Application of ZXL-9 in the preparation of GluA1 postsynaptic localization modulators and antidepressants

Abstract

This invention discloses the application of ZXL-9 in the preparation of GluA1 postsynaptic localization modulators and antidepressants, relating to the field of biomedical technology. The invention found that ZXL-9 can reduce the pathological protein coupling between GluA1 and calpain-1, decrease the binding level of calpain-1 in the GluA1 immunoprecipitation complex, and regulate the postsynaptic localization of GluA1 in the piriform cortex. This invention provides an antidepressant intervention strategy based on GluA1 postsynaptic localization regulation and GluA1-calpain-1 protein coupling uncoupling, which can be used to prepare GluA1 postsynaptic localization modulators, GluA1-calpain-1 uncoupling agents, and drugs for the prevention and / or treatment of depression.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of ZXL-9 in the preparation of GluA1 postsynaptic localization modulators and antidepressants. Background Technology

[0002] Depression is a common and highly disabling mental disorder, and chronic stress is one of the important risk factors for its development. While existing antidepressants can improve symptoms in some patients, they still suffer from slow onset of action, insufficient efficacy, high relapse rates, and poor treatment response in some patients.

[0003] Abnormal glutamatergic synaptic transmission and altered postsynaptic receptor localization are important foundations for chronic stress-induced mood disorders. AMPAR is a key receptor mediating rapid excitatory synaptic transmission, with the GluA1 subunit involved in receptor postsynaptic localization and maintenance of synaptic plasticity. If chronic stress leads to a decrease in the postsynaptic localization of GluA1 in the piriform cortex, it may cause local excitatory synaptic dysfunction, thereby promoting depressive-like behaviors. This invention aims to develop antidepressant intervention strategies based on novel synaptic molecular mechanisms. Summary of the Invention

[0004] The purpose of this invention is to provide the application of ZXL-9 in the preparation of GluA1 postsynaptic localization modulators and antidepressants, thereby addressing the problems existing in the prior art. This invention achieves an antidepressant intervention based on GluA1-calpain-1 protein coupling-uncoupling and GluA1 postsynaptic localization regulation, providing a novel treatment strategy not only at the level of postsynaptic protein interactions but also demonstrating an effect on improving depression-related behavioral phenotypes in a chronic, unpredictable, mild stress-related depression-like model.

[0005] Calpain-1 is a calcium-dependent cysteine ​​protease involved in synaptic protein remodeling and receptor localization regulation. The inventors found that chronic stress enhances the pathological binding of GluA1 to calpain-1, promotes the recruitment of calpain-1 into the GluA1-related postsynaptic complex, and is accompanied by a decrease in the postsynaptic localization level of GluA1 in the piriform cortex. This invention modulates the postsynaptic localization of GluA1 in the piriform cortex via ZXL-9 and reduces the aberrant interaction between GluA1 and calpain-1, thereby improving chronic stress-induced depression-related behavioral phenotypes.

[0006] Based on this, the present invention provides the following solution: This invention provides the application of ZXL-9 in the preparation of GluA1 postsynaptic localization modulators, wherein the structural formula of ZXL-9 is: .

[0007] Furthermore, the GluA1 postsynaptic localization modulator is used to regulate the GluA1 postsynaptic localization level in the piriform cortex.

[0008] Furthermore, the GluA1 postsynaptic localization regulator works by reducing the pathological protein coupling between GluA1 and calpain-1.

[0009] The present invention also provides a GluA1 postsynaptic localization modulator, the active ingredient of which includes ZXL-9; The structural formula of the ZXL-9 is: .

[0010] This invention also provides the application of ZXL-9 in the preparation of uncoupling agents for GluA1 and calpain-1, wherein the structural formula of ZXL-9 is: .

[0011] Furthermore, the uncoupling agent is used to reduce the pathological protein coupling between GluA1 and calpain-1.

[0012] The present invention also provides an uncoupling agent for GluA1 and calpain-1, the active ingredient of which includes ZXL-9; The structural formula of the ZXL-9 is: .

[0013] This invention also provides the application of ZXL-9 in the preparation of antidepressant drugs, wherein the structural formula of ZXL-9 is: .

[0014] Furthermore, the drug exerts its antidepressant effect by modulating the postsynaptic localization of GluA1 and reducing the pathological protein coupling between GluA1 and calpain-1.

[0015] Furthermore, the antidepressant targets depression including chronic stress-related depression, chronic unpredictable mild stress-related depressive-like state, depressive state with anhedonia, or major depressive disorder.

[0016] The present invention discloses the following technical effects: 1. Provides a new target direction for antidepressant intervention: Unlike traditional monoamine antidepressants or non-specific glutamate receptor modulation strategies, this invention targets chronic stress-related GluA1 postsynaptic localization abnormalities and achieves postsynaptic localization regulation by reducing GluA1-calpain-1 pathological protein coupling.

[0017] 2. It has a clear postsynaptic molecular basis: Experimental results show that ZXL-9 can reduce the binding level of calpain-1 in the GluA1 immunoprecipitation complex at the neuronal level, and can reduce the level of calpain-1 bound to GluA1 in the piriform cortex in a chronic stress model, indicating that it has the effect of uncoupling GluA1 and calpain-1.

[0018] 3. Ability to modulate GluA1 postsynaptic localization: ZXL-9 can modulate the level of GluA1 postsynaptic localization in the piriform cortex under chronic stress, suggesting that it can be used to improve chronic stress-related postsynaptic AMPAR localization abnormalities.

[0019] 4. It has antidepressant-like behavioral effects: ZXL-9 can improve tail suspension immobility time, forced swimming immobility time and sucrose preference rate in mice with chronic unpredictable mild stress model.

[0020] 5. The antidepressant-like effect is not caused by non-specific motor excitation: Behavioral results showed that while ZXL-9 improved depression-related behavioral phenotypes, it did not significantly change the total open-field motor distance, suggesting that its effect was not caused by general motor ability enhancement or non-specific excitation.

[0021] 6. Prospects for drug development and translational application: This invention demonstrates the application value of ZXL-9 in the preparation of GluA1 postsynaptic localization modulators, uncoupling agents of GluA1 and calpain-1, antidepressants, and related drug screening tools, and has the prospect of further development into drugs for the treatment of depression.

[0022] Therefore, this invention realizes an antidepressant intervention based on GluA1-calpain-1 protein coupling-uncoupling and GluA1 postsynaptic localization regulation. It not only provides a new treatment strategy at the level of postsynaptic protein interaction, but also shows the effect of improving depression-related behavioral phenotypes in a chronic unpredictable mild stress-related depression-like model. It has clear scientific significance, drug development value and clinical translation prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Here is the chemical structural diagram of compound ZXL-9; Figure 2A schematic diagram of the experimental procedure for ZXL-9 treatment and GluA1 immunoprecipitation of primary cortical neurons; Figure 3 Western blotting results (A) and quantitative statistics (B) of calpain-1 detected after GluA1 immunoprecipitation; Figure 4 Western blotting results (A) and quantitative statistics (B) of calpain-1 and GluA1 postsynaptic localization-related indicators detected by immunoprecipitation in piriform cortex samples; Figure 5 The graph shows the results of antidepressant-like behaviors of ZXL-9 in a chronic unpredictable mild stress model; where A is the immobility time statistics of the tail suspension test; B is the immobility time statistics of the forced swimming test; C is the sucrose preference rate statistics; and D is the total movement distance statistics of the open field test. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those referred to herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Terminology Explanation: The structural formula of compound ZXL-9 referred to in this invention is as follows: Figure 1 As shown.

[0031] The “GluA1-calpain-1 protein coupling” referred to in this invention refers to the binding or complex formation between GluA1 and calpain-1 that can be detected by immunoprecipitation, Western blotting or other protein interaction detection methods.

[0032] The term "uncoupling" in this invention refers to reducing the interaction between GluA1 and calpain-1, or reducing the binding level of calpain-1 in the GluA1 immunoprecipitation complex.

[0033] The "GluA1 postsynaptic localization" referred to in this invention includes the GluA1 localization level evaluated by detection methods that can reflect the postsynaptic distribution of GluA1, such as postsynaptic related components, postsynaptic dense related components, membrane localization related components, or other detection methods that can reflect the postsynaptic distribution of GluA1.

[0034] Example 1: Structural basis of ZXL-9 for GluA1-calpain-1 protein coupling and uncoupling This embodiment illustrates that ZXL-9 has a structural basis for the coupling and uncoupling of GluA1-calpain-1 protein.

[0035] Based on the structural prediction results of the GluA1-calpain-1 complex, candidate binding interfaces that interact with the intracellular C-terminal region of GluA1 on calpain-1 were identified, and potential binding pockets near these interfaces were used as small molecule screening regions. Small molecule compounds capable of occupying or approaching the GluA1 binding groove on calpain-1 were screened by performing molecular docking scoring, binding conformation analysis, and interfacial residue interaction analysis on candidate compounds.

[0036] Among the candidate compounds, ZXL-9 was identified as the target small molecule compound for further validation. Structural analysis revealed that ZXL-9 is located near the predicted GluA1 binding groove of calpain-1 and forms hydrophobic and polar interactions with the relevant amino acid residues in this region, thus providing a structural basis for interfering with the binding of GluA1 to calpain-1.

[0037] Therefore, ZXL-9 has a structural basis for the coupling and uncoupling of GluA1-calpain-1 proteins, which can be used for subsequent functional validation at the neuronal level and in vivo in animal models.

[0038] Example 2: ZXL-9 reduces GluA1-calpain-1 interaction at the neuronal level This embodiment is used to verify whether ZXL-9 can reduce the interaction between GluA1 and calpain-1 in the neuronal environment.

[0039] Primary cortical neurons were cultured in vitro. After the neurons formed a certain synaptic network, they were treated with 10 μM ZXL-9 for 24 h; the control group was treated with the corresponding solvent. After treatment, neuronal samples were collected and lysed. The cell lysates were immunoprecipitated with anti-GluA1 antibody, and the binding level of calpain-1 in the GluA1 immunoprecipitation complex was then detected by Western blotting. The experimental procedure is as follows: Figure 2 As shown.

[0040] Experimental results are as follows Figure 3 As shown, compared with the solvent control group, the level of calpain-1 in the GluA1 immunoprecipitation complex was significantly reduced after ZXL-9 treatment.

[0041] The above results indicate that ZXL-9 can reduce the interaction between GluA1 and calpain-1 at the neuronal level and has GluA1-calpain-1 protein coupling and uncoupling activity.

[0042] Example 3: ZXL-9 modulates the postsynaptic localization of GluA1 in the piriform cortex and reduces GluA1-calpain-1 protein coupling in the CUMS model. This embodiment is used to verify whether ZXL-9 can regulate the postsynaptic localization of GluA1 in the piriform cortex and reduce GluA1-calpain-1 protein coupling in a chronic stress-induced depression-like animal model.

[0043] A mouse model of depression was established using chronic unpredictable mild stress (CUMS). After modeling, mice were divided into a normal control group, a CUMS+solvent group, and a CUMS+ZXL-9 group. The CUMS+ZXL-9 group received ZXL-9 via intraperitoneal injection at a dose of 10 mg / kg / day for 3 consecutive days; the CUMS+solvent group received an equal volume of solvent. After administration, piriform cortex tissue was collected from mice to prepare postsynaptic components. GluA1 immunoprecipitation and Western blotting were used to detect the levels of calpain-1 bound to GluA1 and the postsynaptic localization-related levels of GluA1.

[0044] The results are as follows Figure 4 As shown, compared with the normal control group, the CUMS+ solvent group showed an increase in the level of calpain-1 bound by GluA1 in the piriform cortex, accompanied by a decrease in the level of GluA1 postsynaptic localization; compared with the CUMS+ solvent group, ZXL-9 treatment reduced the level of calpain-1 bound by GluA1 in the piriform cortex and modulated the postsynaptic localization of GluA1.

[0045] The results indicate that ZXL-9 can reduce pathological protein coupling of GluA1-calpain-1 in the piriform cortex and regulate the postsynaptic localization of GluA1 in the piriform cortex in vivo.

[0046] Example 4: ZXL-9 improves depressive-like behavior in CUMS model mice This embodiment is used to verify the antidepressant-like effect of ZXL-9 in a chronic unpredictable mild stress model.

[0047] A mouse model of depression was established using CUMS. After modeling, mice were divided into a normal control group, a CUMS + solvent group, and a CUMS + ZXL-9 group. The CUMS + solvent group was administered ZXL-9 via intraperitoneal injection at a dose of 10 mg / kg / day for 3 consecutive days; the CUMS + solvent group was administered an equal volume of solvent. After administration, tail suspension test, forced swimming test, sucrose preference test, and open field test were performed.

[0048] The results are as follows Figure 5 As shown, compared with the normal control group, mice in the CUMS+solvent group exhibited prolonged immobility time in tail suspension, prolonged immobility time in forced swimming, and a decreased sucrose preference rate. Compared with the CUMS+solvent group, ZXL-9 treatment reduced both tail suspension and forced swimming immobility time and restored the sucrose preference rate. Meanwhile, no significant changes were observed in the total movement distance of mice in any group during the open-field experiment. These results indicate that ZXL-9 can improve CUMS-induced depressive-like behavior, and this effect is not due to non-specific motor ability enhancement.

[0049] In summary, ZXL-9 can reduce the interaction between GluA1 and calpain-1 at the neuronal level, decrease pathological protein coupling of GluA1-calpain-1 in the piriform cortex and regulate the postsynaptic localization of GluA1 in the piriform cortex in vivo, while improving CUMS-induced depression-related behavioral phenotypes. Therefore, ZXL-9 can be used to prepare GluA1 postsynaptic localization modulators, as well as GluA1-calpain-1 protein coupling uncoupling agents and drugs for the prevention and / or treatment of depression. This depression includes, but is not limited to, chronic stress-related depression, chronic unpredictable mild stress-related depressive-like state, depressive state with anhedonia, or major depressive disorder. The above applications are particularly suitable for antidepressant interventions based on GluA1 postsynaptic localization regulation and GluA1-calpain-1 pathological protein coupling uncoupling.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of ZXL-9 in the preparation of GluA1 postsynaptic localization modulators, characterized in that, The structural formula of the ZXL-9 is: 。 2. The application according to claim 1, characterized in that, The GluA1 postsynaptic localization modulator is used to regulate the level of GluA1 postsynaptic localization in the piriform cortex.

3. The application according to claim 2, characterized in that, The GluA1 postsynaptic localization regulator works by reducing the pathological protein coupling between GluA1 and calpain-1.

4. A GluA1 postsynaptic localization modulator, characterized in that, The active ingredient includes ZXL-9; The structural formula of the ZXL-9 is: 。 5. The application of ZXL-9 in the preparation of an uncoupling agent for GluA1 and calpain-1, characterized in that, The structural formula of the ZXL-9 is: 。 6. The application according to claim 5, characterized in that, The uncoupling agent is used to reduce the pathological protein coupling between GluA1 and calpain-1.

7. An uncoupling agent for GluA1 and calpain-1, characterized in that, The active ingredient includes ZXL-9; The structural formula of the ZXL-9 is: 。 8. The application of ZXL-9 in the preparation of antidepressant drugs, characterized in that, The structural formula of the ZXL-9 is: 。 9. The application according to claim 8, characterized in that, The drug exerts its antidepressant effect by modulating the postsynaptic localization of GluA1 and reducing the pathological protein coupling between GluA1 and calpain-1.

10. The application according to claim 8, characterized in that, The antidepressants targeted include chronic stress-related depression, chronic unpredictable mild stress-related depressive-like state, depressive state with anhedonia, or major depressive disorder.

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