Application of ULK1 agonist in preparation of anti-depression drugs and ULK1 in preparation of products for diagnosing depression

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of a ULK1 agonist in preparation of an anti-depression medicine and application of ULK1 in preparation of a depression diagnosis product. Experiments show that ULK1 not only can be used as a biomarker for diagnosing depression, but also can be used as an important target for developing drugs for treating / preventing depression. In addition, the invention further indicates that the ULK1 agonist BL-918 has a rapid anti-depression effect and can be used for treating / preventing depression.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of ULK1 agonists in the preparation of antidepressant drugs and ULK1 in the preparation of products for diagnosing depression. Background Art

[0002] Depression is a psychological disorder characterized by a significant and persistent low mood. Its causes are complex and its specific mechanism remains unclear. For a long time, research on depression has focused on the monoamine system (including 5-hydroxytryptamine, norepinephrine, and dopamine systems), and drug treatments developed based on this, such as 5-hydroxytryptamine selective reuptake inhibitors (SSRIs), have an effective rate of only 60%-65%, and the treatment takes a long time to take effect, has serious side effects, and has a high relapse rate after discontinuation of the drug.

[0003] Therefore, there is an urgent need to find new therapeutic targets for depression, and then research and develop more effective and safe drugs to meet the treatment needs of depression. Summary of the invention

[0004] The present invention aims to solve at least one of the problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of ULK1 agonists in the preparation of antidepressant drugs and ULK1 in the preparation of products for diagnosing depression. The experiments of the present invention show that ULK1 can not only be used as a biomarker for diagnosing depression, but also as an important target for developing drugs for treating / preventing depression. In addition, the present invention further points out that the ULK1 agonist BL-918 has a rapid antidepressant effect and can be used to treat / prevent depression.

[0005] The present invention provides use of a ULK1 agonist in preparing an antidepressant drug.

[0006] UNC-51-like kinase 1 (ULK1) is a serine / threonine kinase best known for its role in the initiation of autophagy. ULK1 has been shown to be a direct target of cellular energy and nutrient-sensing kinases that respond to the allocation and utilization of cellular resources and play a key role in a variety of diseases.

[0007] In recent years, people have gradually paid attention to the non-autophagic function of ULK1. Many studies have found that in certain specific cases, the ULK1 signaling network related to non-autophagic functions may also be involved in regulating the progression of various diseases. In addition, a variety of small molecule regulators targeting ULK1 (such as agonists, inhibitors, and indirect regulators) have been gradually developed, showing certain potential in the treatment of various diseases.

[0008] The present invention takes ULK1 as the target and focuses on its regulatory effects on cellular autophagy and depression. It first discovered that the expression level of ULK1 in depression model mice was significantly decreased, and experimentally confirmed that ULK1 agonists have a positive effect on the treatment of depression, and has good prospects for drug development.

[0009] Preferably, the antidepressant drug is a drug for treating or preventing depression.

[0010] Preferably, the ULK1 agonist is BL-918 or a pharmaceutically acceptable salt thereof. BL-918 (CAS No.: 2101517-69-3) is an activator of ULK1. Experiments of the present invention show that BL-918 can quickly and effectively alleviate the depressive-like behavior of CSDS-induced depression-susceptible mice and exert an antidepressant effect.

[0011] The present invention also provides the use of ULK1 in the preparation of a product for diagnosing depression. The research of the present invention shows that ULK1 can be used as a biomarker for diagnosing depression and plays an important role in the field of depression diagnosis.

[0012] The present invention also provides the use of a reagent for detecting ULK1 in preparing a product for diagnosing depression.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The present invention first discovered that compared with wild-type mice, the expression level of ULK1 in depression model mice was significantly reduced, and experiments proved that the downregulation of ULK1 levels is expected to serve as an early warning or clinical diagnostic standard for depression, indicating that ULK1 can be used as a biomarker for diagnosing depression. The experiments of the present invention also confirmed that by increasing the expression level / function of ULK1, depression can be prevented and the effect of treating depression can be achieved, thereby providing a new target and drug for the treatment / prevention of depression. In addition, the present invention further points out that the ULK1 agonist BL-918 has a rapid antidepressant effect, and establishes the use of BL-918 as a drug for the treatment / prevention of depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Figure 2 is the cell autophagy damage in the CSDS mouse model; A is the construction of the chronic social frustration stress model and the behavioral test process; BE is the statistical results of the social interaction test (SIT), sucrose preference test (SPT), tail suspension test (TST) and open field test (OFT) in the control group (Ctrl), depression-resistant group (Res) and depression-susceptible group (Sus); FG is the expression levels of ULK1 protein, p-ULK1 protein, LC3-I protein, LC3-II protein and internal reference in the medial prefrontal cortex (mPFC) and lateral habenula (LHb) of the control group, depression-resistant group and depression-susceptible group.

[0016] Figure 2 Figure 5 shows the construction of ULK1+ / - mice and the results of depressive-like behavior detection; A is a schematic diagram of the construction of transgenic mice; BC is a fluorescence quantitative PCR comparison of the mRNA expression levels of ULK1 in mPFC and LHb of ULK1+ / + mice, ULK1+ / - mice and ULK1- / - mice; DF is a comparison of the SPT, TST and OFT results of ULK1+ / + mice, ULK1+ / - mice and ULK1- / - mice.

[0017] Figure 3 The results of depressive-like behavior detection in wild-type mice with downregulated ULK1 expression; A is a schematic diagram of the process of virus injection and behavioral detection of wild-type mice; B is a schematic diagram of the injection position of shRNA in the LHb region; CD are the expression levels of ULK1 protein, p-ULK1 protein, LC3-I protein, and LC3-II protein in LHb of the ULK1-shRNA group and the ULK1-Ctrl group; EG is a comparison of SPT, TST, and OFT results between the ULK1-shRNA group and the ULK1-Ctrl group.

[0018] Figure 4 Schematic diagram of rAAV-CMV-Ulk1-3XFlag-WPREs recombinant plasmid.

[0019] Figure 5Figure 3 shows the results of depressive-like behavior detection in ULK1+ / - mice with upregulated ULK1 expression; A shows the process of virus injection and behavioral testing of ULK1+ / + mice (i.e. wild-type mice) and ULK1+ / - mice; B shows the schematic diagram of the injection position of AAV-CMV-ULK1&CTB647 in the LHb region; CD shows the expression levels of ULK1 protein, p-ULK1 protein, LC3-I protein and LC3-II protein in the LHb of ULK1+ / ++AAV-Ctrl group, ULK1+ / -+AAV-Ctrl group and ULK1+ / -+AAV-ULK1 group; EG shows the comparison of SPT, TST and OFT results of ULK1+ / ++AAV-Ctrl group, ULK1+ / -+AAV-Ctrl group and ULK1+ / -+AAV-ULK1 group.

[0020] Figure 6 Figure 2 shows the effect of BL-918 on the alleviation of depressive-like behaviors in CSDS-induced depression-susceptible mice; A is a schematic diagram of the experimental process for wild-type mice; B is a schematic diagram of the location of the buried tube in the LHb region; C is a comparison of the immobility time in the tail suspension test of wild-type mice injected with different concentrations of BL-918; D is a schematic diagram of the experimental process for wild-type mice; EH is a comparison of the results of the social experiment, sugar water preference experiment, tail suspension experiment, and open field experiment between the Saline group and the BL-918 group.

[0021] Figure 7 The effect of up-regulated ULK1 expression on the proportion of depression-susceptible mice after CSDS modeling; A is a schematic diagram of the experimental process of wild-type mice; B, D, E, and F are comparisons of SIT, SPT, TST, and OFT experimental results of the AAV-Ctrl group and the AAV-ULK1 group before and after CSDS modeling, respectively. DETAILED DESCRIPTION

[0022] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0023] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0024] Example 1: Role of ULK1 expression levels as a biomarker for depression

[0025] 1. Construction of CSDS mouse model:

[0026] The CSDS mouse model is constructed by exposing mice to social shock for a long period of time (usually 10 days) to induce behavioral responses with depressive symptoms, such as loss of interest and reduced social behavior.

[0027] The specific construction method is ( Figure 1 A): Mice were placed in the same cage with CD-1 mice, which are naturally aggressive, for 24 hours. The two mice were separated by a perforated partition to avoid conflicts during the adaptation stage. After the adaptation, a 10-minute conflict was conducted every day for 10 consecutive days. After the conflict process was over, the mice were separated by a perforated partition again and adapted for 24 hours.

[0028] After the last conflict, mice were allowed to acclimate for 24 h and then assessed using the following behavioral tests:

[0029] 1) Social interaction test (SIT): This test measures the social ability of animals by observing the way and degree of interaction between animals and unfamiliar objects. The ratio of the time spent exploring the social area between the mice with aggressors and the mice without aggressors after the depression model is used to evaluate whether they have social avoidance and screen out depression-susceptible and depression-resistant mice.

[0030] 2) Sucrose preference test (SPT): It is a classic experiment to detect anhedonia, a typical symptom of depression. It is a detection method designed based on the preference of rodents for sweet taste.

[0031] 3) Open field test (OFT): It is a method used to evaluate the autonomous behavior, exploratory behavior and tension of experimental animals in a new environment. The exploratory characteristics of animals will prompt them to move in the central area. If the animal is afraid of the new open environment, it will mainly move in the peripheral area.

[0032] 4) Tail suspension test (TST): It is a classic method that can quickly evaluate the efficacy of antidepressants, stimulants, and sedatives. In this animal model, the tail of the experimental animal is fixed so that its head hangs downward. The animal struggles in this environment, trying to get rid of the predicament. After failing to get rid of it after efforts, it becomes intermittent and immobile, showing a state of "behavioral despair". This behavioral despair model is similar to depression and is sensitive to most antidepressants. Its efficacy is significantly correlated with clinical efficacy, so it is widely used in the preliminary selection of antidepressants.

[0033] Behavioral results such as Figure 1As shown in BE, after 10 days of CSDS treatment, the mice were divided into a depression-resistant group (Res) and a depression-sensitive group (Sus). Compared with the mice in the control group and the depression-resistant group, the mice in the depression-sensitive group showed a significant decrease in the proportion of social exploration and the percentage of sugar water preference, and an increase in the immobility time in the tail suspension test. The above results showed that after 10 days of social failure treatment, the CSDS depression mouse model was successfully established.

[0034] 2. Protein immunoblotting assay

[0035] In order to detect whether cell autophagy in the brain of depression-susceptible mice (CSDS model) is changed, the present invention studies cell autophagy mediated by ULK1. ULK1 is an autophagy initiator, and cell autophagy in mammals starts from the ULK1 complex, so the expression levels of ULK1 and p-ULK1 can reflect the state of cell autophagy; LC3 is an autophagy marker, and LC3-I will be converted to LC3-II during the autophagy process, so the ratio of LC3-II to LC3-I can evaluate the level of autophagy.

[0036] Through protein immunoblotting experiments, the expression levels of ULK1, p-ULK1 and autophagy function-related proteins LC3-I and LC3-II in two depression-related brain regions (mPFC and LHb) of mice in the control group, depression-resistant group, and depression-susceptible group were detected respectively.

[0037] Test results such as Figure 1 As shown in FH, compared with wild-type mice, the expression levels of ULK1 and p-ULK1 in the mPFC and LHb of CSDS-induced depression-susceptible mice were significantly reduced, and the ratio of LC3-II to LC3-I was significantly decreased, indicating that the ULK1 level in these two brain regions was decreased and showed cellular autophagy damage.

[0038] Example 2: Construction of ULK1+ / - mice and detection of depressive-like behavior:

[0039] 1. Construction of ULK1 knockdown (ULK1+ / -) mice ( Figure 2 A)

[0040] ULK1 is the only core protein with serine / threonine kinase activity in the autophagy signaling pathway. Before the assembly of autophagolysosomes, autophagy signaling is mediated by the activation of the ULK complex composed of ULK1, FIP200 and mATG13.

[0041] The mouse Ulk1 (NCBI reference sequence: NM_009469; Ensembl: ENSMUSG00000029512) gene is located on chromosome 5 of the mouse, with a total length of 25637 bp. 28 exons have been identified, of which the ATG start codon is located in the first exon and the TGA stop codon is located in the 28th exon (transcript Ulk1-201: ENSMUST00000031490).

[0042] In this method, exons 4-11 are knocked out, and this region contains 613 bp of coding sequence. The specific method is as follows:

[0043] a. Design gRNA: Design small guide RNA (gRNA) through http: / / crispor.tefor.net / . Choose high-scoring gRNA, which is usually about 20 nucleotides in length. These nucleotides can specifically bind to the target site and determine the precise location where the Cas9 enzyme cuts the DNA.

[0044] The gRNA sequences in this gene editing are as follows: gRNA1 (matching reverse strand of gene): 5'-ACGTGGACCATTGCCTATGAAGG-3' (SEQ ID NO: 1), gRNA2 (matching forward strand of gene): 5'-TTGGAGTACTGGTGCCAGTCAGG-3' (SEQ ID NO: 2), which can specifically bind to both ends of exons 4-11 of ULK1.

[0045] b. Preparation of RNP complex: Mix Cas9 protein and the above-mentioned gRNA to prepare RNP complex.

[0046] c. Transfection or transduction of cells: The prepared RNP complex is introduced into the target cell. This method uses high-throughput electroporation to introduce it into the fertilized egg. The Cas9 protein and gRNA combine to form a complex, recognize and bind to the target DNA sequence, and then cut the double-stranded DNA under the guidance of the gRNA. The cell repairs the broken DNA through non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms.

[0047] d. Screening of knockout cells: Verify the gene knockout effect through PCR, sequencing and other methods, and screen out cells that have successfully knocked out the target gene to obtain Ulk1 knockout mice. The PCR primers used for mouse genotype identification are as follows: (annealing temperature 60.0℃)

[0048] F1: 5'-TGTAGGTTGTTTTAGACCCAGCC-3' (SEQ ID NO: 3);

[0049] R1: 5'-CAAACCCATCACTATGCAGTCC-3' (SEQ ID NO: 4);

[0050] R2N: 5'-AGGTCTGAGTATTCCCATAAGGTTC-3' (SEQ ID NO: 5);

[0051] The bands of each genotype are as follows: homozygous: 530bp; heterozygous: 530bp / 399bp; wild type: 399bp.

[0052] 2. Behavioral testing

[0053] First, the expression level of ULK1 in mPFC and LHb was detected by real-time fluorescence quantitative PCR ( Figure 2 BC), compared with wild-type mice (ULK1+ / +), the expression level of ULK1 in mPFC and LHb of ULK1+ / - mice was significantly reduced. Secondly, behavioral tests were performed to detect the depressive-like behavior of ULK1+ / - mice. The test results are as follows Figure 2 As shown in CF, compared with wild-type mice (ULK1+ / +), ULK1+ / - mice showed significantly decreased social exploration time in SIT and sugar water preference in SPT, and significantly increased immobility time in TST. The results showed that ULK1+ / - mice showed obvious depressive-like behavior, while the total movement distance in OFT remained unchanged, indicating that the mice's exercise ability did not change. The above results indicate that downregulating the expression level of ULK1 can induce depressive-like behavior in mice.

[0054] Example 3: ULK1 protein knockdown in wild-type mice and detection of their depressive-like behavior

[0055] AAV-shRNA was used to specifically knock down the expression level of ULK1 protein in the LHb of wild-type mice. Figure 3 AB), detection of depressive-like behavior and tissue protein immunoblotting experiments ( Figure 3 CG).

[0056] The process of AAV-shRNA knocking down protein expression is as follows:

[0057] 1. Design of shRNA sequence: First, it is necessary to design specific shRNA sequences based on the mRNA sequence of the target protein ULK1. These sequences will specifically bind to the mRNA. The shRNA sequence in this method is as follows: 5'-GCTGCTTAAGGCTGCATTTGGTTCAAGAGACCAAATGCAGCCTTAAGCAGCTTTT-3' (SEQ ID NO: 6)

[0058] 2. Construct AAV vector: clone the designed shRNA sequence into AAV vector. AAV vector contains necessary promoters (such as U6 promoter) and ITRs (inverted terminal repeats) and other elements.

[0059] 3. Virus packaging: The AAV vector containing ULK1-shRNA is co-transfected with the helper plasmid into packaging cells to produce and package into infectious AAV virus particles.

[0060] 4. Infect host cells: AAV virus particles containing ULK1-shRNA are used to infect target host cells. AAV virus has low immunogenicity and a wide range of host cell infection capabilities, and can efficiently deliver shRNA into host cells.

[0061] 5. shRNA expression and RNAi mechanism: In infected host cells, shRNA driven by the U6 promoter will be cleaved into siRNA (small interfering RNA) by the cellular mechanism, and siRNA will then bind to the RNA-induced silencing complex (RISC).

[0062] 6. mRNA degradation: The RISC complex utilizes the guiding effect of siRNA to recognize and bind to the target mRNA through base complementary pairing, leading to the degradation of mRNA, thereby reducing the expression level of ULK1 protein.

[0063] Test results such as Figure 3 As shown, AAV virus was injected into the LHb brain region of mice ( Figure 3 B), Western blotting confirmed that ULK1 protein in the LHb brain region was significantly reduced ( Figure 3 CD), and through SPT and TST behavioral experiments, it was found that the percentage of sugar water preference of ULK1-shRNA mice was significantly reduced ( Figure 3 E), the immobility time in the tail suspension test increased significantly ( Figure 3 F) shows that when ULK1 levels in LHb are downregulated by interfering RNA technology, mice exhibit depressive-like behaviors.

[0064] Example 4: Overexpression of ULK1 in the LHB brain region using recombinant adeno-associated virus

[0065] The control virus rAAV-CMV-3XFlag-WPRE-hGH polyA was injected into the LHb of wild-type mice, and the control virus rAAV-CMV-3XFlag-WPRE-hGH polyA and ULK1 overexpression virus rAAV-CMV-Ulk1-3XFlag-WPREs were injected into the LHb of ULK1+ / - mice, and CTB-594 was injected at the same time to mark the injection site. Depression-like behavior was detected three weeks later.

[0066] The construction method of ULK1 overexpression virus rAAV-CMV-Ulk1-3XFlag-WPREs is as follows:

[0067] 1. Design of expression vector: Select CMV promoter to drive the target gene ULK1.

[0068] 2. Construction of recombinant plasmid: Using rAAV-CMV-3XFlag-WPRE-hGHpolyA as a plasmid vector, PCR amplification or restriction endonuclease cleavage was used to construct a recombinant plasmid containing the target gene, promoter, reporter gene and AAV inverted terminal repeat (ITR) sequence (such as Figure 4 shown):

[0069] 3. Virus packaging: The constructed recombinant plasmid and the auxiliary plasmid are co-transfected into host cells (such as HEK293T cells) and the virus is packaged using cell culture technology.

[0070] 4. Virus purification: Collect and purify virus particles from cell culture medium by ultracentrifugation and other methods to remove possible impurities such as cell debris and unpackaged plasmid DNA.

[0071] 5. Virus titer determination: Use quantitative PCR and other methods to determine the virus titer to ensure that the virus concentration is suitable for subsequent experimental applications.

[0072] 6. Validation analysis: Verify the transduction efficiency, specificity and expression effect of the viral vector in in vitro cell culture or in vivo animal models.

[0073] Test results such as Figure 5 As shown, AAV virus was injected into the LHB brain region of mice, and the successful overexpression of ULK1 protein was proved by western blotting experiments. The results of SPT, TST, and OFT experiments showed that overexpression of ULK1 in ULK1+ / - mice increased the percentage of mouse preference for sugar water, reduced the immobility time in the tail suspension test, and alleviated depressive-like behavior. In summary, the ULK1 level in the LHb of ULK1+ / - mice was upregulated by the ULK1 overexpression virus rAAV-CMV-Ulk1-3XFlag-WPREs, which can alleviate their depressive-like behavior.

[0074] Example 5: Possibility of ULK1 as a drug target for the treatment of depression

[0075] BL-918 (CAS No.: 2101517-69-3) is an activator of ULK1, which can exert its cytoprotective autophagy effect by targeting the ULK complex. Its structural formula is shown below:

[0076]

[0077] First, we screened the optimal concentration of BL918 ( Figure 6 AC), implanted tubes above the bilateral LHb brain regions of wild-type mice, so that the drug can act directly on the LHb brain region. One week later, different concentrations of BL-918 saline solution (0μM, 5μM, 10μM, 20μM) were given for 3 consecutive days, and the changes in the immobility time of wild-type mice were detected by tail suspension experiment. It was found that 20μM BL918 significantly reduced the immobility time of wild-type mice. This result also shows that BL918 may play a role in preventing depressive-like behavior. Secondly, in CSDS-induced depression-susceptible mice, implanted tubes above the bilateral LHb brain regions, and one week later, saline and 20μM BL-918 were given for 3 consecutive days, and then behavioral tests ( Figure 6 DH) found that BL918 increased the social exploration ratio and sugar water preference percentage of depression-prone mice and reduced the immobility time in the tail suspension test, indicating that injection of BL-918 into the LHb can alleviate the depressive-like behavior of depression-prone mice induced by CSDS and play a therapeutic role.

[0078] Example 6: Possibility of ULK1 as a drug target for preventing depression

[0079] Wild-type mice were divided into two groups and injected into their LHb with control virus AAV-CMV-NC-eGFP, ULK1 overexpression virus AAV-CMV-ULK1( Figure 7 A). After three weeks of viral expression, behavioral tests were performed. Then, the two groups of mice were subjected to 10 days of CSDS modeling, and depression-related behavioral tests were performed again after the modeling was completed.

[0080] Test results such as Figure 7As shown, three weeks after viral expression, SPT and TST results showed that neither the ULK1 overexpression group nor the control group showed depressive-like behavior. After CSDS modeling, SPT and TST results showed that the ULK1 overexpression group still did not show depressive-like behavior, but the control group showed obvious depressive-like behavior; SIT results showed that overexpression of ULK1 significantly reduced the proportion of depression-susceptible mice after CSDS modeling. The above experimental results show that upregulating the expression level of ULK1 in LHb can reduce the sensitivity to CSDS-induced depression.

[0081] The embodiments of the present application are described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present application.

Claims

1. Application of ULK1 agonists in the preparation of antidepressant drugs.

2. The use according to claim 1, characterized in that: The antidepressant drug is a drug for treating or preventing depression.

3. The use according to claim 1, characterized in that: The ULK1 agonist is BL-918 or a pharmaceutically acceptable salt thereof.

4. Application of ULK1 in the preparation of products for diagnosing depression.

5. Application of reagents for detecting ULK1 in the preparation of products for diagnosing depression.

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