDAP1-knockdown SH-SY5Y cells.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the application is:

[0007] The application provides the application of BL-918 in the preparation of a drug for treating Charcot-Marie-Tooth disease 2K.

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

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

[0010] In some embodiments, the active ingredient of the drug is one of BL-918 or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph and solvate thereof, and the chemical formula of the BL-918 is C 23 H 15 F8N3OS, CAS No.: 2101517-69-3.

[0011] In some embodiments, the dosage form of the composition is selected from one of a tablet, a capsule, a liquid preparation, a suspension, an emulsion, a cream, an ointment, a gel, a nasal spray, a suppository, a finely dispersed powder or an aerosol for inhalation, a spray, a sterile aqueous or oily solution, a suspension and a sterile emulsion for parenteral administration.

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

[0013] In some embodiments, the liquid preparation comprises an active ingredient, a coloring agent, a flavoring agent, a stabilizer and a thickening agent.

[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 needed. Aqueous suspensions for oral use can be prepared by dispersing a finely dispersed active ingredient in water along with a viscous substance, which may be a natural or synthetic gum, resin, methylcellulose, carboxymethylcellulose, or other suspending agents known in the pharmaceutical field.

[0016] The pharmaceutical composition may be in unit dose form. In these forms, the composition is divided into unit doses containing an appropriate amount of the active ingredient. This unit dose form may be a packaged formulation, comprising a portion of the formulation, such as boxed tablets, capsules, and powders in tubular vials or ampoules. The unit dose form may also be capsules, sachets, or tablets, or may be in the form of an active powder.

[0017] In some embodiments, the peroneal muscular atrophy 2K is a hereditary motor and 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) Restore the damage to mitophagy caused by GDAP1 deficiency;

[0020] (2) Improve the motor phenotype of a zebrafish model of GDAP1-related peroneal muscular atrophy;

[0021] (3) Improve mitochondrial function in skin fibroblasts derived from Gdap1-deficient zebrafish, patients with peroneal muscular dystrophy 2K, and GDAP1 knockdown SH-SY5Y cells.

[0022] The beneficial effects of this invention are:

[0023] This invention reports for the first time the application of the ULK1 agonist BL-918 in the preparation of a drug for treating peroneal muscular dystrophy (PCD) 2K. This invention found that BL-918 can restore impaired mitophagy caused by GDAP1 deficiency and improve the motor phenotype in a GDAP1-related zebrafish model of PCD. Simultaneously, BL-918 significantly improves mitochondrial function in Gdap1-deficient zebrafish, skin fibroblasts derived from PCD 2K patients, and GDAP1 knockdown SH-SY5Y cells. This invention provides a novel intervention method for the treatment of PCD 2K and has significant clinical application potential. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

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

[0026] Figure 2 In this embodiment of the invention, live-cell confocal imaging was performed on control cells expressing mt-Keima and SH-SY5Y cells with GDAP1 knockdown, or on cells treated with a combination of CCCP and BL-918, to observe mitochondrial autophagy flux.

[0027] Figure 3 The image shows the movement trajectory and heat map of zebrafish juveniles with gdap1 knocked down after BL-918 treatment in this embodiment of the invention.

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

[0029] Figure 5 The ATP levels in Gdap1-deficient zebrafish, skin fibroblasts derived from patients with peroneal muscular dystrophy 2K, and GDAP1 knockdown SH-SY5Y cells were obtained after BL-918 treatment in this embodiment of the invention.

[0030] Figure 6 In this embodiment of the invention, the Seahorse extracellular flow analysis method was used to measure the cellular oxygen consumption rate (OCR) in skin fibroblasts and GDAP1 knockdown SH-SY5Y cells derived from GDAP1-deficient peroneal muscular dystrophy patients after BL-918 treatment. Detailed Implementation

[0031] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0032] According to the present invention, the concept of "treatment" refers to any measure applicable to the treatment of peroneal muscular dystrophy 2K and related diseases. The term "treatment" should be understood in the broadest sense and does not necessarily imply treatment until complete recovery. Treatment includes relieving symptoms of a specific condition or reducing the risk of developing a specific condition, or proactively preventing or reducing the occurrence of such diseases or symptoms.

[0033] The subjects referred to are those who have received treatment, observation, or experimentation, including humans and non-human animals such as zebrafish. The "therapeutic effective amount" refers to the amount of the active compound or agent, including the compound of the present invention, that can elicit a biological or medical response in the target tissue system, animal, or human, and the effect observed by researchers, veterinarians, physicians, or other medical personnel, including the reduction or partial reduction of symptoms of the treated disease, syndrome, symptom, or disorder. It must be recognized that the nature of the present invention, as well as the form, route, and site of administration, and the optimal dosage and interval of the therapeutic active ingredient, are determined by the specific animal and external conditions, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the daily dosage and optimal duration of treatment can be determined using methods known in the art to ensure the effective action of the compound within a specified time.

[0034] Example 1

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

[0036] Mitochondrial damage, such as loss of membrane potential, leads to the accumulation of PINK1 kinase in the outer mitochondrial membrane, which in turn phosphorylates serine residue 65 of ubiquitin and the ubiquitin-like domain of Parkin. Activated Parkin then recruits a series of autophagy receptor proteins by ubiquitinizing outer mitochondrial membrane proteins, initiating the formation of autophagosomes.

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

[0038] Cell culture method: Using the cell culture medium described above, SH-SY5Y cells were cultured in a 37℃, 5% CO2 incubator. The culture medium was replaced with fresh medium every 3-4 days, and the cells were passaged at a ratio of 1:2 to 1:3. Trypsin digestion was performed at room temperature for 1-2 minutes, and the passage cycle was usually 4-5 days.

[0039] Construction of a stable GDAP-knockdown human neuroblastoma cell line SH-SY5Y: A lentiviral silencing vector targeting human GDAP1 (pCLenti-U6-shGDAP1-CMV-Puro-WPRE) and a non-targeted control shRNA (shCTRL) were purchased from Heyuan Biotechnology. The shRNA sequences are shown below: 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 lentivirally coated shRNA vector was transfected into SH-SY5Y cells, and selection was performed using puromycin to establish a stable GDAP1-silenced cell line.

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

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

[0042] Medium containing 10 μM CCCP and medium containing 10 μM CCCP and 5 μM BL-918: Medium obtained by adding an additional 10 μM CCCP (diluted with cell culture medium from 100 mM CCCP stock solution) and 5 μM BL-918 (diluted with cell culture medium from 15 mM BL-918 stock solution) to the cell culture medium.

[0043] Prepare the vector solution: Dilute 100% DMSO to 0.01% using cell culture medium as the vector control solution.

[0044] Experimental grouping and experimental procedure:

[0045] SH-SY5Y cells in the shCTRL group were treated with the following methods: single treatment with vector solution, single treatment with 10 μM CCCP, and combined treatment with 10 μM CCCP and 5 μM BL-918.

[0046] The SH-SY5Y cells in the shGDAP1 group were treated in the same way as the SH-SY5Y cells in the shCTRL group.

[0047] Cell samples (SH-SY5Y cells from the shCTRL group and SH-SY5Y cells from the shGDAP1 group) were collected. Total protein was extracted using RIPA lysis buffer (Solepro, Cat#R0020), and protein quantification was performed using the BCA method (Thermo Fisher Scientific, Cat#A55865). Western blotting was then conducted. β-actin was used as an internal control protein to observe phosphorylated Ser65 ubiquitin levels. The results are as follows: Figure 1 As shown.

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

[0049] Example 2

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

[0051] Mito-targeting 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 405 nm; as mitophagy proceeds, the mitochondria are transported to the acidic lysosomes, and its excitation wavelength shifts to 561 nm.

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

[0053] Experimental grouping and experimental procedure:

[0054] SH-SY5Y cells in the shCTRL group 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 a combination of 10 μM CCCP and 5 μM BL-918 for 6 hours.

[0056] like Figure 2 As shown, live-cell imaging revealed that after CCCP-induced mitophagy, the mitophagy rate decreased in the GDAP1 knockdown group compared to the control group, while BL-918 treatment significantly increased mitophagy flux.

[0057] Example 3

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

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

[0060] Zebrafish embryo culture medium containing 1 μM BL-918: The culture medium obtained by adding an additional 1 μM BL-918 to the zebrafish embryo culture medium.

[0061] Experimental grouping and experimental procedure:

[0062] ctrl-MO and gdap1-MO were injected via microinjection into 1-2 cell stage AB line zebrafish embryos, which were then cultured in zebrafish embryo culture medium at a constant temperature of 28°C. Three days post-fertilization, juveniles in the gdap1-MO group were treated with 1 μM BL-918 embryo culture medium, with the medium changed every 24 hours, until 5 days post-fertilization for homing. Swimming behavior of zebrafish juveniles was recorded for 15 minutes in the dark using a DanioVision system (EthoVision XT 14.1324, Noldus).

[0063] like Figure 3 As shown, the control group juveniles exhibited clear swimming paths, while the gdap1 knockdown juveniles exhibited irregular swimming paths. The swimming trajectory heatmap revealed that the control group juveniles tended to swim along the edge of the culture dish, while the gdap1 knockdown juveniles remained in fixed areas for longer periods. BL-918 significantly improved the kinetic defects in the gdap1 knockdown juveniles.

[0064] Example 4

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

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

[0067] The experimental groups and procedures are the same as in Example 3.

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

[0069] Example 5

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

[0071] Culture medium containing 5 μM BL-918: The culture medium obtained by adding an additional 5 μM BL-918 to the cell culture medium; the zebrafish embryo culture medium containing 1 μM BL-918 is the same as in Example 3.

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

[0073] Experimental grouping and experimental procedure:

[0074] Skin fibroblasts were divided into: healthy control skin fibroblasts treated with cell culture medium containing the corresponding concentration of DMSO for 6 hours; skin fibroblasts from patients with Charcot-Marie-Kirchen syndrome carrying the GDAP1 gene mutation treated with cell culture medium containing the corresponding concentration of DMSO for 6 hours; and skin fibroblasts from patients with Charcot-Marie-Kirchen syndrome carrying the GDAP1 gene mutation 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 the corresponding concentration of DMSO for 6 hours; shGDAP1 group SH-SY5Y cells treated with cell culture medium containing the corresponding concentration of DMSO for 6 hours; and shGDAP1 group SH-SY5Y cells treated with 5μM BL-918 for 6 hours.

[0076] Zebrafish were divided into three groups: the ctrl-MO group treated with embryo culture medium containing the appropriate concentration of DMSO for 3 days post-fertilization; the gdap1-MO group treated with embryo culture medium containing the appropriate concentration of DMSO for 3 days post-fertilization; and the gdap1-MO group treated with BL-918, specifically, the gdap1-MO group juveniles were treated with BL-918 embryo culture medium containing 1 μM for 3 days post-fertilization. The zebrafish embryo culture medium was changed every 24 hours until ATP levels were measured 5 days post-fertilization.

[0077] ATP levels were measured using an enhanced ATP assay kit (Beyotime, Cat#S0027). Cell and zebrafish samples were collected and lysed using ATP assay lysis buffer. Specifically, zebrafish samples were homogenized using a glass tissue homogenizer. Samples were then centrifuged to obtain supernatant for ATP assay. Sample protein concentrations were quantified using a BCA protein assay kit (Thermo Fisher Scientific, Cat#A55865), and ATP concentrations were normalized to protein levels.

[0078] like Figure 5As shown, the relative ATP levels of Gdap1-deficient zebrafish, skin fibroblasts derived from patients with peroneal muscular dystrophy 2K, and SH-SY5Y cells were lower than those of the control group, while BL-918 treatment significantly increased the reduced ATP levels.

[0079] Example 6

[0080] Assessment of the impact of BL-918 on mitochondrial bioenergy in an in vitro GDAP1-deficient model

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

[0082] Experimental grouping and experimental procedure:

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

[0084] Oxygen consumption rate (OCR) was assessed using an Agilent Seahorse XFe24 analyzer and the Seahorse XF cell mitochondrial stress assay. Skin fibroblasts and SH-SY5Y cells from each group were seeded into XF24V7 cell culture microplates. In the BL-918 treatment group, 5 hours after seeding, when cells were well adhered, medium containing BL-918 was added to achieve a final concentration of 5 μM. Mitochondrial stress was measured 24 hours post-treatment. After cell culture was 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 concentrations were quantified using a BCA protein assay kit (Thermo Fisher Scientific, Cat#A55865) and used for data normalization. OCR parameters were calculated using Agilent / Seahorse XF report generation software.

[0085] like Figure 6 As shown, compared with the control group, basal respiration and ATP production-related respiration were reduced in skin fibroblasts and GDAP1 knockdown SH-SY5Y cells from patients with peroneal muscular dystrophy 2K, although reserve respiration capacity was not affected. BL-918 treatment significantly improved the impaired mitochondrial bioenergetics caused by GDAP1 deficiency, as evidenced by a significant increase in the reduced basal respiration and ATP production-related respiration rates.

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

[0087] (1) This invention demonstrates for the first time that BL-918 can restore mitochondrial autophagy damage caused by GDAP1 deficiency;

[0088] (2) This invention is the first to demonstrate that BL-918 can significantly improve the motor deficits in a zebrafish model of GDAP1-related peroneal muscular atrophy;

[0089] (3) This invention is the first to demonstrate that BL-918 can significantly improve mitochondrial function in skin fibroblasts and SH-SY5Y cells derived from Gdap1-deficient zebrafish, patients with peroneal muscular dystrophy 2K.

[0090] Finally, it should be noted that the above description is merely 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of BL-918 in the preparation of a 2K drug for treating peroneal muscular dystrophy, characterized in that, The drug has one or more of the following effects: (1) Restore the damage to mitophagy caused by GDAP1 deficiency; (2) Improve the motor phenotype of a GDAP1-related zebrafish model of peroneal muscular atrophy; (3) Improve mitochondrial function in skin fibroblasts derived from Gdap1-deficient zebrafish, patients with peroneal muscular dystrophy 2K, and GDAP1 knockdown SH-SY5Y cells.

2. The application as described in claim 1, characterized in that, The drug is a composition.

3. The application as described in claim 2, characterized in that, The composition comprises the active ingredient of a drug and its pharmaceutically acceptable carrier or medium.

4. The application as described in claim 3, characterized in that, The active ingredient of the drug is BL-918 or a pharmaceutically acceptable salt thereof.

5. The application as described in claim 3, characterized in that, The dosage form of the composition is selected from one of the following: tablets, capsules, liquid preparations, creams, ointments, gels, nasal sprays, suppositories, finely dispersed powders or aerosols for inhalation, and sprays.

6. The application as described in claim 5, characterized in that, The solvent used in the liquid formulation is one of sterile water, water-propylene glycol solution, and ethylene glycol aqueous solution.

7. The application as described in claim 5, characterized in that, The liquid formulation includes active ingredients, Coloring agents, flavoring agents, stabilizers, and thickeners.

8. The application as described in claim 1, characterized in that, The drug is administered orally.

9. The application as described in claim 1, characterized in that, The aforementioned peroneal muscular atrophy 2K is a hereditary motor and sensory neuropathy caused by a mutation in the GDAP1 gene.

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