阅读说明：本技术 一种小分子化合物在制备帕金森病中药物的应用 (Application of small molecular compound in preparation of medicine for treating Parkinson's disease ) 是由 陈生弟 潘泓 徐进 丁健青 李红霞 于 2021-08-24 设计创作，主要内容包括：本发明涉及一种小分子化合物在制备帕金森病中药物的应用。本发明的小分子化合物ChemJ可以通过促进DJ-1的表达,降低帕金森病的氧化应激水平,而达到保护多巴胺能神经元的效果。因此,ChemJ可能被制作成一种新的帕金森病的疾病修饰治疗药物。(The invention relates to application of a small molecular compound in preparing a medicine for treating Parkinson's disease. The micromolecular compound ChemJ disclosed by the invention can be used for reducing the oxidative stress level of the Parkinson disease by promoting the expression of DJ-1, so that the effect of protecting dopaminergic neurons can be achieved. Therefore, ChemJ may be made as a new disease modifying therapeutic agent for parkinson's disease.)

1. The application of the small molecular compound in preparing the medicine for treating the Parkinson disease is characterized in that: the structural formula of the small molecule compound is as follows:

2. use according to claim 1, characterized in that: the target of the medicine is DJ-1.

3. Use according to claim 1, characterized in that: the medicine comprises small molecular compounds and pharmaceutically available auxiliary materials.

4. Use according to claim 3, characterized in that: the auxiliary materials comprise a filling agent, a bonding agent, a lubricating agent, a dispersing agent, a flow aid, a wetting agent, a disintegrating agent, a spice or a pigment.

5. Use according to claim 1, characterized in that: the dosage form of the medicine is oral preparation or injection.

6. Use according to claim 5, characterized in that: the oral preparation can be in the form of tablet, hard or soft capsule, lozenge, dripping pill, pellet, aqueous or oil suspension, emulsion, powder, granule, oral liquid or syrup.

7. Use according to claim 5, characterized in that: the injection is in the form of: sterile aqueous or oily solutions, sterile powders, liposomes, emulsions or microcapsules.

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of a small molecular compound in preparation of medicines for treating Parkinson's disease.

Background

Parkinson's Disease (PD) is the second most common neurodegenerative disease, the prevalence rate and age of which are positively correlated, and the clinical manifestations include motor symptoms such as resting tremor, bradykinesia, muscular rigidity, abnormal posture and gait, and non-motor symptoms such as hyposmia, autonomic dysfunction, sleep disorder, and mental disorder, and these symptoms affect the working ability and life quality of patients chronically and seriously. By 2016, the number of PD patients worldwide has exceeded 610 thousands, with 300 thousands in china. Especially in countries and regions where aging is progressing, the number of PD patients will continue to increase, which will cause a heavy social burden.

The main pathological feature of PD is the loss of dopaminergic neurons at the substantia nigra of the midbrain, with the formation of Lewy bodies. Most of the currently clinically used PD treatment medicines target dopamine pathways, can relieve clinical symptoms, but cannot prevent the progress of the disease course of PD, and even have obvious side effects.

(1) Levodopa. Supplementing the missing dopamine neurotransmitter level. Side effects: hypoefficacy, fluctuation of symptoms, dyskinesia, anorexia, tachycardia, ventricular premature beat, etc.

(2) Dopamine receptor agonists (Pramipexole, Ropinirole, Rotigotine, etc.). Enhancing the reactivity of neurons to dopamine to compensate for the loss or absence of dopaminergic function. Side effects: anorexia, dyspepsia, hallucinations, lethargy, compulsive behavior, etc.

(3) catechol-O-transferase (COMT) inhibitors (tolcapone, ntacapone, opiopone). Inhibit degradation of dopamine, and can be used in combination with levodopa. Side effects: aggravate the side effects of levodopa, liver damage, affect sleep, etc.

(4) Monoamine oxidase B inhibitors (Selegiline, rasagine, safinamide). Inhibition of dopamine degradation may have some neuroprotective effect (this is still subject to further clinical trials). Side effects: aggravate the side effects of levodopa, delirium, hallucinations, anxiety, insomnia, orthostatic hypotension, etc.

In addition, there have been several new drug development efforts for PD, which have focused mainly on drugs targeting SNCA and LRRK 2. However, PD is a heterogeneous disease and a drug directed to a single target may not solve the problem for all PD patients.

A strategy for new drugs targeting α -synuclein is to inhibit the deposition of α -synuclein to ameliorate the symptoms of PD. Specific methods include antibodies, vaccines, small molecule compounds, and the like. Improvement of PD motor symptoms by these drugs was observed in transgenic mouse models of PD, and corresponding clinical trials were in progress. However, this strategy also presents some problems: is the deposition of alpha-synuclein responsible for the root cause or concomitant phenomenon of PD? What form of alpha-synuclein was chosen as a therapeutic target? Since α -synuclein also has some physiological functions, do inhibition of α -synuclein produce side effects by interfering with its physiological functions? These problems require a more thorough understanding of the role of α -synuclein.

The strategy for new drugs targeting LRRK2 is to inhibit the kinase activity of LRRK 2. In a mouse model of PD, it was observed that LRRK2 inhibitors act to reduce alpha-synuclein deposition and neuroprotection, and corresponding clinical trials are in progress, however the current difficulty is the lack of methods for activity detection of LRRK2 and the difficulty in recruiting sufficient LRRK 2-associated PD patients.

Therefore, the development of new modified PD disease treatment drugs, especially drugs aiming at other targets, is still a current urgent task.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of the small molecular compound in preparing the medicine for treating the Parkinson's disease, wherein the target point of the small molecular compound is DJ-1, and the small molecular compound is expected to make up the limitation of the existing PD treatment medicine.

The invention provides an application of a small molecular compound in preparing a medicine for treating Parkinson's disease, wherein the structural formula of the small molecular compound is as follows:hereinafter referred to as chemJ.

The target of the medicine is DJ-1.

The medicine comprises small molecular compounds and pharmaceutically available auxiliary materials.

Further, the auxiliary materials comprise a filling agent, a bonding agent, a lubricating agent, a dispersing agent, a flow aid, a wetting agent, a disintegrating agent, a spice or a pigment.

The dosage form of the medicine is oral preparation or injection.

Further, the form of the oral preparation includes tablets, hard or soft capsules, troches, dripping pills, pellets, aqueous or oil suspensions, emulsions, powders, granules, oral liquids or syrups.

Further, the injection is in the form of: sterile aqueous or oily solutions, sterile powders, liposomes, emulsions or microcapsules.

Advantageous effects

The micromolecular compound ChemJ disclosed by the invention can be used for reducing the oxidative stress level of the Parkinson disease by promoting the expression of DJ-1, so that the effect of protecting dopaminergic neurons can be achieved. Therefore, ChemJ may be made as a new disease modifying therapeutic agent for parkinson's disease. In addition, because DJ-1 also plays a protective role in Inflammatory Bowel Disease (IBD) against oxidative stress and apoptosis inhibition, ChemJ could theoretically play a therapeutic role in improving IBD by increasing the expression of DJ-1in the intestinal tract.

Drawings

FIG. 1 shows a screening strategy for small molecule compounds.

FIG. 2 is a screening process of small molecule compounds.

FIG. 3 is a graph demonstrating the effect of small molecule compound ChemJ on DJ-1 expression; wherein A is a chemical structural formula of ChemJ; detecting the expression quantity of DJ-1 by using Western blot, taking alpha-tubulin as an internal reference, and using ChemJ with the concentration of 0.25-5 mu M to treat an SH-SY5Y cell line; c is treatment of primary mouse cerebral cortical neurons with ChemJ at concentrations of 0.1. mu.M to 2.5. mu.M. P <0.05, one-way anova-multiple comparison test.

FIG. 4 shows detection of superoxide anion (O)₂ ^-) With Reactive Oxygen Species (ROS) to determine the presence of small molecule compound ChemJ versus MPP⁺Intervention of oxidative stress levels in PD cell models treated with SH-SY 5Y; wherein, A is 1 μ M ChemJ treatment given for 24 hours after 6 hours of 0.5mM MPP + treatment of SH-SY5Y cells; b is useSH-SY5Y cells were treated with 1 μ M ChemJ for 24 hours and then treated with 0.5mM MPP + for 2 hours; ctr: without MPP addition⁺(ii) a DMSO, DMSO: DMSO solvent treatment at 0.1% concentration without compound; j: treatment with 1 μ M ChemJ. P<0.05，**P<0.01, one-way anova-multiple comparison test.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

8000 small molecule compounds used in the high-flux drug screening stage are all from the small molecule compound library of ChemDiv company. The ChemJ used was synthesized by MCE.

Example 1

In the early work, the expression regulation mechanism of DJ-1 was explored from the transcriptional level and the post-transcriptional level respectively [ Tan Y, Wu L, Li D, Liu X, Ding J, Chen S.methylation status of DJ-1in leukcyte DNA of Parkinson's disease activities. Transl neurogene.2016; 5:5, doi:10.1186/s40035-016-0052-6 ]; [ Chen Y, Gao C, Sun Q, Pan H, Huang P, Ding J, et al. MicroRNA-4639is a regulator of DJ-1expression and a potential early diagnostic marker for Parkinson's disease front Aging neurosci.2017; 9:1-9.doi:10.3389/fnagi.2017.00232 ]. On the basis of the above, the present example constructed a luciferase reporter system based on DJ-1 promoter activity to screen for small compounds that could promote the expression of DJ-1 (see FIG. 1).

The preparation method of the luciferase reporter system comprises the following steps:

(1) the DJ-1 promoter fragment is inserted in front of the luciferase gene of a luciferin reporter plasmid 'pLuc 0' (the plasmid is provided by professor of the institute of neuroscience, Chinese academy of sciences), so that the expression of the luciferase gene is regulated by the activity of the DJ-1 promoter. The plasmid has a Neo gene, so the plasmid has G418 resistance.

(2) This plasmid was transfected into SH-SY5Y cells (this cell line was purchased from the cell bank of the Central department) using lipofectamine2000 (purchased from ThermoFischer Co.) and a cell line stably expressing the above-mentioned report was obtained by G418 as a reporter system for drug screening.

(3) This cell line was inoculated into 384 well plates and 24 hours after treatment with a small molecule compound, a long half-life luciferase substrate, Steady-Glo (available from Premega corporation) was added to react with luciferase in the cells and emit light with an intensity reflecting the activity of the DJ-1 promoter. The luminescence intensity of each treatment group was measured using a microplate reader, and analyzed in combination with a negative control group (solvent treatment without compound).

8000 primary screening works of small molecular compounds which are not prepared into medicines are carried out in a domestic compound sample library, and 11 candidate compounds are obtained through secondary screening verification (see figure 2). In order to verify whether the compounds can effectively improve the expression of DJ-1, 8 available compounds are selected, SH-SY5Y cell lines are respectively treated, and the expression quantity of DJ-1 protein is detected by Westernblot. The effect of the ChemJ on promoting the expression of the DJ-1 protein is most obvious, and the expression amount of DJ-1 reaches about 2 times of that of a solvent control group when the ChemJ with the concentration of 1-2 mu M is treated (see figure 3B). After this time primary neurons from mice were treated with ChemJ, similar results were obtained, i.e. the expression of DJ-1 reached around 2-fold of that of the solvent control group at ChemJ treatment concentrations of 0.25 μ M to 0.5 μ M (see figure 3C).

In order to verify whether the ChemJ has a relieving effect on the excessive oxidative stress of PD, MPP is selected⁺SH-SY 5Y-stimulated PD oxidative stress cell model. The results show that the level of oxidative stress is relieved whether ChemJ treatment is given after molding or before molding (see fig. 4).

MPP currently in Parkinson's disease⁺In the cell model, the present invention treated SH-SY5Y cell line with a culture medium containing ChemJ at 1. mu.M or other concentration. Wherein, DMSO is used as solvent to prepare 10mM solution, on the basis of which DMSO can be continuously diluted to 1mM solution or other concentration solution. Thereafter, the corresponding cells can be usedThe culture medium (such as DMEM + 10% fetal calf serum) is diluted 1000 times to prepare a ChemJ culture medium with a final concentration of 1 μ M or other concentrations, and the cells are treated by changing the culture medium.

These results suggest that small molecule compound ChemJ can increase the expression of DJ-1 protein and alleviate excessive oxidative stress of PD.