Analog peptides, drugs for neurodegenerative diseases and uses thereof

Abstract

The present disclosure belongs to the field of medicine, and particularly relates to a mimetic peptide, a drug for neurodegenerative diseases and application, wherein a central amino acid sequence of a domain of the mimetic peptide is selected from (a) and / or (b), (a) LIR2: a.a.Y 127 CYI 130 ; (b) LIR3: a.a.Y 136 ERI 139 Considering that the LIR2-3 mimetic peptide has the advantages of fast delivery speed, high efficiency, wide spectrum, simple operation, no cell damage caused by the body environment within a certain range, and the ability to pass through the blood-brain barrier, the LIR2-3 mimetic peptide is applied to the drug research and development of neurodegenerative diseases, and particularly Alzheimer's disease.

Description

Technical Field

[0001] This disclosure pertains to the pharmaceutical field, specifically relating to mimicry peptides, drugs for neurodegenerative diseases, and their applications. Background Technology

[0002] Neurodegenerative diseases are primarily characterized by the degeneration of neurons, including Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia. Currently, there are still no effective drugs or treatments for these diseases. Although the clinical symptoms of these diseases vary, they share a very common pathological mechanism: autophagy disorder. Autophagy is a highly conserved mechanism for clearing misfolded proteins and damaged organelles within cells. These damaged proteins and organelles are encapsulated in autophagosomes, and as the autophagosomes fuse with lysosomes, these proteins and organelles degrade. With aging, the level of autophagy declines in various organs, including the brain. Studies show a positive correlation between autophagy levels and lifespan. Autophagy disorders caused by a lack of autophagy genes lead to shortened lifespan and neurodegenerative diseases in model animals. Autophagy disorders have been found in the brains of patients with various neurodegenerative diseases such as AD and PD, as well as in animal models. Drugs that enhance autophagy can also improve the pathological manifestations in AD and PD model animals. These studies suggest that autophagy disorder is a common target of neurodegenerative diseases.

[0003] Selective autophagy plays a crucial role in maintaining cellular homeostasis. Notably, selective autophagy impairment or abnormalities have been found in the brains of various degenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). For drug development, selective autophagy is a more precise target than bulk autophagy. Ignoring selective autophagy and simply improving autophagy disorders overlooks the precision of cellular substrate degradation, which may be a significant reason why molecules targeting bulk autophagy have not yet been successfully translated into drugs for treating neurodegenerative diseases. Summary of the Invention

[0004] Firstly, the present invention provides a mimic peptide, which has the advantages of not being affected by the body's environment and causing cell damage within a certain range, and being able to cross the blood-brain barrier, and can be applied to the drug development of neurodegenerative diseases, especially Alzheimer's disease.

[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solutions.

[0006] A mimic peptide, wherein the central amino acid sequence of the structural domain of the mimic peptide is selected from (a) and / or (b).

[0007] (a)LIR2:aaY 127 CYI 130 ;

[0008] (b)LIR3: aaY 136 ERI 139 .

[0009] In some publications, (a) and (b) are both derived from GPR50.

[0010] G protein-coupled receptors (GPCRs) play a significant role in drug development. These receptors possess specific signaling pathways, offering a considerable advantage in drug screening. Currently, 40% of drugs on the market target GPCRs. GPR50 (G-protein coupled receptor 50) is a solitary G protein-coupled receptor GPCR located on chromosome Xq28, belonging to the GPCR family, and is expressed in both the hippocampus and cortex of the brain. Studies have found that GPR50 expression levels are decreased in brain regions of Alzheimer's disease (AD) patients. GPR50 can inhibit BACE1 activity and reduce BACE1 expression, thereby inhibiting the production of Aβ40 and Aβ42. GPR50 can be used to develop drugs for treating Alzheimer's disease.

[0011] We performed amino acid sequence alignment of GPR50 from different species and found that human GPR50 has two potentially conserved LIR domains. These two potential LIR domains of GPR50 are LIR2(aaY) 127 CYI 130 ), LIR3(aaY 136 ERI 139 Based on GPR50 promoting endoplasmic reticulum autophagy and BACE1 degradation; mutations in LIR2 and LIR3 or deletions of LIR2-3 can lead to increased GPR50-mediated endoplasmic reticulum autophagy and BACE1 degradation. We developed and constructed the LIR2-3peptide mimetic, which can increase the binding of GPR50 to LC3, promote endoplasmic reticulum autophagy, and promote BACE1 degradation.

[0012] Currently, there are still no effective drugs for treating Alzheimer's disease (AD). Considering the advantages of LIR2-3 peptide mimetic, such as rapid, efficient, broad-spectrum delivery, simple operation, and minimal impact from the body's environment on cell damage within a certain range, as well as its ability to cross the blood-brain barrier, LIR2-3 peptide mimetic is being applied to drug development.

[0013] In some publications, (a) and (b) are LIR2-3, and the LIR2-3 sequence is: aa125-143.

[0014] In some disclosures, the mimic peptide is composed of an LIR2-3 sequence fused to the C-terminus of the protein transduction domain (YGRKKRRQRRR) of the human immunodeficiency virus (HIV) TAT protein, wherein the sequence of the mimic peptide is YGRKKRRQRRRNRYCYICHSLQYERIFSVR.

[0015] Secondly, this invention provides an expression vector. The expression vector contains a mimic peptide, which, within a certain range, is not affected by the body's environment and will not cause cell damage, and has the advantage of being able to cross the blood-brain barrier. This allows it to be applied in the development of drugs for neurodegenerative diseases, especially Alzheimer's disease.

[0016] To achieve the above-mentioned objectives, the present invention employs the following technical solutions.

[0017] An expression vector, characterized in that it contains at least one copy of the mimic peptide as described in the first aspect.

[0018] Thirdly, the present invention provides a host cell. The host cell contains an expression vector containing a mimic peptide. This mimic peptide has advantages such as being unaffected by the body's environment and thus not causing cell damage, and being able to cross the blood-brain barrier. These advantages make it suitable for application in drug development for neurodegenerative diseases, particularly Alzheimer's disease.

[0019] To achieve the above-mentioned objectives, the present invention employs the following technical solutions.

[0020] A host cell containing the expression vector described in the second aspect.

[0021] Fourthly, the present invention provides the use of an active substance in the preparation of an inhibitor of BACE1 protein, said active substance comprising any of the following aspects:

[0022] (1) The mimetic peptide as described in any of the first aspects;

[0023] (2) The expression carrier as described in the second aspect;

[0024] (3) Host cells as described in the third aspect.

[0025] Inhibitors are applied to BACE1 protein inhibition, and the inhibitory properties of BACE1 protein are extended to the development of drugs for neurodegenerative diseases, especially Alzheimer's disease.

[0026] Fifthly, the present invention provides the use of an effective substance in the preparation of an accelerator for endoplasmic reticulum autophagy, said effective substance comprising any of the following aspects:

[0027] (1) The mimetic peptide as described in any of the first aspects;

[0028] (2) The expression carrier as described in the second aspect;

[0029] (3) Host cells as described in the third aspect.

[0030] The promoters are applied to promote endoplasmic reticulum autophagy, and the promoting properties of endoplasmic reticulum autophagy are extended to the development of drugs for neurodegenerative diseases, especially Alzheimer's disease.

[0031] Sixthly, the present invention provides the use of an active substance in the preparation of a medicament for neurodegenerative diseases, said active substance comprising any of the following aspects:

[0032] The active substance comprises any of the following:

[0033] (1) The mimetic peptide as described in any one of claims 1-4;

[0034] (2) The expression vector as described in claim 5;

[0035] (3) The host cell as described in claim 6. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0037] Figure 1 Schematic diagram showing two potential LIR sites on GPR50, among which Figure 1 The 'a' in the GPR50 is a human-derived compound with two potentially more conservative LIR domains. Figure 1 b in the sequence is the LIR site sequence on human GPR50.

[0038] Figure 2 The specific construction and sequence of the LIR2-3 mimic peptide are shown in the figure; where the white box represents the TAT short peptide sequence and the gray box represents the GPR50-LIR2-3 short peptide sequence.

[0039] Figure 3 This is a schematic diagram illustrating how LIR2-3 peptide mimetic promotes BACE1 degradation;

[0040] Figure 4 This is a schematic diagram illustrating how LIR2-3 peptide mimetic enhances the level of endoplasmic reticulum autophagy (ERphagy).

[0041] Figure 5 This is a schematic diagram showing how LIR2-3 peptide mimetic can cross the blood-brain barrier; where a: schematic diagram of the location of brain tissue regions bd, bd: FITC+ signal regions of the mouse brain in the hippocampus (b), cortex (c), and corpus callosum (d) as captured by confocal microscopy.

[0042] Figure 6 LIR2-3 peptide mimetic reduces BACE1 protein levels in the brains of AD mice. a: Seven-month-old mice were injected with LIR2-3 peptide mimetic using stereotactic injection, and brain tissue proteins were extracted to detect BACE1 protein levels in the hippocampus. b: Seven-month-old mice were injected with LIR2-3 peptide mimetic via intraperitoneal injection, and brain tissue proteins were extracted to detect BACE1 protein levels in the cortical region.

[0043] Figure 7 This is a schematic diagram illustrating how LIR2-3 peptide mimetic reduces Aβ levels in APP / PS1 mice. Detailed Implementation

[0044] The technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0045] GPR50 (Gprotein coupled receptor 50) is a solitary G protein coupled receptor GPCR located on Xq28 of the X chromosome.

[0046] Based on the fact that GPR50 promotes endoplasmic reticulum autophagy and BACE1 degradation; mutations in LIR2 and LIR3, or deletion of LIR2-3, can lead to increased GPR50-mediated endoplasmic reticulum autophagy and BACE1 degradation, we developed and constructed an LIR2-3 peptidemimetic. This mimic peptide can increase the binding of GPR50 to LC3 and promote endoplasmic reticulum autophagy. Figure 4 ), promotes BACE1 degradation ( Figure 3 LIR2-3 mimic peptide was injected into the brains of 7-month-old APP / PS1 mice via stereotactic injection. Within just 7 days, it was able to downregulate BACE1 levels in the brains of APP / PS1 mice. Figure 6In addition, this mimic peptide can cross the blood-brain barrier and enter the brain. Figure 5 Intraperitoneal injection of the LIR2-3 mimic peptide successfully reduced BACE1 levels and Aβ plaque density in the brains of APP / PS1 mice. Figure 6 and Figure 7 Currently, there are no effective drugs for treating Alzheimer's disease (AD). Considering the advantages of LIR2-3 peptide mimicry, such as rapid, efficient, broad-spectrum delivery, simple operation, and minimal cellular damage due to environmental factors within a certain range, as well as its ability to cross the blood-brain barrier, it is promising to apply LIR2-3 peptide mimicry to drug development.

[0047] Example 1:

[0048] There are two potential LIR sites on GPR50.

[0049] We compared the amino acid sequences of GPR50 from different species and found that human GPR50 has two potentially conserved LIR domains. Figure 1 a). The two potential LIR domains of GPR50 are LIR2 (aaY127CYI130) and LIR3 (aaY136ERI139) ( Figure 1 b).

[0050] We performed amino acid sequence alignment on GPR50 from different species, using the classic LIR site formula (-D / EW / F / YxxL / I / VD / E-) to identify two potential LIR sites: LIR2 (aa127-130, YCYI) and LIR3 (aa136-139, YERI) (labeled) (a), and the LIR site sequence on human GPR50 (b). In subsequent experiments, we mutated these LIR sites to alanine, which was marked in orange as an acidic amino acid.

[0051] LIR2-3 peptide mimetic treatment for Alzheimer's disease

[0052] Constructing LIR2-3 peptide mimetic

[0053] Based on the characteristics of GPR50 as an endoplasmic reticulum autophagy receptor and the fact that the presence of LIR2-3 inhibits its binding to LC3, we believe that the LIR2-3 sequence (aa125-143) is a key sequence regulating GPR50 as an autophagy receptor. To enhance the delivery efficiency and permeability of the peptide within cells, we fused the LIR2-3 sequence (aa125-143) to the C-terminus of the protein transduction domain (YGRKKRRQRRR) of the human immunodeficiency virus (HIV) TAT protein. The specific construction and sequence of the LIR2-3 mimic peptide are shown in the figure (…). Figure 2 ).

[0054] in Figure 2 LIR2-3 peptide mimetic sequence. White box: TAT short peptide sequence; gray box: GPR50-LIR2-3 short peptide sequence.

[0055] LIR2-3 peptide mimetic promotes BACE1 degradation

[0056] LIR2-3 peptide mimetic enhances the binding of GPR50 to LC3. Our research indicates that GPR50 promotes BACE1 degradation via autophagy. Therefore, we investigated whether LIR2-3 peptide mimetic could promote BACE1 degradation. We transfected HEK-293T cells with the BACE1-HA plasmid, and added 0, 5, and 10 μM of LIR2-3 peptide mimetic 26 hours before cell harvest. Cells were harvested 48 hours after transfection, and proteins were extracted for Western blotting. The results showed that the degree of BACE1 degradation increased with increasing peptide concentration. Figure 3 This indicates that the LIR2-3 peptide mimetic promotes BACE1 degradation.

[0057] in Figure 3 LIR2-3 peptide mimetic promotes BACE1 degradation. HEK-293T cells were transfected with the BACE1-HA plasmid. After 22 hours, cells were treated with different concentrations of LIR2-3 peptide mimetic for 26 hours. Protein samples were collected after 26 hours, and BACE1-HA protein levels were measured. Results are expressed as mean ± SEM. One-way ANOVA was used for comparisons among multiple groups, and LSD analysis was performed. *: p < 0.05. n = 3 independent replicates.

[0058] LIR2-3 peptide mimetic enhances endoplasmic reticulum autophagy (ERphagy) levels.

[0059] Does the LIR2-3 peptide mimetic play a role in GPR50-mediated endoplasmic reticulum autophagy? We transfected the endoplasmic reticulum autophagy reporter plasmid pCW57-CMV-ssRFP-GFP-KDEL into Hela-GPR50-FLAG cells to detect the level of endoplasmic reticulum autophagy. Four hours after transfection with the pCW57-CMV-ssRFP-GFP-KDEL plasmid, cells were treated with 0.5 mg / ml doxycycline (DOX) to initiate KEDL plasmid expression. At 0, 0.5, 1.0, and 2.0 hours before sample collection, 10 μM of the LIR2-3 peptide mimetic was added. Subsequently, cells were mounted and GFP was detected. - RFP + The number of red fluorescent dots was used to measure the level of endoplasmic reticulum autophagy. Results showed that with increasing LIR2-3 peptide mimetic concentration, GFP... - RFP + The number of red fluorescent dots also increased significantly. Figure 4 The results indicate that LIR2-3 peptide mimetic enhances endoplasmic reticulum autophagy, and the increase in endoplasmic reticulum autophagy is positively correlated with the concentration of short peptides.

[0060] in, Figure 4 LIR2-3 peptide mimetic enhances endoplasmic reticulum autophagy (ERphagy) levels. Hela-GPR50-FLAG cells were transfected with the pCW57-CMV-ssRFP-GFP-KDEL plasmid and treated with 10 μM LIR2-3 peptide mimetic for 0, 0.5, 1.0, and 2.0 hours before mounting. GFP levels were then statistically analyzed. - RFP + Fluorescent dotted signals. Results were expressed as mean... + SEM analysis showed that univariate ANOVA was used for comparisons between multiple groups, with LSD analysis, **: p < 0.01. n = 40 cells were obtained from 3 independent replicates. Scale bar: 10 μm. LIR2-3 peptide mimetic can cross the blood-brain barrier.

[0061] Neurodegenerative diseases are a class of chronic, progressive neurological disorders. Currently, there are few drugs available for treating neurodegenerative diseases, especially Alzheimer's disease. While some small molecule compounds and short peptides have been developed for treating neurodegenerative diseases, their instability in vivo and difficulty crossing the blood-brain barrier present significant challenges in drug development. Considering the role of LIR2-3 peptide mimetic in in vitro experiments, we conducted in vivo experiments in mice to verify its potential application in treating AD model mice. First, we verified whether LIR2-3 peptide mimetic could cross the blood-brain barrier and directly enter the brain to exert its effects. We administered 10 mg / kg of FITC-labeled LIR2-3 peptide mimetic to 3-month-old ICR mice via tail vein injection, twice consecutively with a 30-minute interval. Immediately after injection, the mice underwent cardiac perfusion, and frozen sections of brain tissue were prepared to observe the distribution of FITC+ in the brain. The results showed that, compared with mice injected with saline, mice injected with FITC-labeled LIR2-3peptide mimetic had a significantly lower incidence of hippocampal abnormalities. Figure 5 b) There were obvious FITC positive signals in the cortical area and the corpus callosum region, indicating that LIR2-3 peptide mimetic can cross the blood-brain barrier and directly enter the mouse brain to exert its function.

[0062] in, Figure 5 LIR2-3 peptide mimetic can cross the blood-brain barrier. Three-month-old ICR mice were injected twice via tail vein with either saline or LIR2-3 peptide mimetic (10 mg / kg), with a 30-minute interval between injections. Brain tissue sections were then photographed using fluorescence imaging after each injection. a: Schematic diagram of the location of the brain tissue regions b and d. bd: FITC+ signal regions in the mouse brain, including the hippocampus (b), cortex (c), and corpus callosum (d), as photographed using confocal microscopy. Scale bar: 20 μm. In vivo injection of LIR2-3 peptide mimetic reduced BACE1 protein levels in AD mice.

[0063] We administered short peptides to 7-month-old APP / PS1 mice and control C57BL / 6J mice over a 7-day period using two injection methods (intraperitoneal injection and stereotactic brain injection), at concentrations of 0, 0.5, and 1.0 mg / kg. At a concentration of 1.0 mg / kg, mice injected stereotactically showed a decrease in BACE1 levels in the hippocampus due to the high concentration of short peptides. Figure 6 a) Mice injected intraperitoneally with a short peptide showed a decrease in BACE1 protein levels in the cortical region. Figure 6 b). The above results indicate that LIR2-3 peptide mimetic at a concentration of 1.0 mg / kg can reduce the level of BACE1 protein in the brains of AD mice.

[0064] in, Figure 6 LIR2-3 peptide mimetic reduced BACE1 protein levels in the brains of AD mice. a: Seven-month-old mice were injected with LIR2-3 peptide mimetic via stereotactic injection, and brain tissue proteins were extracted to detect BACE1 protein levels in the hippocampus. b: Seven-month-old mice were injected with LIR2-3 peptide mimetic via intraperitoneal injection, and brain tissue proteins were extracted to detect BACE1 protein levels in the cortical region. Results were expressed as mean... + SEM analysis was performed, and multiple group comparisons were conducted using univariate ANOVA and LSD analysis. *: p < 0.05. n = 3 mice / group.

[0065] In vivo injection of LIR2-3 peptide mimetic: LIR2-3 peptide mimetic (low dose: 0.5 mg / kg / day, high dose: 1 mg / kg / day) was injected into 7-month-old male APP / PS1 transgenic mice and their wild-type mice via intraperitoneal injection and stereotactic brain injection. For mice injected intraperitoneally, different doses of the short peptide were injected continuously for 7 days, followed by brain tissue analysis of Aβ and BACE1 levels. For stereotactic brain injection, LIR2-3 peptide mimetic was injected into mice using a miniature osmotic pump (RWD, model 1002W, Shenzhen, China; flow rate 0.267 μl / h, continuous for 7 days) and subcutaneously implanted according to the manufacturer's instructions for BACE1 level analysis. The detailed procedure for implanting the osmotic pump is as follows: Before implantation, 200 μl of PBS containing 0.9% sodium chloride or LIR2-3 peptide mimetic was filled into the miniature osmotic pump, and then incubated overnight in PBS at 37°C. Implantation was performed the following day. After anesthesia, a small incision was made on the back of the mouse, and a micro-osmotic pump was subcutaneously implanted. The drug was injected into the dorsal hippocampus of the mouse brain (2.0 mm anterior and posterior to the cerebral horn, 2.15 mm medial to the cerebral horn, and 1.25 mm below the skull surface) via a catheter (RWD, SK-BIC-3). Subsequently, brain tissue was collected to detect changes in BACE1 protein levels. LIR2-3 peptide mimetic sequence: The LIR2-3 peptide mimetic sequence was designed based on short peptide design principles. Nanjing Genscript Biotech Co., Ltd. was commissioned to fuse the GPR50-LIR2-3 sequence (aa125-143, NRYCYICHSLQYERIFSVR) into the C-terminus of the protein transduction domain (YGRKKRRQRRR) of the human immunodeficiency virus (HIV) TAT protein. In vivo injection of the LIR2-3 peptide mimetic reduced Aβ levels in AD mice.

[0066] Aβ is the most prominent pathological marker in Alzheimer's disease (AD), and Aβ accumulation is a key initiating factor in AD pathogenesis. BACE1, as a key rate-limiting enzyme in Aβ production, exerts its enzymatic activity by cleaving the amyloid precursor protein APP, thereby generating Aβ. The above results indicate that LIR2-3 peptide mimetic can reduce BACE1 protein levels; therefore, we investigated whether LIR2-3 peptide mimetic could reduce Aβ accumulation. We administered different concentrations (0, 0.5, 1.0 mg / ml) of LIR2-3 peptide mimetic to 7-month-old APP / PS1 mice via intraperitoneal injection for 7 consecutive days. Subsequently, brain tissue was perfused from the mice for immunofluorescence staining to detect the number of Aβ plaques. The results showed that the number of Aβ plaques in the cortical area decreased after treatment with 1.0 mg / kg of the short peptide. Figure 7 In summary, LIR2-3 peptidemimetic reduces the number of Aβ plaques in AD mice.

[0067] in, Figure 7 LIR2-3 peptide mimetic reduced Aβ levels in APP / PS1 mice. Seven-month-old APP / PS1 mice were injected intraperitoneally with LIR2-3 peptide mimetic, and the cortical area was stained with immunofluorescence for Aβ, and the number of Aβ plaques was counted. Results are expressed as mean + SEM. One-way ANOVA was used for comparisons among multiple groups, and LSD analysis was performed. *: p < 0.05. n = 3 mice / group. Scale bar: 200 μm.

[0068] Conclusion: LIR2-3 peptide mimetic, as a small-molecule transmembrane peptide carrying a target short peptide, can promote the degradation of BACE1 protein by transferring the target short peptide into the cell. In in vivo experiments in mice, LIR2-3 peptide mimetic reduced the level of BACE1 protein in the mouse brain and partially cleared the number of Aβ plaques in the mouse brain. Therefore, we hope that its structure can be further optimized and applied to the development of corresponding AD drugs.

[0069] Description of experimental materials and methods:

[0070] Plasmids: Wild-type cDNA sequences encoding GPR50-Human and cDNA sequences with LIR site mutations are inserted into the C-terminus with a FLAG sequence and cloned into the pcDNA3.1(+) vector between the BamHI and NotI sites. CDNA sequences encoding BACE1-Human or cDNA sequences with ubiquitination site mutations are inserted into the C-terminus with a HA sequence and cloned into the pcDNA3.1(+) vector between the BamHI and NotI sites.

[0071] Mice: GPR50 - / - Mice were obtained by replacing the second exon with Lacz-neo, and the C57BL / 6N background was maintained by crossing heterozygous transgenic mice with C57BL / 6N mice. APP / PS1 transgenic mice co-expressing the mutant human APP and PS1 genes and with a C57BL / 6J background were purchased from Jackson Laboratories (catalog number: 004462). Control mice were male mice of the same sex and age with a C57BL / 6J background. Animals were housed at the SPF Experimental Animal Center of Soochow University. Animal care and surgical procedures were approved by the Animal Care and Use Committee of Soochow University.

[0072] Immunofluorescence staining (IF): For cell samples, cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes and infiltrated with 0.3% Triton X-100 for 5 minutes. For mouse tissues, mouse brain slices were washed three times with PBS containing 0.5% Triton X-100, and then blocked with PBS containing 10% fetal bovine serum (FBS) at room temperature for 1 hour to prevent nonspecific binding. Next, diluted primary antibody was added to the brain slices and incubated overnight at 4°C. The primary antibody was washed three times with PBS, and the secondary antibody was incubated at room temperature for 1 hour. The secondary antibody was washed three times with PBS, and the slices were mounted with mounting medium containing DAPI. Images were captured using a laser confocal microscope (Zeiss, LSM 710). Images were analyzed using image ProPlus 6.0 image analysis software (Media-Conternetics, MD, USA).

[0073] The sequence of the GPR50-LIR short peptide used in the experiment: LIR2 WT: 125 NRYCYICHSL 134 LIR2mutant: 125 NRAAAACHSL 134 LIR3 WT: 131 CHSLQYERIFSVR 143 LIR3 mutant: 131 CHSLQAAAAFSVR 143 ...

[0074] Intracellular detection of endoplasmic reticulum autophagy: Cells were transfected with the pCW57-CMV-ssRFP-GFP-KDEL plasmid. Four hours after transfection, cells were treated with 0.5 mg / ml doxycycline (DOX) for 36 hours. Cell protein lysates were collected for Western blotting or immunofluorescence staining. The RFP / RFP-GFP protein ratio or the GFP-RFP-GFP protein ratio was statistically analyzed. - RFP + The number of red fluorescent dots is used to measure the level of endoplasmic reticulum autophagy.

[0075] Statistical methods: All statistical analyses were performed using SPSS 20.0. Data are presented as mean... + SEM was used to represent the data. Comparisons between two groups were performed using independent samples t-tests. Data from multiple groups were analyzed using one-way ANOVA or two-way ANOVA, followed by Fischer LSD post-hoc tests. All analyses were considered significant at a p-level of <0.05. *P<0.05**P<0.01***P<0.001

[0076] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this disclosure. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0077] The foregoing has shown and described the basic principles, main features, and advantages of this disclosure. Those skilled in the art should understand that this disclosure is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of this disclosure. Various changes and modifications can be made to this disclosure without departing from its spirit and scope, and all such changes and modifications fall within the scope of this disclosure as claimed.

Claims

1. A peptide mimic, characterized in that, The mimic peptide is composed of the LIR2-3 sequence of human GPR50 fused to the C-terminus of the protein transduction domain of human immunodeficiency virus TAT ​​protein. The LIR2-3 sequence is aa125-143 of human GPR50, and its amino acid sequence is NRYCYICHSLQYERIFSVR. The amino acid sequence of the protein transduction domain of the human immunodeficiency virus TAT ​​protein is YGRKKRRQRRR. The amino acid sequence of the mimic peptide is YGRKKRRQRRRNRYCYICHSLQYERIFSVR.

2. The use of the mimic peptide of claim 1 in the preparation of a BACE1 protein inhibitor for Alzheimer's disease.

3. The use of the mimic peptide of claim 1 in the preparation of an endoplasmic reticulum autophagy promoter for Alzheimer's disease.

4. The use of the mimetic peptide according to claim 1 in the preparation of Alzheimer's disease drugs.

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