Application of Compound C108 in preparation of medicine for treating autism spectrum disorder

Abstract

The invention discloses application of Compound C108 in preparation of a medicine for treating autism spectrum disorder, and belongs to the field of biomedicine. Experimental research proves that SGs is a key factor participating in formation of autism caused by genetic mutation and environmental factors, and Compund C108 can inhibit the content of SGs by intervening G3BP2 protein expression, and finally alleviate social behaviors of autism. Therefore, the Compound C108 can be used as a novel compound for preparing the medicine and / or the health care product for resisting the autism social disorder.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to the application of Compound C108 in the preparation of a drug for treating autism spectrum disorders. Background Art

[0002] Autism spectrum disorder (ASD) is a neurodevelopmental psychiatric disorder characterized by poor social skills and repetitive stereotyped behaviors, often accompanied by language communication disorders and cognitive deficits. The number of ASD patients is increasing year by year, placing a great burden on families and society. However, ASD has an insidious onset and its pathogenesis is not fully understood. Existing treatments are mainly based on rehabilitation training, which has very limited effects. Therefore, research on new and effective methods for treating ASD is of great scientific and clinical value.

[0003] According to relevant literature reports, stress granules (SGs) are involved in the formation of various neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis, but there are no reports on the relationship between SGs and ASD.

[0004] Compound C108, molecular formula C 15 H 14 N2O3, with a molecular weight of 270.28, is a white or off-white powder-like reagent that is easily soluble in DMSO. It can significantly reduce the overexpression of G3BP2 (a key factor in the formation of SGs) and has been used to inhibit the metastasis of esophageal squamous cell carcinoma (ESCC) cells. There are currently no reports on the use of Compound C108 in alleviating social disorders in autism. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide the use of Compound C108 in the preparation of drugs for treating autism spectrum disorders.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention discloses the use of Compound C108 in the preparation of a drug for treating autism spectrum disorders.

[0008] Preferably, Compound C108 is a drug that reduces the content of SGs.

[0009] Preferably, Compound C108 is a drug that inhibits the expression of G3BP2.

[0010] Preferably, Compound C108 is a drug that alleviates social disorders by inhibiting G3BP2 and regulating the content of SGs.

[0011] Preferably, Compound C108 is a drug capable of increasing the frequency of social interaction.

[0012] Preferably, Compound C108 is a drug capable of increasing social time.

[0013] Preferably, the medicament contains one or more pharmaceutically acceptable carriers or excipients.

[0014] Preferably, the drug is in the form of powder, tablet, capsule or injection.

[0015] Preferably, the drug concentration of Compound C108 is 10 μM, the unilateral administration volume is 500 nL, and the drug is administered to bilateral ACC brain regions.

[0016] The second aspect of the present invention discloses a composition for treating autism spectrum disorder, comprising Compound C108 and other drugs for treating autistic social disorders.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The use of Compound C108 provided by the present invention in the preparation of drugs for treating autism spectrum disorders, by finding that the SGs content is significantly increased in the relevant brain regions and primary neurons of ASD mice caused by Shank3 mutation (genetic factors) and VPA exposure during pregnancy (environmental factors). Compound C108 can reduce the SGs content by inhibiting G3BP2 (a key factor in the formation of SGs), thereby alleviating the symptoms of autism in mice and improving the social ability of autistic mice, which fully supports the use of Compound C108 in the preparation of drugs and / or health products for treating autism social disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of the determination of SGs content in neurons of ASD mice are shown in Figure 1; A is the immunofluorescence staining results (upper row) and Western-Blotting results (lower row) of Shank3 mice, B is the immunofluorescence staining results (upper row) and Western-Blotting results (lower row) of VPA mice, n=15 (neurons from 3 mice), Bar=4μm;

[0020] Figure 2The experimental results of Compound C108 intervening G3BP2 to regulate SGs; A is a schematic diagram of the uniform injection of Compound C108 into the bilateral ACC brain regions of mice, B is the Western-Blotting result (left) and result statistics (right), and C is the immunofluorescence staining result;

[0021] Figure 3 This is a graph showing the behavioral characterization results of Compound C108 alleviating autism; A is the result of the three-box experiment, from left to right are the trajectory of social preference, interval time and social preference score, B is the result of the exploration time of the social novelty mouse (S2), from left to right are the trajectory of social preference, interval time and social score of the novelty mouse, C is the result of the residence-invasion experiment, the left side is the social time, the right side is the contact frequency, and D is the duration of social contact per unit time. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0023] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0024] Herein, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0025] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0026] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0027] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0028] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art.

[0029] 1. Determination of the content of SGs in the relevant brain regions of two ASD mouse models and normal mice:

[0030] 1) The Shank3 mouse model with ASD caused by common genetic factors and the VPA mouse model with ASD caused by environmental factors during pregnancy were selected as ASD mouse models (6-week young and middle-aged male mice). The key ASD anterior cingulate cortex (ACC) brain tissues of the Shank3 mouse model, VPA mouse model and normal mice (C57BL6J mice) or neurons isolated from the fetal mouse cortex and cultured for 7 days were obtained;

[0031] 2) Take 2×10 6 Primary neurons or 0.2 g mouse ACC tissue were used to purify the protein components in SGs of mouse primary neurons or ACC tissue in an RNase-free environment, and then the mRNA components in SGs were purified using the Trizol extraction method.

[0032] 3) Immunofluorescence technology was used to detect the co-labeling of SGs marker protein TIA-1 and different cell markers in ACC; the expression of TIA-1 / Tuj1 in primary neurons was detected to reflect the distribution of SGs, and the data were analyzed using Imgae J software to analyze the changes in the number and area of ​​SGs.

[0033] Results of the determination of SGs content in neurons of Shank3 and VPA ASD mice Figure 1 As shown, Figure 1 The immunofluorescence staining results of Shank3 mice in A showed that Shank3 mutation (genetic factor) caused the primary neurons of ASD mice with typical SGs formation (arrows), and Western-Blotting results showed that G3BP2 was significantly increased in the cortex of Shank3 mutant mice. Figure 1The immunofluorescence staining results of VPA mice in middle B showed that VPA exposure during pregnancy (environmental factor) caused typical SGs to form in primary neurons of ASD mice (arrows), and Western-Blotting results showed that G3BP2 was significantly increased in the cortex of VPA-exposed mice. The above results indicate that the number and area of ​​SGs in neurons of the two ASD mice are abnormally increased.

[0034] 2. Compound C108 regulates SGs formation:

[0035] 1) If Figure 2 As shown in middle A, two types of ASD mice (Shank3 mice, VPA mouse model) were injected with Compound C108 reagent (10 μM, 500 nL, for 3 consecutive days) or control placebo (normal saline) into the bilateral ACC brain regions;

[0036] 2) After 3 weeks, the protein expression level of G3BP2, a key factor in SGs formation, and the content of SGs were detected by SGs extraction technology and Western-blot. The specific steps of SGs extraction technology are as follows: 2×10 6 Primary neurons or 0.2g mouse ACC tissue were extracted with SGs lysis buffer (50mM Tris-HCl, 100mM KOAc, 2mMMgOAc and 0.5mM DTT) in an RNase-free environment. 1mL SGs lysis buffer was added to each sample, and the tissue was physically ground and digested, then placed on ice for 15min and centrifuged at 4℃2000×g for 5min. 50μL of supernatant was taken for total protein or total RNA extraction, 950μL of supernatant was taken for centrifugation at 4℃18000×g for 20min, repeated twice to obtain a precipitate, and then 200-300μL SGs lysis buffer was added to resuspend the precipitate, and then centrifuged at 4℃850×g for 2min. The supernatant was the protein component of SGs and was used for subsequent RNA extraction or protein experiments.

[0037] 3) Immunofluorescence technology was used to detect the formation of SGs in cortical neurons.

[0038] The experimental results of Compound C108 regulating SGs by intervening in G3BP2 are as follows Figure 2 As shown in Figure 2, Western-Blotting results showed that the expression of G3BP2, a key factor in the formation of SGs, was significantly reduced, indicating that Compound C108 has the effect of inhibiting the expression of G3BP2 ( Figure 2 Middle B); Immunofluorescence staining results showed that the content of SGs in the ACC brain region decreased, indicating that Compound C108 has the effect of inhibiting the formation of SGs ( Figure 2 Middle C).

[0039] 3. Test the social behavior of ASD mice after injection of Compound C108 reagent:

[0040] 1) Compound C108 reagent (10 μM, 500 nL, for 3 consecutive days) or control placebo (normal saline) was injected into the bilateral ACC brain region of Shank3 mice;

[0041] 2) The three-box experiment and housing-invasion experiment in animal behavior techniques were used to observe changes in the social behavior and other ASD-like behaviors (such as repetitive stereotyping, anxiety, etc.) of mice.

[0042] Compound C108 alleviates autism behavioral manifestations Figure 3 As shown, in the three-box experiment, the time that ASD mice injected with Compound C108 explored the social preference mouse (S1) was significantly increased ( Figure 3 Middle A), the exploration time of the social novelty mouse (S2) also increased significantly ( Figure 3 In the housing-invasion test, the ASD mice injected with Compound C108 showed a significant increase in the duration and frequency of contact with stranger mice of the same sex ( Figure 3 C and D).

[0043] The above results indicate that Compound C108 can intervene in G3BP2 expression, thereby inhibiting SGs formation and ultimately effectively alleviating the social disorders of ASD mice.

[0044] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Application of Compound C108 in the preparation of drugs for the treatment of autism spectrum disorders.

2. The use of Compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorders, characterized in that: Compound C108 is a drug that reduces the content of SGs.

3. The use of Compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorders, characterized in that: Compound C108 is a drug that inhibits the expression of G3BP2.

4. The use of Compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorders, characterized in that: Compound C108 is a drug that alleviates social disorders by inhibiting G3BP2 and regulating the content of SGs.

5. The use of Compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorders, characterized in that: Compound C108 is a drug that can increase the frequency of social interaction.

6. The use of Compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorders, characterized in that: Compound C108 is a drug that can increase social time.

7. The use of Compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorders, characterized in that: The medicament contains one or more pharmaceutically acceptable carriers or excipients.

8. The use of Compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorders, characterized in that: The dosage form of the medicine is powder, tablet, capsule or injection.

9. Use of Compound C108 according to any one of claims 1 to 8 in the preparation of a drug for treating autism spectrum disorders, characterized in that: The concentration of Compound C108 was 10 μM, the unilateral administration volume was 500 nL, and the administration was performed in bilateral ACC brain regions.

10. A composition for treating autism spectrum disorder, characterized in that: Including Compound C108 and other drugs for treating social disorders in autism.

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