TRPML1 specific micromolecule agonist ML-SA5 and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a TRPML1 specific small molecule agonist ML-SA5 and application thereof. The structural formula of the TRPML1 specific small molecule agonist ML-SA5 is as shown in the specification. The TRPML1 specific micromolecule agonist ML-SA5 has the beneficial effects that the TRPML1 specific micromolecule agonist ML-SA5 can be used for remarkably reducing the central nervous inflammation level of a depression model mouse; the mouse neuron excitability of the depression model is improved; depression-like behaviors of depression model mice are improved; the brain function of a depression model mouse is comprehensively protected; the TRPML1 specific micromolecule agonist ML-SA5 provided by the invention provides a new medicine for treating depression, and has good market value and clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a TRPML1 specific small molecule agonist ML-SA5 and application thereof. BACKGROUND

[0002] Major Depressive Disorder is a mental disorder disease that plagues modern humans, and its main feature is significant and persistent low mood. Some patients may have self-injury, suicidal behavior, and even may be accompanied by delusions, hallucinations and other psychotic symptoms. The causes of depression are very complex. In the past, it was usually believed to be caused by neurotransmitter imbalance, neuron function decline or psychosocial factors. However, recent studies have found that the levels of inflammatory factors in most patients with depression, especially in blood and cerebrospinal fluid, are significantly elevated. This suggests that depression may not only be a "mood" problem, but also be closely related to the body's immune system response. Neuroinflammation refers to a series of reactions triggered by the activation of microglial cells, astrocytes and other non-neuronal cells in the central nervous system under the stimulation of infection, toxins, stress or metabolic disorders, and the release of inflammatory factors (such as TNF-alpha, IL-1 beta). Many studies have shown that neuroinflammation not only participates in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, but also is closely related to the occurrence of depression. Therefore, controlling or reversing neuroinflammation may be a potential path to improve depressive symptoms. SUMMARY

[0003] The application provides a TRPML1 specific small molecule agonist ML-SA5 and application thereof.

[0004] The first aspect of the application provides a TRPML1 specific small molecule agonist ML-SA5, and the structural formula of the TRPML1 specific small molecule agonist ML-SA5 is as follows:

[0005]

[0006] The TRPML1 specific small molecule agonist ML-SA5 described in the application can significantly reduce the central neuroinflammation level of a depression model mouse, improve the neuron excitability of a depression model mouse, improve the depression-like behavior of a depression model mouse, and has a comprehensive protective effect on the brain function of a depression model mouse.

[0007] The second aspect of the application provides application of the TRPML1 specific small molecule agonist ML-SA5 described in the first aspect of the application in the preparation of a drug for treating depression.

[0008] The third aspect of the present application provides a medicament for treating depression, comprising the TRPML1 specific small molecule agonist ML-SA5 of the first aspect of the present application and a pharmaceutically acceptable adjuvant thereof.

[0009] According to some embodiments of the medicament for treating depression of the present application, the effective content of the TRPML1 specific small molecule agonist ML-SA5 in the medicament for treating depression is 0.1-1 mg / kg.

[0010] According to some embodiments of the medicament for treating depression of the present application, the effective content of the TRPML1 specific small molecule agonist ML-SA5 in the medicament for treating depression is 0.3 mg / kg.

[0011] According to some embodiments of the medicament for treating depression of the present application, the dosage form of the medicament for treating depression comprises injection, tablet, capsule, granule, aerosol or oral liquid agent.

[0012] The beneficial effects of the present application include that the TRPML1 specific small molecule agonist ML-SA5 of the present application can significantly reduce the central nervous inflammation level of depression model mice, improve the neuronal excitability of depression model mice, improve the depression-like behavior of depression model mice, and has a comprehensive protective effect on the brain function of depression model mice.

[0013] The TRPML1 specific small molecule agonist ML-SA5 of the present application provides a new medicament for treating depression, and has good market value and clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Preparation of chronic restraint stress depression model mice and time mode diagram of ML-SA5 administration intervention;

[0015] Figure 2a Depression model mouse cerebral prefrontal cortex (mPFC) section microglia (Iba-1; red) and interleukin 1-beta (IL-1β; green) staining diagram after administration of the TRPML1 specific small molecule agonist ML-SA5 of the present application;

[0016] Figure 2b Statistical diagram of interleukin 1-beta (IL-1β) level in depression model mouse cerebral prefrontal cortex (mPFC) section microglia (Iba-1; red) after administration of the TRPML1 specific small molecule agonist ML-SA5 of the present application, to evaluate the central inflammation (IL-1β) level of the mice;

[0017] Figure 2cThe Iba-1 (red) staining and 3D reconstruction of the single microglia cells in the prefrontal cortex (mPFC) section of the depressive model mice after administration of the TRPML1 specific small molecule agonist ML-SA5 described in the present application, comprehensive evaluation of the central nervous inflammation level of the mice;

[0018] Figure 2d The quantitative analysis of the microglia morphology (branch point number and total branch length) in the prefrontal cortex (mPFC) section of the depressive model mice after administration of the TRPML1 specific small molecule agonist ML-SA5 described in the present application, comprehensive evaluation of the central nervous inflammation level of the mice (the fewer branches and shorter length, the higher the activation state of microglia cells) ;

[0019] Figure 3a The prefrontal cortex (mPFC) calcium imaging heat map of the depressive model mice after administration of the TRPML1 specific small molecule agonist ML-SA5 described in the present application;

[0020] Figure 3b The prefrontal cortex (mPFC) calcium imaging statistical chart of the depressive model mice after administration of the TRPML1 specific small molecule agonist ML-SA5 described in the present application, evaluation of the neuronal excitability of the depressive model mice (the stronger the calcium signal, the higher the neuronal excitability) ;

[0021] Figure 4a The sugar water preference index analysis chart of the depressive model mice after administration of the TRPML1 specific small molecule agonist ML-SA5 described in the present application, evaluation of the depressive-like behavior change (the lower the preference, the higher the depressive mood) ;

[0022] Figure 4b The tail suspension test result analysis chart of the depressive model mice after administration of the TRPML1 specific small molecule agonist ML-SA5 described in the present application, evaluation of the depressive-like behavior change (the longer the immobility time, the higher the depressive mood) ;

[0023] Figure 4c The movement trajectory chart and total distance statistics of the depressive model mice in the open field after administration of the TRPML1 specific small molecule agonist ML-SA5 described in the present application, evaluation of the depressive-like behavior change (the shorter the total distance, the higher the depressive mood). DETAILED DESCRIPTION

[0024] The embodiments of the present application are described in detail below, and the examples of the embodiments and the drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0025] In this disclosure, the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" mean that a particular feature, structure, material, or characteristic is included in at least one embodiment or example of the present disclosure. Illustrative expressions of the above terms do not necessarily refer to the same embodiment or example in this specification. Moreover, the particular features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples. In addition, different embodiments or examples described in this specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0026] The present application provides a TRPML1 specific small molecule agonist ML-SA5, and the structural formula of the TRPML1 specific small molecule agonist ML-SA5 is as follows:

[0027]

[0028] The TRPML1 specific small molecule agonist ML-SA5 can significantly reduce the central nervous inflammation level of a depression model mouse, improve the neuronal excitability of a depression model mouse, improve the depression-like behavior of a depression model mouse, and has a comprehensive protective effect on the brain function of a depression model mouse.

[0029] The present application also provides a use of the TRPML1 specific small molecule agonist ML-SA5 of the first aspect of the present application in the preparation of a drug for treating depression.

[0030] The present application also provides a drug for treating depression, which comprises the TRPML1 specific small molecule agonist ML-SA5 of the first aspect of the present application and a pharmaceutically acceptable adjuvant thereof. The TRPML1 specific small molecule agonist ML-SA5 provides a new drug for treating depression, and has good market value and clinical application prospect.

[0031] In some embodiments of the present application, the effective content of the TRPML1 specific small molecule agonist ML-SA5 in the drug for treating depression is 0.1-1 mg / kg, such as 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1.0 mg / kg, etc.

[0032] In some embodiments of the present application, the effective content of the TRPML1 specific small molecule agonist ML-SA5 in the drug for treating depression is 0.3 mg / kg.

[0033] In some embodiments of the present application, the dosage form of the drug for treating depression includes injections, tablets, capsules, granules, aerosols or oral liquid agents.

[0034] The technical solutions of the present application are further described below in conjunction with specific embodiments.

[0035] Example 1

[0036] A TRPML1-specific small molecule agonist ML-SA5, the structural formula of which is as follows:

[0037]

[0038] Microglia-released inflammatory factors such as IL-1β and TNF-α mediate neuroinflammation, which is one of the main pathological bases of depression, but the mechanism of excessive production of these inflammatory factors has not been clear. The inventors have proved that in intestinal macrophages, lysosomal ion channel TRPML1 mediates the release of iron ions in lysosomes to the cytoplasm, thereby inhibiting the activation of NF-κB signaling pathway and limiting the excessive production of IL-1β in macrophages. Then, does TRPML1 also limit the excessive production of IL-1β in the central nervous system "macrophages", i.e. microglia, thereby participating in the pathological process of depression? Through experiments, the inventors found that the use of TRPML1-specific small molecule agonist ML-SA5 to activate the TRPML1 channel can significantly reduce the inflammation level (IL-1β) of the prefrontal cortex (mPFC; the most important brain region related to depression) of the depression model mice; inhibit the excessive activation of mPFC microglia; improve the excitability of mPFC neurons; and significantly improve the depression-like behavior of the model mice. It is thus speculated that the down-regulation of TRPML1 channel is one of the key factors leading to neuroinflammation in depression. This study will provide a solid theoretical basis for more accurately understanding the pathological mechanism of neuroinflammation in depression, and provide a new target—TRPML1 for the clinical treatment of depression. The following is the experiment of the present application to verify the therapeutic effect of TRPML1-specific small molecule agonist ML-SA5 on depression model mice.

[0039] 1. Establishment of depression model mice and intervention of ML-SA5

[0040] The intervention mode diagram of ML-SA5 for chronic restraint stress (CRS) depression model mice / sham operation group is as shown in Figure 1

[0041] 1.1 Materials SPF C57BL / 6J mice

[0042] ​1.2 Animal model Before the formal experiment, C57BL / 6J mice were placed in the behavior room for 1 week, 12-hour day-night cycle, free drinking and eating. After the initial screening of the mice by weight, sugar water preference, open field test, etc., 20 mice with similar behavior scores were selected and randomly divided into normal control (Sham) group and CRS group, 10 mice in each group. The CRS group mice were placed in the restraint tube made of a 50ml centrifuge tube (the centrifuge tube was sterilized in advance to make air holes, which were scattered throughout the tube body, and a small hole was made in the center of the cover, the mouse tail was inserted into it and exposed to the air, and the mice were restrained for 28 days, 4 hours a day, and the CRS group mice were fasted and watered during the restraint period. The Sham group mice were free to drink and eat. The mice were grouped as follows:

[0043] Sham+Saline group: Sham operation group control mice were given physiological saline 200 microliters intraperitoneally every two days;

[0044] Sham+ML-SA5 group: Sham operation group control mice were given ML-SA5 (0.3mg / kg) intraperitoneally every two days;

[0045] CRS+Saline group: modeling group; given physiological saline 200 microliters intraperitoneally every two days;

[0046] CRS+ML-SA5 group: modeling group mice were given ML-SA5 (0.3mg / kg) intraperitoneally every two days.

[0047] 2, The effect of TRPML1 specific small molecule agonist ML-SA5 administration on IL-1β level and microglial cell activation in mPFC brain region of depression model mice.

[0048] 2.1 Measurement method:

[0049] After modeling for 28 days, the control group (Sham+Saline), modeling group (CRS+Saline), modeling combined with ML-SA5 group (CRS+ML-SA5), and control combined with ML-SA5 (Sham+ML-SA5) mice were anesthetized, and the left ventricle was punctured and perfused with PBS buffer.

[0050] Complete brain mPFC tissue was collected. First, the tissue was fixed overnight in paraformaldehyde, dehydrated with 30% sucrose, and then frozen at -20°C using embedding medium. The frozen tissue blocks were trimmed and pre-cut to the appropriate thickness according to different tissue types; the thickness of brain tissue patches was 15-25 μm. The sections were air-dried at room temperature before subsequent staining. Before staining, antigen retrieval was performed: the samples were thoroughly soaked in 0.01M citrate buffer (pH 6.0), heated in a microwave oven on low heat, maintaining a gentle boil for 5 minutes. The sections were then permeated with 0.5% Triton X-100 (prepared with 1xPBS) at room temperature for 20 minutes and then blocked. Incubation with IL-1β and Iba-1 primary antibodies was then performed overnight at 4°C. Incubation with fluorescent secondary antibody at room temperature for 2 hours was then performed. Cell nuclei were stained with DAPI.

[0051] Under a microscope, Iba-1 appeared red (third row), marking microglia; IL-1β appeared green (second row); and DAPI appeared blue, marking cell nuclei. The results are as follows: Figure 2a and Figure 2b As shown.

[0052] from Figure 2a As can be seen, compared with the model group (CRS+Saline; third column), the ML-SA5 administration group (CRS+ML-SA5; fourth column) significantly reduced the IL-1β (green) level in the mPFC microglia (Iba-1; red) of the model mice, indicating that ML-SA5 administration can significantly reduce the degree of inflammation in the mPFC brain region of the depressed model mice. The sham-operated group (Sham+Saline; first column) and the sham-operated + ML-SA5 group (Sham+ML-SA5; second column) served as negative controls for the model mice, and the IL-1β level in the mouse mPFC tissue was extremely low. The scale bar in the staining images is 20 micrometers. Figure 2b Statistical graphs showing the relative IL-1β levels in microglia of the mPFC brain region of mice in each group, N=3-6.

[0053] from Figure 2a Typical microglia (Iba-1 positive; red) were selected from the captured images for 3D reconstruction, followed by quantitative analysis of microglia morphology, including the number and length of microglia branches. Results are as follows: Figure 2c and Figure 2d As shown.

[0054] from Figure 2cAs can be seen, compared with the model group (CRS+Saline; third column), the ML-SA5 administration group (CRS+ML-SA5; fourth column) significantly improved the amoeboid state of microglia in the mPFC tissue of model mice (the morphology of activated microglia changed from quiescent branched to activated amoeboid, characterized by enlarged cell bodies and shortened or disappeared cell processes), indicating that ML-SA5 administration can significantly reduce the degree of inflammation in the mPFC brain region of depressed model mice. The sham-operated group (Sham+Saline; first column) and the sham-operated + ML-SA5 group (Sham+ML-SA5; second column) served as negative controls for the model mice, and the microglia in the mouse mPFC tissue were in a quiescent branched state. The scale bar of the stained images is 10 micrometers. Figure 2d Quantitative analysis of microglial morphology (number of branching points and total branch length) in mPFC sections of mice in each group was performed. The ML-SA5-treated group showed increased microglial branch number and shorter branch length, indicating that microglial activation was inhibited by ML-SA5 intervention. N = 3–6.

[0055] 3. Effects of ML-SA5 administration on the excitability of glutamatergic neurons in the mPFC region of a mouse model of depression.

[0056] After injecting the calcium ion indicator GCaMP6, which labels glutamatergic neurons, into the mPFC brain region of mice, various models were established. Twenty-eight days after modeling, mice in the control group (Sham+Saline), model group (CRS+Saline), model combined with ML-SA5 group (CRS+ML-SA5), and control combined with ML-SA5 group (Sham+ML-SA5) underwent in vivo calcium imaging tests. The calcium signal intensity of glutamatergic neurons in the mPFC brain region of each group was analyzed. The results are as follows: Figure 3a As shown.

[0057] from Figure 3a As can be seen, compared with the model group (CRS+Saline; lower left figure), the ML-SA5 administration group (CRS+ML-SA5; lower right figure) significantly increased the calcium ion signal in the mPFC region of the model mice (the redder the color, the stronger the calcium signal), indicating that ML-SA5 administration can significantly increase the excitability of glutamatergic neurons in the mPFC region of the depression model mice. The sham-operated group (Sham+Saline; upper left figure) and the sham-operated + ML-SA5 group (Sham+ML-SA5; upper right figure) served as negative controls for the model mice, and the excitability of glutamatergic neurons in the mPFC region of these mice was at a high level. Figure 3b Statistical graph of calcium ion signal levels in glutamatergic neurons in the mPFC brain region of mice in each group. N=13.

[0058] 4. Effects of administration of the TRPML1-specific small molecule agonist ML-SA5 on depressive-like behavior in a mouse model of depression.

[0059] Twenty-eight days after modeling, the mice in the control group (Sham+Saline), the model group (CRS+Saline), the modeling combined with ML-SA5 group (CRS+ML-SA5), and the control combined with ML-SA5 group (Sham+ML-SA5) underwent behavioral tests.

[0060] The sugar solution preference test (SPT) used 50ml centrifuge tubes to prepare test drinking (sugar) solution bottles, each containing 25ml of 1% sucrose solution or pure water. Rats had free access to sugar solution and food throughout the test, lasting 4 days. Before the test, each rat was individually acclimatized in its own cage for 48 hours, with two bottles of water provided. To prevent positional preference in the drinking (sugar) solution, the positions of pure water and sugar solution were exchanged at 24-hour intervals during both the pre-acclimatization and the actual test. The weight of the bottles before and after the test was used to calculate the sugar solution, pure water, and total liquid consumption, as well as the percentage of sugar solution preference: Sugar solution preference percentage = Sugar solution consumption (g) / (Sugar solution consumption + Pure water consumption) × 100% Figure 4a ).

[0061] In the tail suspension test (TST), mice are gently removed from their cages and their tails are secured as quickly as possible, avoiding any unnecessary stress or pressure. Specifically, medical tape is used to attach the tail 1 cm from the tip, suspending the animal's tail from the suspension rod. The height between the tail tip and the ground is approximately 30 cm, placing the mouse in a head-down position. The camera lens must be pre-adjusted to be horizontal and aimed at the mouse's body to ensure a complete field of view during recording. The experiment requires a total of 6 minutes of video recording, documenting the animal's behavior during the experiment, but including the last 4 minutes of immobility. Figure 4b ).

[0062] At the start of the Open Field Test (OPT) experiment, the mouse is gently placed in the center or a corner of the open field apparatus.

[0063] Behavioral observation: Begin recording the behavior of rats in an open field. The observation time is usually 5 to 10 minutes, which is the standard duration in most studies.

[0064] Behavioral indicators: Record the rats’ behavior, including time spent in the center and edge of the open field, distance traveled, cleaning behavior, standing behavior, etc.

[0065] Data recording: The behavior of rats was recorded and analyzed using cameras and motion tracking software to improve the accuracy and objectivity of the data. Figure 4c ).

[0066] Data Analysis

[0067] Behavioral analysis: Based on video recordings and motion tracking software data, the behavioral patterns of rats were analyzed, including movement trajectories, lingering areas, etc. (trajectory diagram; 4c).

[0068] As shown in the figure, ML-SA5 administration (CRS+ML-SA5) significantly improved depression-like behaviors in a mouse model of depression (CRS+Saline), including increasing sucrose preference (SPT) in the mouse model of depression. Figure 4a ), and reduce immobility time (TST) in a mouse model of depression. Figure 4b ) and increase the open field distance (OPT) of mice in a depression model. Figure 4c N = 17-20.

[0069] The above experimental results demonstrate that the TRPML1-specific small molecule agonist ML-SA5 can significantly reduce the level of central nervous system inflammation in mice with depression; improve neuronal excitability in mice with depression; improve depressive-like behavior in mice with depression; and provide comprehensive protection for brain function in mice with depression. The application of the TRPML1-specific small molecule agonist ML-SA5 described in this application to the preparation of drugs for treating depression provides a new drug for the treatment of depression patients, and has good market value and clinical application prospects.

[0070] Although the above embodiments have been shown and described, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made to the above embodiments by those skilled in the art are within the protection scope of the present invention.

Claims

1. A TRPML1-specific small molecule agonist, ML-SA5, characterized in that, The structural formula of the TRPML1-specific small molecule agonist ML-SA5 is shown below:

2. The use of the TRPML1-specific small molecule agonist ML-SA5 as described in claim 1 in the preparation of a drug for treating depression.

3. A drug for treating depression, characterized in that, Includes the TRPML1-specific small molecule agonist ML-SA5 as described in claim 1 and its pharmaceutically acceptable excipients.

4. The drug for treating depression according to claim 3, characterized in that, The effective content of the TRPML1-specific small molecule agonist ML-SA5 in the aforementioned drug for treating depression is 0.1-1 mg / kg.

5. The medicament for treating depression according to claim 3, characterized in that, The effective content of the TRPML1-specific small molecule agonist ML-SA5 in the aforementioned drug for treating depression is 0.3 mg / kg.

6. The medicament for treating depression according to claim 3, characterized in that, The dosage forms of the medication for treating depression include injections, tablets, capsules, granules, aerosols, or oral liquids.

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