Targeting polypeptide and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a targeted polypeptide and application thereof. It is found that in a mouse obesity model, hypothalamic arcuate nucleus and para-ventricular nucleus overexpression DEC2 can significantly induce leptin resistance and obesity; a key binding region of interaction of DEC2 and STAT3 is further analyzed, and a polypeptide (Tat-DEC2) targeting DEC2 is constructed. In-vitro experiments prove that the Tat-DEC2 polypeptide can effectively improve the leptin reactivity of DEC2 overexpression mouse hypothalamic neurons, and the effect of the Tat-DEC2 polypeptide has time dependence and concentration dependence; in-vivo experiments prove that the Tat-DEC2 polypeptide can significantly improve leptin resistance and play an effective weight reducing role. The polypeptide is used for intervening key molecules caused by leptin resistance, so that the occurrence mechanism of obesity induced by leptin resistance is understood more deeply, and meanwhile, the polypeptide has important significance in developing effective clinical treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a targeting polypeptide and its application. Background Art

[0002] The World Health Organization (WHO) defines overweight and obesity as abnormal or excessive fat accumulation that can damage health. With the development of society, overweight or obesity has not only become a serious public health problem, but its incidence rate is also showing an increasing trend year by year. Obesity is a modifiable risk factor for many diseases. In particular, visceral adipose tissue can regulate pro-inflammatory cytokines (such as interleukin-6), leading to inflammation, oxidative stress, and insulin resistance, thus increasing the risk of chronic diseases (Thomas DM, Bredlau C, Bosy-Westphal A, et al. Relationships between body roundness with body fat and visceral adipose tissue emerging from a new geometrical model. Obesity (Silver Spring), 2013, 21(11): 2264-2271.). In the past 40 years, the number of obese people has doubled. It is estimated that by 2035, nearly one-half of adults will be obese (Ostrominski JW, Powell-Wiley TM. Risk Stratification and Treatment of Obesity for Primary and Secondary Prevention of Cardiovascular Disease. Cur Atherseler Rep, 2024, 26(1): 11-23.). Obesity can not only pose a serious threat to human health, but also trigger serious psychosomatic diseases, thus causing great losses to people's bodies and imposing huge pressure on the world economy (World Obesity Federation. The economic impact of overweight & obesity in 2020 and 2060. Second edition with estimates for 161 countries. 2022.). Therefore, it is of great significance to carry out research on the pathogenic mechanism of obesity and weight loss strategies.

[0003] Obesity is not only the accumulation of body fat, but more importantly, its harm lies in causing metabolic disorders, ultimately leading to abnormal functions of organs and tissues. Many domestic and foreign research teams have conducted a series of studies on the mechanisms of metabolic disorders during the occurrence and development of obesity: The team led by Professor Qi Ling elucidated the specific mechanism by which the SEL1L-HRD1 protein complex-mediated endoplasmic reticulum stress is involved in leptin resistance in POMC neurons (Mao H, Kim GH, Pan L, et al. Regulation of leptin signaling and diet-induced obesity by SEL1L-HRD1 ER-associated degradation in POMC expressing neurons. Nat Commun. 2024 Sep 29;15(1):8435.); Sass F et al. developed a long-acting targeted NK2R agonist for regulating leptin-independent insulin sensitivity (Sass F, Ma T, Ekberg JH, et al. NK2R control of energy expenditure and feeding to treat metabolic diseases. Nature. 2024 Nov;635(8040):987-1000.); Offermanns et al. revealed the role of PTP1B-mediated endothelial cell insulin resistance in obesity-related diabetes (Cho H, Lai CC, Bonnavion R, et al. Endothelial insulin resistance induced by adrenomedullin mediates obesity-associated diabetes. Science. 2025 Feb 7;387(6734):674-682.); A series of studies by the team led by Professor Wang Qiang revealed the inflammatory mechanism by which abnormal metabolism of microglia and astrocytes mediates insulin resistance (Li Q, Zhao Y, Guo H, et al. Impaired lipophagy induced-microglial lipid droplets accumulation contributes to the buildup of TREM1 in diabetes-associated cognitive impairment. Autophagy. 2023 Oct;19(10):2639-2656; Jiang T, Li Y, He S, et al.Reprogramming astrocytic NDRG2 / NF-κB / C3 signaling restores the diabetes-associated cognitive dysfunction. (EBioMedicine. 2023 Jul; 93: 104653.) These studies provide important theoretical and experimental bases for understanding the pathogenic mechanisms of obesity and its metabolic disorders. It is not difficult to see that leptin resistance and insulin resistance, as the most prominent metabolic disorders during the occurrence and development of obesity, play important roles in the process of obesity development.

[0004] Marc Claret et al. ingeniously designed a 4-day Western diet (high-fat and high-sugar) feeding for mice. This feeding protocol does not cause weight gain and metabolic disorders but can significantly induce spatial memory impairment in mice. This result excludes the confounding factors of obesity and glucose homeostasis imbalance (Ramírez S, Haddad-Tóvolli R, Radosevic M, et al. Hypothalamic pregnenolone mediates recognition memory in the context of metabolic disorders. Cell Metab. 2022 Feb 1; 34(2): 269-284.e9.). Studies have found that measures to improve leptin resistance, such as appropriately reducing circulating leptin levels, using leptin-neutralizing antibodies or leptin sensitizers, etc., can effectively play the roles of weight loss and improving insulin resistance (Zhao S, Zhu Y, Schultz RD, et al. Partial Leptin Reduction as an Insulin Sensitization and Weight Loss Strategy. Cell Metab. 2019 Oct 1; 30(4): 706-719.e6; Fernandes C, Forny-Germano L, Andrade MM, et al. Leptin receptor reactivation restores brain function in early-life Lepr-deficient mice. Brain. 2024 Aug 1; 147(8): 2706-2717.). The above results suggest that leptin resistance precedes insulin resistance and mediates the occurrence and development of obesity. However, most weight loss drugs developed targeting leptin have failed clinically. The key bottleneck is that the problem of leptin resistance has not been effectively solved, and new ideas are urgently needed to deeply analyze the neural mechanism of leptin resistance promoting obesity.

[0005] DEC2 is an important rhythm-regulating gene (encoding the gene Bhlhe41), and is known as the famous "short sleep gene" because the P384R mutation of this gene affects the circadian rhythm and sleep duration (Honma S, Kawamoto T, Takagi Y, et al. Dec1 and Dec2 are regulators of the mammalian molecular clock. Nature. 2002 Oct 24; 419(6909):841-4; He Y, Jones CR, Fujiki N, et al. The transcriptional repressor DEC2 regulates sleep length in mammals. Science. 2009 Aug 14; 325(5942):866-70; Hirano A, Hsu PK, Zhang L, et al. DEC2 modulates orexin expression and regulates sleep. Proc Natl Acad Sci U S A. 2018 Mar 27; 115(13):3434-3439.). In addition, as a transcriptional regulator, the role of DEC2 in tumorigenesis and development has also attracted much attention (Montagner M, Enzo E, Forcato M, et al. SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors. Nature. 2012 Jul 19; 487(7407):380-4.). Interestingly, the latest research also found that DEC2 may also affect the cholesterol clearance and lysosomal degradation processes of macrophages and microglia, thus mediating the occurrence of Alzheimer's disease (AD) ( A, Novikova G, Liu Y, et al. BHLHE40 / 41 regulate microglia and peripheral macrophage responses associated with Alzheimer's disease and other disorders of lipid-rich tissues. Nat Commun. 2024 Mar 6; 15(1):2058.).

[0006] Small molecule drugs, polypeptides, and biological macromolecules (such as antibodies, viral vectors, etc.) are currently the three most promising directions for clinical prevention, treatment, and translational applications. Although gene editing technology is one of the effective targeted prevention and treatment means, it may face risks such as uncertain transfection efficiency, off-target effects, and potential activation of proto-oncogenes in clinical translation, which greatly restricts its clinical translational applications. Polypeptides have characteristics intermediate between small molecule drugs and biological macromolecules and have the following advantages in clinical translation: ① High targeting and specificity: They can accurately recognize targets through specific sequence design, reducing off-target toxicity; ② Good biocompatibility and low toxicity: Composed of natural amino acids, their metabolites are non-toxic, with good biocompatibility and fewer side effects, especially suitable for the long-term treatment of chronic diseases; ③ High affinity and potency: The binding of polypeptides to targets usually involves multiple sites, with high affinity, capable of effectively blocking protein-protein interactions; ④ Targeting complex disease mechanisms: They can target targets that are difficult for traditional drugs to act on, such as protein-protein interaction interfaces and intracellular targets; ⑤ Flexible chemical modification: Stability can be enhanced, half-life can be extended, and penetrability can be improved through chemical modification or fusion protein technology; multifunctionalization can be achieved through coupling technology; combined with new delivery technologies, diverse administration routes can be realized. Therefore, the development of targeted polypeptide inhibitors is of great significance for enriching weight loss means. Summary of the Invention

[0007] The present invention discovers that overexpression of DEC2 in the arcuate nucleus (ARC) and paraventricular nucleus (PVH) in a mouse obesity model can significantly induce leptin resistance and obesity; further analyzes the key binding region of the interaction between DEC2 and STAT3, constructs a polypeptide targeting DEC2 (Tat-DEC2), and verifies in vitro experiments that this Tat-DEC2 polypeptide can effectively improve the leptin responsiveness of DEC2-overexpressing GT1-7 cells and can play a role in improving leptin resistance and weight loss before effective weight loss. Based on this, the present invention is completed.

[0008] In a first aspect, the present invention provides a polypeptide targeting DEC2 or a pharmaceutically acceptable salt thereof, and the amino acid sequence of the polypeptide targeting DEC2 is as shown in SEQ ID NO.1.

[0009] Furthermore, the polypeptide targeting DEC2 can be conjugated with a cell-penetrating peptide (Tat) to guide DEC2 to penetrate the cell membrane, and the amino acid sequence of the Tat-conjugated targeting polypeptide (Tat-DEC2) is as shown in SEQ ID NO.2.

[0010] Even further, the Tat-conjugated targeting polypeptide can also be conjugated with a CTM tail to guide DEC2 to be degraded through the chaperone-mediated autophagy pathway, and the amino acid sequence of the Tat- and CTM-conjugated targeting polypeptide (Tat-DEC2-CTM) is as shown in SEQ ID NO.3.

[0011] Furthermore, the amino acid sequence of the polypeptide targeting DEC2 or a pharmaceutically acceptable salt thereof has at least 70% or 75% or 80% or 85% or 90% or 95% sequence identity with the amino acid sequence of SEQ ID No. 1.

[0012] Preferably, the amino acid sequence of the polypeptide targeting DEC2 or a pharmaceutically acceptable salt thereof has 95% sequence identity with the amino acid sequence of SEQ ID No. 1.

[0013] In a second aspect, the present invention provides a nucleic acid molecule that encodes the polypeptide described in the first aspect.

[0014] In a third aspect, the present invention provides a gene expression vector that includes nucleotides encoding the polypeptide described in the first aspect.

[0015] In a fourth aspect, the present invention provides a host cell that contains the nucleic acid molecule described in the second aspect and / or the expression vector described in the third aspect, and the host cell is transformed or transfected by the nucleic acid molecule described in the second aspect and / or the expression vector described in the third aspect.

[0016] Furthermore, the host cell includes bacteria, fungi, and / or animal cells.

[0017] In a fifth aspect, the present invention provides a pharmaceutical composition that contains the polypeptide described in the first aspect and, optionally, pharmaceutically acceptable excipients.

[0018] Furthermore, one or more pharmaceutically acceptable carriers can also be added to the pharmaceutical composition.

[0019] Furthermore, the pharmaceutical composition can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, and / or suppositories.

[0020] Furthermore, the preparation can be one or more of an ordinary preparation, a sustained-release preparation, and / or a controlled-release preparation.

[0021] Furthermore, various preparations can also add coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, or other materials to the pharmaceutical preparation as needed.

[0022] Furthermore, the pharmaceutical composition can be administered by injection, via a cavity, or via the respiratory tract.

[0023] Even further, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; the respiratory tract administration includes nasal administration.

[0024] In a sixth aspect, the present invention provides the use of the polypeptide described in the first aspect in the preparation of a drug for treating obesity.

[0025] Furthermore, the drug plays a role in weight loss by improving leptin resistance.

[0026] Beneficial effects

[0027] 1. The present invention found that in a mouse obesity model induced by a high-fat diet, the expression of DEC2 in the arcuate nucleus (ARC) of the hypothalamus was significantly increased; overexpression of DEC2 in the ventromedial nucleus (VMH) and dorsomedial nucleus (DMH) of the hypothalamus did not affect food intake and body weight, while overexpression of DEC2 only in the ARC and paraventricular nucleus (PVH) significantly induced leptin resistance and obesity.

[0028] 2. Based on AlphaFold artificial intelligence-assisted protein-protein interaction prediction, the present invention analyzed the key binding regions of the interaction between DEC2 and STAT3 (351-358AA of DEC2 and 146-161AA of STAT3), and constructed a Tat-conjugated polypeptide (Tat-DEC2) targeting DEC2.

[0029] 3. The present invention verified in vitro experiments that the Tat-DEC2 polypeptide can effectively improve the leptin responsiveness of DEC2-overexpressing GT1-7 cells (mouse hypothalamic neurons), manifested as an increase in the basal expression level of POMC and an increase in the expression of p-STAT3 and POMC after leptin stimulation.

[0030] 4. The present invention verified in vitro experiments that the effect of the Tat-DEC2 polypeptide is time-dependent and concentration-dependent, and the treatment peak is reached 4 hours after a single incubation; the treatment peak is reached at a single dose of 100 nM.

[0031] 5. Continuous administration into the third ventricle of obese mice in the present invention can significantly improve leptin resistance and play an effective role in weight loss, manifested as an improvement in leptin-mediated reduction of food intake and body weight, an increase in the expression of p-STAT3 in ARC neurons mediated by leptin, and an enhancement of the discharge of ARC neurons after leptin perfusion. POMC neurons, and an increase in the expression of p-STAT3 in ARC POMC neurons and enhanced discharge of ARC neurons after leptin perfusion.

[0032] The above results suggest that DEC2 may be another important molecular target for improving leptin resistance; using polypeptides to intervene in key molecules caused by leptin resistance not only provides a deeper understanding of the pathogenesis of leptin resistance-induced obesity, but also has important significance for the development of effective clinical therapeutic drugs. Therefore, the Tat-conjugated targeting polypeptide (Tat-DEC2) constructed by the present invention has advantages in the clinical transformation of weight loss. Brief description of the drawings

[0033] Figure 1 Hypothalamic transcriptome analysis of high-fat diet-induced obese mice. A. Volcano plot of differentially expressed genes; B. Heatmap of differentially expressed genes; C. Functional enrichment of differentially expressed genes.

[0034] Figure 2 Effect of overexpressing DEC2 in ARC on obesity in mice. A. Schematic diagram of stereotaxic positioning; B. Stereotaxic example diagram of overexpressing DEC2 in ARC and statistical analysis of its effect on mouse body weight; C. Stereotaxic example diagram of overexpressing DEC2 in VMH and statistical analysis of its effect on mouse body weight; D. Stereotaxic example diagram of overexpressing DEC2 in DMH and statistical analysis of its effect on mouse body weight; E. Stereotaxic example diagram of overexpressing DEC2 in PVH and statistical analysis of its effect on mouse body weight. ***P<0.001.

[0035] Figure 3 Screening and verification of the interacting protein STAT3 of DEC2. A. Schematic diagram of the DEC2 motif; B. Schematic diagram of the STAT3 motif; C. Prediction of the binding sites of DEC2 transcriptional regulatory genes to promoters; D. Scoring of transcriptional regulatory genes of the POMC promoter; E. Statistical analysis of luciferase reporter gene experiments on leptin-induced Pomc transcription; F. Statistical analysis of leptin-induced STAT3 binding enrichment in the POMC promoter; G. Verification of the interaction between DEC2 and STAT3 in GT1-7 cells stably transfected with LV-Bhlhe41; H. Verification of the interaction between DEC2 and STAT3 in GT1-7 cells stably transfected with LV-Bhlhe41; I. Verification of the interaction between DEC2 and STAT3 in ARC tissues overexpressing DEC2; J. GST-Pull down verification of DEC2-STAT3 interaction; K. PLA verification of the promotion of DEC2-STAT3 interaction by overexpressing DEC2; L. Molecular docking to analyze the key sites of DEC2-STAT3 interaction.

[0036] Figure 4 Purification and detection of synthetic polypeptides by HPLC and MS. A. HPLC purification of the target peptide segment; B. MS detection of the molecular weight of the target peptide segment.

[0037] Figure 5Effects of Tat-DEC2 on leptin resistance and body weight in mice. A. Experimental flow chart; B. Schematic diagram of polypeptide; C. Statistical analysis of the effects of Tat-DEC2 on the expression of DEC2 and POMC in LV-Bhlhe41-stably transfected GT1-7 cells; D. Statistical analysis of the effects of Tat-DEC2 on leptin-induced p-STAT3 expression in LV-Bhlhe41-stably transfected GT1-7 cells; E. Experimental flow chart; F. Statistical analysis of the effects of Tat-DEC2 at different times on the expression of DEC2 in LV-Bhlhe41-stably transfected GT1-7 cells; G. Experimental flow chart; H. Statistical analysis of the effects of Tat-DEC2 at different concentrations on the expression of DEC2 in LV-Bhlhe41-stably transfected GT1-7 cells. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0038] Figure 6 Effects of Tat-DEC2 on leptin resistance and body weight in mice. A. Experimental flow chart; Statistical analysis of cumulative body weight gain in DIO mice with gradient doses of Tat-DEC2: B.; C. Statistical analysis of cumulative food intake; D. Experimental flow chart; Effects of Tat-DEC2 on DIO mice: E. Statistical analysis of leptin-mediated body weight loss; F. Statistical analysis of leptin-mediated reduction in food intake; G. Leptin-mediated ARC POMC Representative diagram and statistical analysis of p-STAT3 expression; H. ARC of two groups of mice POMC Examples of spontaneous discharge before and after leptin perfusion; I. ARC of two groups of mice POMC Number of cells with different discharge patterns recorded; ARC of two groups of mice POMC Before and after leptin perfusion: J. Statistical analysis of changes in action potential frequency; K. Statistical analysis of changes in action potential amplitude; L. Statistical analysis of the degree of cell membrane depolarization; M. Statistical analysis of changes in the ratio of spontaneous discharge frequencies. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Specific implementation manners

[0039] The specific implementation manners of the present invention will be further described below. It should be noted here that the description of these implementation manners is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the following described implementation manners can be combined with each other as long as they do not conflict with each other.

[0040] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional channels unless otherwise specified.

[0041] The DEC2 of the present invention (UniProt No. Q99PV5): is an important rhythm-regulating gene (encoding gene Bhlhe41), and is known as the famous "short sleep gene" because the P384R mutation of this gene affects the circadian rhythm and sleep time. In addition, as a transcriptional regulator, the role of DEC2 in tumorigenesis and development has also attracted much attention.

[0042] The complete amino acid sequence of DEC2 of the present invention is shown in SEQ ID NO. 4:

[0043] MDEGIPHLQERQLLEHRDFIGLDYSSLYMCKPKRSLKRDDTKDTYKLPHRLIEKKRRDRI

[0044] NECIAQLKDLLPEHLKLTTLGHLEKAVVLELTLKHLKALTALTEQQHQKIIALQNGERSLK

[0045] SPVQADLDAFHSGFQTCAKEVLQYLARFESWTPREPRCAQLVSHLHAVATQLLTPQVPSG

[0046] RGSGRAPCSAGAAAASGPERVARCVPVIQRTQPGTEPEHDTDTDSGYGGEAEQGRAAVK

[0047] QEPPGDSSPAPKRPKLEARGALLGPEPALLGSLVALGGGAPFAQPAAAPFCLPFYLLSPSAA

[0048] AYVQPWLDKSGLDKYLYPAAAAPFPLLYPGIPAAAAAAAAAAFPCLSSVLSPPPEKAGAT

[0049] AGAPFLAHEVAPPGPLRPQHAHSRTHLPRAVNPESSQEDATQPAKDAP

[0050] The amino acid sequence of the polypeptide targeting DEC2 of the present invention is shown in SEQ ID NO. 1: QHLQDVRKRVQDLEQK

[0051] The amino acid sequence of the targeting polypeptide conjugated with Tat (Tat-DEC2) of the present invention is shown in SEQ ID NO. 2: YGRKKRRQRRR-QHLQDVRKRVQDLEQK

[0052] The amino acid sequence of the targeting polypeptide (Tat-DEC2-CTM) coupling Tat and CTM of the present invention is as shown in SEQ ID NO.3: YGRKKRRQRRR-QHLQDVRKRVQDLEQK-KFERQKILDQRFFE

[0053] AAV-CMV: As a control virus for AAV-CMV-Bhlhe41, it has the same vector and promoter structure as AAV-CMV-Bhlhe41 and does not carry the gene sequence for overexpressing Bhlhe41.

[0054] Example 1 Main Experimental Techniques and Methods

[0055] 1. High-fat diet-induced obese mouse model (DIO)

[0056] C57BL / 6J mice at 8 - 10 weeks of age were fed a high-fat diet (Research Diets, D12492) for 8 weeks, and the average body weight gain was 10.8 ± 5.1 g.

[0057] 2. Taking POMC-tdTomato fluorescently labeled transgenic mice as an example

[0058] POMC-Cre mice were mated with Rosa26-LSL-tdTomato mice to produce POMC-tdTomato mice, and the Cre / 1oxP system was used to achieve tdTomato fluorescent labeling of POMC neurons in the brain.

[0059] 3. Leptin sensitivity experiment

[0060] Mice were pre-adapted to single-cage housing for 1 week. Mice were intraperitoneally injected with normal saline twice a day (10 am and 6 pm) for 3 days, and then received intraperitoneal injection of 1 mg / kg recombinant leptin for 3 days. Body weight and food intake were measured once a day.

[0061] 4. Metabolic monitoring

[0062] Metabolic monitoring was performed using a comprehensive experimental animal monitoring system (Columbus Instruments, CLAMS). After mice were adapted to the metabolic monitoring system for 3 days, feeding, drinking, and metabolic parameters were continuously monitored for 3 days, including: oxygen consumption, carbon dioxide production, respiratory exchange rate, heat production, horizontal and vertical movement amounts, food intake, water intake, etc.

[0063] 5. Stereotaxic injection into the brain

[0064] After anesthetizing the mice with 2% isoflurane, the heads were shaved and the mice were fixed supine on a stereotaxic apparatus. After disinfection, the scalp on the top of the skull was incised and the skull was fully exposed. The plane of the top of the skull was corrected according to the Bregma and Lamda points. Coordinates were determined with reference to the standardized mouse brain atlas. A dental drill was used to drill a hole in the skull and the dura mater was removed. The AAV virus was stereotaxically injected into the bilateral ARC (Bregma -1.70 mm AP, ±0.20 mm ML, -5.80 mm DV; 200 nL, 40 nL / min). After leaving the needle in place for 10 min, the area was disinfected and the scalp on the top of the skull was sutured layer by layer. 0.5 ml of normal saline was subcutaneously injected into the necks of the mice, and they were kept warm and individually caged until they woke up.

[0065] 6. Single-cell transcriptomics

[0066] The bilateral ARC was stored in liquid nitrogen, and total RNA was extracted to construct a library. Sequencing was performed on the machine to obtain data. The quality of the raw data was controlled and compared with the reference sequence. After exon quantification, gene expression quantification was performed. According to the differential gene expression of different cells, dimensionality reduction and clustering were carried out, and gene differential analysis was performed on each type of cell and cell subset (including: GO enrichment analysis, Pathway enrichment analysis, clustering analysis, specific expression and co-expression analysis, protein network interaction analysis, transcription factor analysis, etc.).

[0067] 7. Screening of DEC2 interacting proteins by CO-IP combined with mass spectrometry

[0068] GT1-7 cells were routinely cultured, and LV-Bhlhe41 was added to transfect the cells to overexpress DEC2. Positive clones were screened by the resistance gene. Protein samples were collected, and the bait protein antibody was used to precipitate the bait protein. SDS-PAGE was used to separate the proteins, and the target band was cut after Coomassie brilliant blue staining. After trypsin digestion, electroelution was used to load the sample for liquid chromatography-mass spectrometry separation / analysis. The peptides were collected and sequenced by Edman degradation for screening and analysis of the target protein.

[0069] 8. GST-Pull down

[0070] After IPTG induction, the coding DEC2-GST fusion protein and the control GST protein were expressed in HEK293T cells. The GST fusion protein was captured using glutathione agarose 4B magnetic beads, and then Flag-STAT3 was added. Binding was carried out at 4 °C for 3 hours. After washing the magnetic beads with PBS, SDS loading buffer was added and boiled. The bound proteins were loaded onto a 10% SDS-PAGE gel for Western blot analysis.

[0071] 9. Protein-protein interaction proximity ligation technique

[0072] Proximity ligation assay (PLA) is a highly sensitive immunoassay method mainly used to detect the interaction of target proteins - protein interactions and visualize protein interactions at the single-cell level. This method uses a pair of probes (PLA probes) labeled with a segment of oligodeoxynucleotide (single-stranded DNA) monoclonal or polyclonal antibodies to recognize the target protein. When these two probes recognize the same protein, the distance between the two probes approaches, and a proximity effect can be generated. At the same time, by adding a segment of ligation oligodeoxynucleotide complementary to the DNA linked to the antibody, the DNA on the PLA probe will be complementary to this segment of DNA through complementary base pairing, and then under the action of ligase, the fragment DNA on the PLA probe is ligated together to form a circular structure, generating a detectable signal through rolling circle amplification, and quantitatively evaluating the interaction of the target protein through the presence, absence, and intensity of the fluorescence signal.

[0073] 10. Molecular docking

[0074] Obtain the crystal structures of the target proteins DEC2 and STAT3 from the AlphaFold3 protein database and the RCSB PDB database. For the obtained protein crystals, use the Protein Preparation Wizard module of Schrödinger software to perform protein pretreatment, natural ligand charge state regeneration, hydrogen bond assignment optimization, protein energy minimization, dehydration, etc. respectively. Perform protein-protein interaction simulation (using the protein-protein docking module) on the processed proteins, set the Number of ligand rotations to probe to 70000 and set the Maximum poses to return to 30. The lower the interaction score, the lower the binding free energy of the ligand and the receptor, and the higher the binding stability. Label different chains of the protein-protein interaction complex with the lowest interaction score with different colors and add Surface to display the 3D stereoscopic view. In addition, use the Protein Interaction Analysis module to determine the specific region where STAT3 binds to DEC2.

[0075] 11. Luciferase reporter gene assay

[0076] Use bioinformatics methods to analyze and predict the possible binding sites of DEC2 and / or STAT3 in the POMC promoter region, design primers and clone the required target promoter fragment from genomic DNA by PCR. Insert this fragment into the Luciferase reporter gene plasmid, and co-transfect the reporter gene plasmid and the transcription factor expression plasmid into GT1-7 cells. Extract proteins and use them for luciferase detection. After adding the substrate, measure the activity of luciferase, calculate the relative fluorescence intensity and compare it with the empty vector control.

[0077] 12. Electrophysiology

[0078] A. Preparation of ex vivo brain slices: After anesthetizing the mice, they were decapitated. The whole brain tissue was quickly dissected and rinsed with pre-cooled oxygenated solution. Brain slices with a thickness of about 400 μm were cut along the coronal plane using a vibratome and then incubated in artificial cerebrospinal fluid (ACSF) saturated with a mixture of 95% O2 and 5% CO2.

[0079] B. Spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs): One brain slice was placed at the bottom of a submerged recording chamber (0.5 - 1.0 mL) and fixed with a U-shaped metal frame nylon net above. ACSF was continuously perfused at a rate of 3 mL / min. Neuronal discharge activity was recorded in the voltage clamp mode. The voltage was clamped at -70 mV, and tdTomato + ARC POMC neurons were subjected to patch clamp recording to record sEPSCs; 0.5 mM TTX was added to the perfusion solution, and mEPSCs were recorded 10 minutes later.

[0080] C. Spontaneous action potential (AP): tdTomato + ARC POMC neurons were found to record the discharge activity. 100 nM leptin was added to the perfusion solution, and the changes in AP of POMC neurons 5 minutes before and after leptin perfusion were recorded.

[0081] Example 2 Transcriptome Analysis of Hypothalamus in High-Fat Diet-Induced Obese Mice

[0082] After 8 weeks, the hypothalamus of high-fat diet-induced obese (DIO) WT mice was removed for transcriptomic sequencing, and bioinformatics analysis was combined to analyze the differential gene expression and its related biological function changes.

[0083] The results showed that the expression of the differential gene Bhlhe41 in the hypothalamus of high-fat diet-induced obese mice increased significantly, and its related differential functions were mostly enriched in biological functions such as synapse formation, vesicle release, and synaptic transmission ( Figure 1 ). These results preliminarily found that hypothalamic DEC2 may play an important role in the occurrence and development of obesity.

[0084] Example 3 Stereotactic Injection of AAV-CMV-Bhlhe41 into Different Hypothalamic Nuclei of WT Mice to Induce Obesity in Mice

[0085] To further study the role of the protein DEC2 encoded by the Bhlhe41 gene in the hypothalamus, in this invention, WT mice were respectively stereotactically injected with AAV-CMV-Bhlhe41 or control virus (AAV-CMV) into different hypothalamic nuclei ARC, VMH, DMH, and PVH, and the body weight changes of the two groups of mice were observed.

[0086] The results showed that overexpression of DEC2 only in the ARC significantly induced obesity in mice, while overexpression in other hypothalamic nuclei such as VMH, DMH, and PVH did not show significant obesity phenotypes( Figure 2 ). This part of the results proved that the hypothalamic ARC is the key nucleus for DEC2-mediated obesity.

[0087] Example 4 Screening and verification of the DEC2 interacting protein STAT3

[0088] Previous studies reported that the biological function of DEC2 is mainly as a transcription factor that can recognize the E-box element in the promoter region of target genes. Therefore, the present invention first verified whether it directly binds to the E-box sequence of POMC and inhibits mRNA transcription. Through analysis of the JASPAR transcription factor binding profile database (https: / / jaspar.elixir.no / ), it was found that there was no obvious interaction between DEC2 and the promoter regions of POMC and STAT3.

[0089] Next, the present invention used the POMC-luciferase reporter gene experiment and found that both the basal and leptin-induced POMC luciferase activities were significantly reduced in the DEC2 overexpression cell group; the ChIP combined with quantitative PCR experiment found that overexpression of DEC2 significantly reduced the binding of leptin-induced p-STAT3 to the POMC promoter.

[0090] In addition, the present invention also verified through CO-IP, GST-Pull down, and PLA experiments that DEC2 can directly interact with STAT3, and the molecular docking results showed that the key binding regions for the DEC2-STAT3 interaction are 351-358AA of DEC2 and 146-161AA of STAT3( Figure 3 ).

[0091] This part of the results suggests that DEC2 inhibits leptin-mediated STAT3 phosphorylation and POMC transcription by directly binding to STAT3.

[0092] Example 5 Construction of Tat-DEC2 polypeptide

[0093] Based on the interaction sites of DEC2-STAT3 analyzed by molecular docking (the key binding region of the interaction between DEC2 and STAT3 (351-358 AA of DEC2 and 146-161 AA of STAT3)), a cell-penetrating peptide (Tat)-conjugated targeting polypeptide (YGRKKRRQRRR-QHLQDVRKRVQDLEQK, Tat-DEC2) was constructed, and a CTM tail (KFERQKILDQRFFE) was conjugated to guide the degradation of DEC2 through the chaperone-mediated autophagy pathway to observe the direct effect of the polypeptide on DEC2. The specific synthesis steps of the polypeptide are as follows:

[0094] 1. Calculate the weight of each raw material according to the weight of the target polypeptide (all raw materials are protected amino acids, and there are corresponding amino acid raw materials at the end, as shown in Table 1);

[0095] 2. Put dichloride resin (RINK resin for amidated polypeptide) into a polypeptide solid-phase synthesis tube and soak it with appropriate DCM;

[0096] 3. Wash the resin with DMF and then drain it. Repeat this to drain the resin;

[0097] 4. Weigh the first amino acid at the C-terminus + DCM + DIEA and add them to the reactor, and then react the reactor;

[0098] 5. Block with a methanol solution (methanol:DIEA:DCM = 1:1:2), then wash with DMF and drain;

[0099] 6. Add 20% piperidine solution to the reactor, react to remove the Fmoc protecting group; after deprotection, wash with DMF and then drain;

[0100] 7. Take the resin and detect it by the ninhydrin method. If the resin has color, it means the deprotection is successful; if there is no color, repeat step 6;

[0101] 8. Weigh the second amino acid at the C-terminus (molar mass 3 times that of the first amino acid) + HOBT + DIC and add them to the reactor to react;

[0102] 9. Take the resin for detection and detect it by the ninhydrin method. If the resin has color, it means the condensation is incomplete, and extend the reaction time; if the resin is colorless, it means the reaction is complete; after the reaction is complete, wash the resin with DMF and then drain;

[0103] 10. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and react to remove the Fmoc protecting group on the resin; after deprotection, wash with DMF and then drain and detect whether the protection is removed;

[0104] 11. Detect the resin by the ninhydrin method. If the resin has color, it indicates that the deprotection is successful; if there is no color, repeat step 6.

[0105] 12. Connect the remaining amino acids successively according to steps 8 - 11.

[0106] 13. Use a cleavage reagent to remove all the polypeptide protecting groups and the resin, and send it for purification.

[0107] 14. Separate the target peptide segment from impurities by a high - performance liquid chromatography instrument (HPLC), and send it for lyophilization.

[0108] 15. After the target peptide segment is lyophilized into powder, perform QC inspections on HPLC and mass spectrometry (MS).

[0109] Figure 4 The results show that the targeted polypeptide (Tat - DEC2) of the present invention was successfully synthesized and purified. Its theoretical molecular weight is 3562.08, the detected actual molecular weight is 3562.00, and the actual purity is 97.78%.

[0110] Table 1 Amino acids and their corresponding raw materials

[0111]

[0112]

[0113] Example 6 Effect of Tat - DEC2 polypeptide on the leptin responsiveness of LV - Bhlhe41 - stably transfected GT1 - 7 cells

[0114] Use LV - Bhlhe41 - stably transfected GT1 - 7 cells, incubate with polypeptides at different concentrations (20, 50, 100, 200, 500 nM) and at 200 nM for different times (1, 2, 4, 6, 8 h) for 30 min, then collect the cells for western blot detection. Add 100 nM recombinant leptin for stimulation 30 min before sampling, and detect the changes in the expression of DEC2, p - STAT3, STAT3, and POMC after polypeptide incubation.

[0115] The results show that: polypeptide treatment significantly increased the expression of p - STAT3 and POMC in LV - Bhlhe41 - stably transfected GT1 - 7 cells, and showed time (peaking at 4 h) and concentration (optimal at 100 nM) dependence ( Figure 5 )

[0116] Example 7 Effect of Tat - DEC2 polypeptide on the body weight of high - fat diet - induced obese mice

[0117] The DIO model mice were cannulated in the third ventricle for 30 consecutive days, and different concentrations (10, 20, 30, 100, 200 μM, volume 5 μL) of Tat-DEC2 polypeptide were administered once a day (at 5 pm). The changes in leptin responsiveness and body weight of the mice were observed.

[0118] The results showed that Tat-DEC2 significantly improved leptin resistance in mice and effectively played a role in weight loss, manifested as improved leptin-mediated reduction in food intake and body weight, and increased expression of p-STAT3 in ARC POMC neurons, and enhanced firing of ARC POMC neurons after leptin perfusion. More importantly, the body weight of the mice did not show significant rebound after drug withdrawal ( Figure 6 ).

Claims

1. A polypeptide targeting DEC2 or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide targeting DEC2 is shown in SEQ ID NO.

1.

2. The polypeptide targeting DEC2 as claimed in claim 1, wherein the polypeptide targeting DEC2 can be coupled to a cell-penetrating peptide to guide DEC2 to penetrate the cell membrane, and the amino acid sequence of the Tat-coupled targeting polypeptide is shown in SEQ ID NO.

2.

3. The polypeptide targeting DEC2 as claimed in claim 2, wherein the Tat-coupled targeting polypeptide can also be coupled with a CTM tail to guide the degradation of DEC2 through a molecular chaperone-mediated autophagy pathway, and the amino acid sequence of the Tat- and CTM-coupled targeting polypeptide is shown in SEQ ID NO.

3.

4. The polypeptide targeting DEC2 according to claim 1, wherein the amino acid sequence of the polypeptide targeting DEC2 or a pharmaceutically acceptable salt thereof has at least 70% or 75% or 80% or 85% or 90% or 95% sequence identity with the amino acid sequence of SEQ ID No.

1. A nucleic acid molecule encoding the polypeptide of claim 1.

6. A gene expression vector comprising a nucleotide encoding the polypeptide according to claim 1.

7. A host cell comprising the nucleic acid molecule according to claim 5 and / or the expression vector according to claim 6, wherein the host cell is transformed or transfected with the nucleic acid molecule according to claim 5 and / or the expression vector according to claim 6.

8. A pharmaceutical composition comprising the polypeptide according to claim 1 and optional pharmaceutically acceptable excipients.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

10. Use of the polypeptide targeting DEC2 as claimed in claim 1 in the preparation of a drug for treating obesity, wherein the drug exerts a weight loss effect by improving leptin resistance.

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