CN111249281A

CN111249281A - Novel application of TRPML1 specific small molecule inhibitor ML-SI3

Info

Publication number: CN111249281A
Application number: CN202010226543.0A
Authority: CN
Inventors: 王午阳; 张鹏
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2020-03-26
Filing date: 2020-03-26
Publication date: 2020-06-09
Anticipated expiration: 2040-03-26
Also published as: CN111249281B

Abstract

The application discloses a new application of a TRPML1 specific small molecule inhibitor ML-SI3, and an application of the inhibitor in preparing a medicament for preventing and treating Parkinson's disease. The invention has the advantages of providing a new medicine for preventing and treating the Parkinson disease and having good market value and clinical application prospect.

Description

Novel application of TRPML1 specific small molecule inhibitor ML-SI3

Technical Field

The application relates to the technical field of medicines, in particular to a new application of a TRPML1 specific small molecule inhibitor ML-SI 3.

Background

Parkinson's Disease (PD) is a common nervous system degenerative disease, and is common in the elderly, with the average age of about 60 years, and the onset of juvenile Parkinson's disease below 40 years being rare. The prevalence rate of PD in people over 65 years old in China is about 1.7%. Most parkinson's disease patients are sporadic cases, with less than 10% of patients having a family history. The most prominent pathological change of parkinson's disease is the degenerative death of mesocerebral Dopaminergic (DA) neurons, which causes a marked reduction in striatal DA content and causes disease. At present, common drugs are anticholinergic drugs, amantadine drugs, MAO-B inhibitors, DR agonists, and the like.

At present, no literature reports that a TRPML1 specific small molecule inhibitor ML-SI3 can act on the treatment of Parkinson's disease.

Disclosure of Invention

The main object of the present application is to provide a novel use of TRPML 1-specific small molecule inhibitor ML-SI3 to solve the problems in the related art.

To achieve the above objects, in a first aspect, the present application provides a new use of TRPML 1-specific small molecule inhibitor ML-SI 3.

The new use of TRPML1 specific small molecule inhibitor ML-SI3 according to the present application: the application of TRPML1 specific small molecule inhibitor ML-SI3 in preparing medicine for preventing and treating Parkinson's disease.

Further, when the inhibitor is used for preparing a medicament for preventing and treating Parkinson's disease, the inhibitor is found to improve the loss of dopaminergic neurons in substantia nigra.

On the other hand, the concentration of the inhibitor is 0.15mg/kg, which is found to be the best for preventing and treating Parkinson's disease.

By combining the technical scheme, a new medicine is provided for preventing and treating the Parkinson's disease, and the medicine has good market value and clinical application prospect.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, serve to provide a further understanding of the application and to enable other features, objects, and advantages of the application to be more apparent. The drawings and their description illustrate the embodiments of the invention and do not limit it. In the drawings:

FIG. 1 is a chemical structural diagram of a TRPML1 specific small molecule inhibitor ML-SI3 of the present invention;

FIG. 2 shows the preparation and administration time of Parkinson's mice;

FIG. 3 shows that ML-SI3 combined administration significantly improves the pole-climbing dyskinesia of Parkinson model mice;

FIG. 4 shows that ML-SI3 combination significantly improves rotarod dyskinesia in Parkinson's model mice;

FIG. 5 shows that ML-SI3 combined administration significantly improved the field trial dyskinesia in Parkinson's model mice;

FIG. 6 is a fluorescence detection graph of ML-SI3 combination administered to significantly improve dopaminergic neuron loss in mice;

FIG. 7 is a fluorescence detection analysis chart of the lack of dopaminergic neurons in mice significantly improved by the combined administration of ML-SI 3.

Detailed Description

In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data so used may be interchanged under appropriate circumstances such that embodiments of the application described herein may be used. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

In this application, the terms "upper", "lower", "left", "right", "front", "rear", "top", "bottom", "inner", "outer", "middle", "vertical", "horizontal", "lateral", "longitudinal", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings. These terms are used primarily to better describe the present application and its embodiments, and are not intended to limit the indicated devices, components or elements to a particular orientation or to be constructed and operated in a particular orientation.

Moreover, some of the above terms may be used to indicate other meanings besides the orientation or positional relationship, for example, the term "on" may also be used to indicate some kind of attachment or connection relationship in some cases. The specific meaning of these terms in this application will be understood by those of ordinary skill in the art as appropriate.

In addition, the term "plurality" shall mean two as well as more than two.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.

The application discloses a new application of a TRPML1 specific small molecule inhibitor ML-SI3 and an application of the inhibitor in preparing medicines for preventing and treating Parkinson's disease. Particularly, when the inhibitor is used for preparing a medicine for preventing and treating the Parkinson's disease, the inhibitor can improve the deficiency of dopaminergic neurons in substantia nigra. The optimal concentration of the inhibitor is 0.15 mg/kg.

Our previous studies indicate that the lysosomal cation channel TRPML1 is a specific target for the regulation of autophagy, and that autophagy activity can be specifically regulated by altering the activity of the TRPML1 channel using agonists or inhibitors of TRPML 1. And a defect in autophagy may be one of the factors of neurodegenerative change in parkinson's disease. Therefore, the following is an experiment of the present application to demonstrate that the specific small molecule inhibitor of TRPML1, ML-SI3 (see figure 1 for its chemical structure), has preventive and therapeutic effects on parkinson's disease:

a Parkinson mouse model was first prepared using C57B/L mice, and the total was divided into 4 groups, i.e., a control group (control), a 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) group [4], an MPTP-associated ML-SI3 group, and an ML-SI3 group. The intervention method was as follows (fig. 2):

the behavioural training was started three days before the start of the experiment for 3 days for each group of mice. Mice were given intraperitoneal injections (ip.) of PBS, 1-Methyl-4- (2' -methylphenenyl) -1,2,3,6-tetrahydropyridine (MPTP; 30mg/kg), ML-SI3(0.15mg/kg) for 5 days in the control group, MPTP group, and ML-SI3 group, respectively. The MPTP (30mg/kg) in combination with ML-SI3(0.15mg/kg) group was administered to mice ML-SI3 as a pretreatment (0.15mg/kg) daily 2 days earlier, followed by intraperitoneal injection simultaneously with MPTP for 5 days. The mice of each group were subjected to behavioral and immunofluorescence tests on

days

6 and 7, respectively. The behavioral tests included pole climbing (fig. 3), rod rotation (fig. 4), and open field (fig. 5) tests to test the motor nerve function of mice. After the behavioral testing, the mouse substantia nigra is taken out, and the dopaminergic neurons in the substantia nigra region are respectively detected by an immunofluorescence experiment (fig. 6 and 7).

Method used in the climbing rod experiment (shown in the results of fig. 3): fixing a small foam ball with the diameter of 2.5cm on the top end of a wood pole with the length of 60cm and the thickness of 1cm, then placing a mouse on the long-pole ball, recording the time that the head is turned upwards to be downwards, and recording the time that the mouse climbs to the bottom of the pole; we used the method of pole climbing experiments to evaluate the motor function of each group of mice. The time for the mice of the MPTP administration group to climb to the bottom of the pole is obviously longer than that of the control group (P <0.001), which indicates that the mice dyskinesia is caused by MPTP intervention and the preparation of the mouse model of Parkinson disease is successful. When MPTP is combined with ML-SI3 to intervene, the time for the mouse to climb to the bottom of the pole is obviously shortened compared with that of the MPTP group (P <0.01), which shows that the dyskinesia of the Parkinson disease model mouse of the combined ML-SI3 group is effectively improved. The mice in the ML-SI3 independent administration group showed no significant change in the time to climb to the bottom of the pole compared with the control group.

Method used in the rotarod experiment (results see FIG. 4): the test was carried out using an XR1514 model rat and mouse rotary rod fatigue tester (purchased from Shanghai Xin soft information technology Co., Ltd.) at a rotation speed of 24r/min for 5 min. The time of movement of the rats on the rotarod was recorded and 3 tests were averaged. Rats were subjected to rotarod training 3 times prior to model creation. We evaluated the motor capacity of each group of mice using a rotarod experiment. The continuous movement time of the mice in the MPTP administration group on the rotating rod before falling is obviously shorter than that of a control group (P <0.001), which indicates that the mice dyskinesia is caused by MPTP intervention, and the preparation of a Parkinson disease mouse model is successful. When MPTP and ML-SI3 are combined to intervene, the continuous movement time of the mice on the rotating rod is obviously increased compared with that of MPTP group mice (P <0.001), which shows that the dyskinesia of ML-SI3 group Parkinson disease model mice is effectively improved. The ML-SI3 single administration group mice continued to move on the rotating rod for a period of time which did not significantly change from the control group.

Method used in the mine field experiments (results see fig. 5): detection was performed using an XR-XZ301 model 4-channel open field video analysis system and high throughput animal behavioural video analysis system Super Maze + software (both available from shanghai soft information technology ltd). The test time is 3min, and the movement distance and the resting time of the rat are mainly recorded. Figure 5 is a method of assessing motor function in groups of mice using a mine site experimental approach. After MPTP intervention, the moving distance and the moving speed of the mouse in a mine field are obviously inferior to those of a control group (P <0.001), which indicates that MPTP induces the dyskinesia of the mouse and the preparation of the Parkinson disease model is successful. When ML-SI3 is combined, the moving distance and moving speed of the mouse in the mine are obviously improved compared with those of MPTP group (P < 0.001). Compared with the control group, the ML-SI3 independent intervention group has no obvious change in the movement distance and speed in the mine field.

Impairment of nigral dopaminergic neurons: preparing brain tablets: perfusion, fixation and slicing: mice were anesthetized and 4% paraformethylene was perfused through the heart, brains were fixed, equilibrated with 20% and 30% sucrose in succession, sectioned with a cryomicrotome to a thickness of 12um, and brain slices containing a substantia nigra nucleus were selected for immunological staining.

Immunofluorescence: the brain slices were stained with a rabbit anti-Tyrosine Hydroxylase (TH) antibody (purchased from Sigma) and a fluorescent secondary antibody. The main experimental steps are as follows: washing the slices with 0.0lmol/LPBS for 3 times, each time for l0 min; incubating PBS buffer containing 5% sheep serum in the kit for 2h at room temperature; rabbit anti-TH antibody (1:1000)40C incubation for 24 h; 0.0lmo1/LPBS film washing for 3 times, each time l0 min; incubating the secondary antibody for 2h at room temperature; washing with 0.0lmol/LPBS for 3 times, l0min each time; and (5) carrying out fluorescence microscope shooting, and counting the number of green neurons for statistics.

FIGS. 6 and 7 We collected the substantia nigra from each group of mice after behavioral assessment, and performed the immunofluorescence detection (green) of tyrosine hydroxylase (dopaminergic neuron marker), and observed the loss of dopaminergic neurons in the substantia nigra of each group of mice. Compared with the control group, the dopaminergic neurons in the substantia nigra of the mice of the MPTP administration group show a green marked dopaminergic neuron deletion condition (P <0.001), which indicates that the mice of the Parkinson disease model are successfully prepared. After the combination of ML-SI3, the dopaminergic neuron deletion condition is improved to a certain extent (P is less than 0.001) compared with the MPTP group, the green marked dopaminergic neuron is obviously increased compared with the MPTP group, and the result shows that the dopaminergic neuron deletion phenomenon of a mouse in a Parkinson disease model can be obviously improved by ML-SI3 administration.

Compared with the dyskinesia of Parkinson's disease model mice in the MPTP group, the dyskinesia of the mice in the group which is combined with the MPTP and ML-SI3 is obviously improved (figure 3-figure 5). Meanwhile, we detected dopaminergic neurons in the substantia nigra region by using an immunofluorescence method, and found that the loss of dopaminergic neurons in the ML-SI3 administration group is significantly better than that in the MPTP group (FIGS. 6 and 7).

The above experimental results prove that: ML-SI3 small molecular compound can obviously improve various dyskinesias and nigra dopaminergic neuron deletion conditions of a Parkinson disease model mouse. The invention provides a new medicine for preventing and treating the Parkinson's disease, and has better market value and clinical application prospect.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

A new use of TRPML1 specific small molecule inhibitor ML-SI3, characterized in that the inhibitor is used for preparing the medicine for preventing and treating Parkinson's disease.
2. The use according to claim 1, wherein the inhibitor is capable of ameliorating the loss of dopaminergic neurons of the substantia nigra when used for the preparation of a medicament for the prevention and treatment of parkinson's disease.
3. Use according to claim 1, wherein the inhibitor is present at a concentration of 0.15 mg/kg.