Targeted degradation of tmem59 polypeptides and uses thereof

Abstract

The application discloses a targeted degradation TMEM59 polypeptide and application thereof, which is composed of 17-33 continuous amino acids of an ATG16L1 protein and contains HVVSVDKGXXAVLWAQX (SEQ ID NO. 09), wherein X is C, K, S, R, Y or P. The targeted degradation TMEM59 polypeptide and the fusion protein in the application can significantly reduce the expression of TMEM59, and thus can be used for treating diseases (such as nervous system diseases like Alzheimer's disease) related to abnormal expression of TMEM59, and have great clinical value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a targeted degradation peptide for TMEM59 and its applications. Background Technology

[0002] TMEM59 is an autophagy-related protein widely expressed in various tissues and organs. It interacts with ATG16L1, thereby promoting LC3 activation and autophagy. Studies have shown that the methylation level of the TMEM59 gene promoter region in the frontal cortex of patients with late-onset Alzheimer's disease (AD) is significantly lower than in controls, and this methylation is correlated with TMEM59 mRNA and protein expression levels. Gene chip analysis also shows that TMEM59 gene expression is higher in AD patients than in controls. Our previous research found that TMEM59 can regulate microglial activity and neuroinflammatory responses by interacting with the AD risk factor TREM2, and downregulating TMEM59 can improve microglial dysfunction caused by TREM2 deficiency. Furthermore, we found that in the 5xFAD AD model mouse, TMEM59 expression increases with pathological progression, and heterozygous loss of TMEM59 significantly improves learning and memory function and pathological features in 5xFAD mice. Another research group also found that TMEM59 deficiency can reduce amyloid plaque deposition and alleviate cognitive impairment in the APP / PS1 AD model mouse.

[0003] Induced protein degradation is an emerging drug development strategy used to regulate the expression of intracellular "drug-incompatible" target proteins. In eukaryotic cells, damaged proteins or organelles can be cleared by proteasomes or lysosomes. The concept of protein degradation-targeting chimeras (PROTACs) was first proposed by Crews et al. in 2001. PROTACs utilize the body's naturally occurring protein clearance system to induce ubiquitination of target proteins, which are then degraded via the ubiquitin-proteasome system, thereby exerting a therapeutic effect. PROTACs primarily target intracellular proteins, while lysosome-targeting chimeras (LYTACs), as a complement to PROTACs, induce the degradation of extracellular and membrane proteins via the endosome-lysosome pathway. LYTAC molecules can simultaneously bind to the protein-binding domain (PBD) of the target protein and the lysosome-targeting receptor (LTR) located on the cell surface, after which the target protein is degraded by lysosomes.

[0004] In conclusion, reducing TMEM59 expression may be a novel strategy for treating various neurological disorders and other diseases. This strategy may offer new insights into the treatment of neurodegenerative diseases such as Alzheimer's disease by modulating pathways related to autophagy and neuroinflammation. Summary of the Invention

[0005] The purpose of this invention is to provide a peptide that targets and degrades TMEM59.

[0006] Another object of the present invention is to provide the use of the above-mentioned targeted degradation of TMEM59 peptide.

[0007] The technical solution of the present invention is as follows:

[0008] A peptide that targets and degrades TMEM59, consisting of 17-33 consecutive amino acids of the ATG16L1 protein, and containing HVVSVDKGXXAVLWAQX (SEQ ID NO.09), wherein X is C, K, S, R, Y or P.

[0009] Preferably, the targeted TMEM59 degradation peptide is composed of 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18 or 17 consecutive amino acid residues.

[0010] In a preferred embodiment of the present invention, the amino acid sequence comprises HVVSVDKGCKAVLWAQY (human source: SEQ ID NO.07) or HVVSVDKGSRAVLWAQP (mouse source: SEQ ID NO.08).

[0011] A conjugate characterized in that it has the above-mentioned targeted TMEM59 degradation peptide and a modification moiety, the modification moiety being selected from other peptides, detectable labels (for detection), or any combination thereof, the other peptide being selected from CPP (to enhance its ability to penetrate cell membranes), a targeting moiety (to make it targeted), and / or a protein tag (to facilitate its expression, detection, tracing, and / or purification).

[0012] A fusion protein having the above-mentioned targeted TMEM59 degradation peptide and another peptide selected from CPP (to enhance its ability to penetrate cell membranes), a targeting moiety (to make it targeted), a protein tag (to facilitate its expression, detection, tracing and / or purification) or any combination thereof.

[0013] Preferably, the modified portion is optionally connected to the N-terminus or C-terminus of the targeted TMEM59 degrading peptide via a linker.

[0014] More preferably, the CPP is a TAT-derived peptide; for example, the CPP has the sequence shown in SEQ ID NO.11.

[0015] Further preferred options include targeting ligands, receptors, or antibodies.

[0016] Further preferred, the detectable label is a fluorescent dye, such as FITC.

[0017] More preferably, the protein tag is HA, myc, GFP, or biotin.

[0018] An isolated nucleic acid molecule having the nucleotide sequence of the above-mentioned targeted degradation TMEM59 polypeptide or the above-mentioned fusion protein.

[0019] A vector having the aforementioned isolated nucleic acid molecules.

[0020] Preferably, the vector includes plasmids, granules, bacteriophages, and Cos plasmids.

[0021] More preferably, the vector is capable of expressing the above-mentioned targeted degradation TMEM59 peptide or the above-mentioned fusion protein in a subject (e.g., a mammal, such as a human).

[0022] A host cell having the above-described isolated nucleic acid molecules or the above-described carrier.

[0023] Preferably, the host cell is selected from prokaryotic cells and eukaryotic cells.

[0024] Prokaryotic cells include Escherichia coli cells.

[0025] Eukaryotic cells include yeast cells, insect cells, plant cells, and animal cells. Animal cells are preferably mammalian cells, and more preferably mouse cells and human cells.

[0026] The host cell can also be a cell line, such as 293T cells.

[0027] The method for preparing the above-mentioned targeted TMEM59 degradation peptide or the above-mentioned fusion protein includes: culturing the above-mentioned host cells under suitable conditions, and recovering the targeted TMEM59 degradation peptide or the above-mentioned fusion protein from the culture of the host cells.

[0028] A pharmaceutical composition comprising the above-described targeted TMEM59 degrading peptide, the above-described conjugate, the above-described fusion protein, the above-described isolated nucleic acid molecule, the above-described carrier or the above-described host cell, and a pharmaceutically acceptable carrier and / or excipient.

[0029] The use of the above-mentioned targeted TMEM59 degradation peptide, the above-mentioned conjugate, the above-mentioned fusion protein, the above-mentioned isolated nucleic acid molecule, the above-mentioned carrier or the above-mentioned host cell in the preparation of a pharmaceutical composition for the treatment of diseases associated with overexpression of TMEM59 or the expression of TMEM59.

[0030] The diseases associated with overexpression of TMEM59 mentioned above include neurological disorders.

[0031] The aforementioned neurological diseases are characterized by abnormal activation of the TMEM59 signaling pathway, with Alzheimer's disease being the preferred example.

[0032] Preferably, the pharmaceutical composition may further comprise an additional pharmaceutically active ingredient; the additional pharmaceutically active ingredient is a drug with therapeutic activity for neurological diseases (e.g., ischemic stroke). Alternatively, the additional pharmaceutically active ingredient is selected from ω-3 fatty acids, vitamin B12, intravenous immunoglobulins, hyperbaric oxygen therapy, glutamate receptor antagonists, catechol-O-methyltransferase (COMT) inhibitors, dopa decarboxylase inhibitors, or any combination thereof.

[0033] A method for reducing the expression of TMEM59 in vitro for non-diagnostic therapeutic purposes, comprising contacting the above-mentioned targeted TMEM59-degrading peptide, the above-mentioned conjugate, or the above-mentioned fusion protein with cells in need.

[0034] Preferably, the cells are neuronal cells or glial cells (the glial cells are preferably microglia).

[0035] The aforementioned targeted TMEM59 degradation peptides, fusion proteins, or pharmaceutical compositions can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, and injections (including injectable solutions and lyophilized powders). In some embodiments, the aforementioned targeted TMEM59 degradation peptides, fusion proteins, or pharmaceutical compositions can be formulated into injectable solutions or lyophilized powders.

[0036] Furthermore, the aforementioned targeted degradation TMEM59 peptides or fusion proteins can be present in the pharmaceutical composition in unit dose form for ease of administration.

[0037] The aforementioned targeted TMEM59 degrading peptide, fusion protein, or pharmaceutical composition can be administered by any suitable method known in the art, including oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In a preferred embodiment, the aforementioned targeted TMEM59 degrading peptide, fusion protein, or pharmaceutical composition is administered by intravenous infusion or injection.

[0038] The targeted TMEM59-degrading peptides, fusion proteins, or pharmaceutical compositions provided by this invention can be used alone or in combination, or in combination with other pharmaceutically active components (e.g., drugs with activity in treating neurological diseases). In some preferred embodiments, the aforementioned targeted TMEM59-degrading peptides or fusion proteins are used in combination with other drugs with activity in treating neurological diseases to prevent and / or treat diseases associated with overexpression of TMEM59 (e.g., neurological diseases). This additional pharmaceutically active component can be administered before, simultaneously with, or after the administration of the aforementioned targeted TMEM59-degrading peptides, fusion proteins, or pharmaceutical compositions.

[0039] The aforementioned pharmaceutical composition may include a “therapeutic effective amount” or a “preventative effective amount” of the aforementioned targeted degrading TMEM59 peptide or fusion protein. A “preventative effective amount” refers to an amount sufficient to prevent, stop, or delay the onset of disease (e.g., diseases associated with overexpression of TMEM59). A “therapeutic effective amount” refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutic effective amount of the aforementioned targeted degrading TMEM59 peptide or fusion protein may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of drug administration, and other concurrent treatments, etc.

[0040] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0041] The typical non-limiting range of the therapeutic or preventative effective dose of the aforementioned targeted degradation of TMEM59 peptides or fusion proteins is 0.001-100 mg / kg body weight, for example 0.01-50 mg / kg body weight, 0.1-25 mg / kg body weight.

[0042] It should be noted that the dosage may vary depending on the type and severity of the symptoms that need to be treated.

[0043] Furthermore, those skilled in the art will understand that for any given patient, a specific dosing regimen should be adjusted over time based on the patient's needs and the physician's professional evaluation; the dosage ranges given herein are for illustrative purposes only and do not limit the use or scope of the pharmaceutical compositions of the present invention.

[0044] In this invention, the subject can be a mammal, such as a human.

[0045] Terminology Definition

[0046] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this invention are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0047] As used herein, the terms “Proteolysis targeting chimeras (PROTACs),” “Lysosome-targeting chimeras (LYTACs),” “cell penetrating peptide (CPP),” “cell-penetrating peptide,” “lysosome-targeting receptor (LTR),” “protein-binding domain (PBD),” etc., refer to polypeptides capable of promoting cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids; for example, the TMEM59 targeted degradation polypeptide or variants thereof of the present invention). Such polypeptides are well known in the art or can be obtained by methods known in the art and are described, for example, in Fan, et al., 2014 and Tong, et al., 2023 (all of which are incorporated herein by reference).

[0048] In this invention, examples of the CPP include: (i) protein-derived CPPs: such as sequences derived from genes controlling antennae (Antennapedia), e.g., pAntp (43-58); sequences derived from HIV-1, e.g., TAT-derived peptides, e.g., amino acid residues 37-72, 37-60, 47-60, or 47-57 of TAT; hCT (9-32); pVEC; plSL; mouse PRP (1-28); E ms (194-220); or Restricocin L3 (60-73), etc.; (ii) model peptides: such as VT5; MAP; or arginine stretch, etc.; (iii) designed CPPs: such as MPG; Transportan; Transportan 10; Pep-1; peptides selected from KALA; or peptides selected from Bulforin2, etc.

[0049] Furthermore, the CPP used in the conjugates of the present invention may also be selected from polypeptide sequences having approximately 60, 70, 80, 90, 95, 99% or 100% sequence identity with any polypeptide sequence as described above, provided that the polypeptide sequence still retains its biological activity, i.e., promoting cellular uptake of the isolated polypeptide (or its variants) of the present invention and / or promoting the isolated polypeptide (or its variants) of the present invention across the blood-brain barrier.

[0050] As used herein, the term "targeting portion" refers to a domain capable of guiding the targeted degradation TMEM59 peptide (or a variant thereof) of the present invention to a desired location, which may be a specific tissue, a specific cell, or even a specific intracellular location (e.g., the nucleus, ribosome, endoplasmic reticulum, lysosome, or peroxisome). Those skilled in the art know how to design corresponding targeting domains based on the characteristics of the desired location.

[0051] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include: plasmids; phage particles; Cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0052] As used in this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0053] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using, for example, a computer program such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoIBiol. 48:444-453 (1970) algorithm in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0054] As used herein, the term "isolated" means that the target analyte (e.g., a peptide) has been purified from contaminants present in a sample, such as a sample containing the target analyte obtained from a natural source. The term "isolated" does not necessarily exclude the presence of other components intended to work in conjunction with the isolated analyte. For example, the targeted degradation TMEM59 peptide of the present invention can be described as isolated, although it may be linked to cell-penetrating peptides.

[0055] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes: pH adjusters, surfactants, ionic strength enhancers, agents for maintaining osmotic pressure, agents for delaying absorption, diluents, adjuvants, preservatives, stabilizers, etc. For example, pH adjusters include phosphate buffers. Surfactants include cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include sodium chloride. Agents for maintaining osmotic pressure include sugars, NaCl, and the like. Agents for delaying absorption include monostearates and gelatin. Diluents include water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Adjuvants include aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), etc. Preservatives include various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which stabilize the desired activity of the active ingredient in a drug (e.g., reduced expression of TMEM59), including monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0056] As used in this invention, the term "treatment" means treating or curing a disease (e.g., a neurological disease), delaying the onset of symptoms of a disease (e.g., a neurological disease), and / or delaying the progression of a disease (e.g., a neurological disease).

[0057] As used in this invention, the term "prevention" means preventing, inhibiting, or delaying the onset of a disease (e.g., a neurological disease).

[0058] As used herein, the term "effective amount" refers to an amount that can effectively achieve the intended purpose. For example, a therapeutically effective amount can be an amount that effectively or sufficiently treats or cures a disease (e.g., a neurological disorder), delays the onset of symptoms of a disease (e.g., a neurological disorder), and / or delays the progression of a disease (e.g., a neurological disorder). A preventatively effective amount can be an amount that effectively or sufficiently prevents, inhibits, or delays the occurrence of a disease (e.g., a neurological disorder). Such effective amounts can be readily determined by those skilled in the art or by a physician and can be related to the intended purpose (e.g., treatment or prevention), the general health condition of the subject, age, sex, weight, severity of the disease to be treated, complications, method of administration, etc. The determination of such effective amounts is entirely within the capabilities of those skilled in the art.

[0059] As used in this invention, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from a disease associated with overexpression of TMEM59 (e.g., a neurological disorder), or is at risk of suffering from a disease associated with overexpression of TMEM59 (e.g., a neurological disorder).

[0060] As used in this invention, the biological functions of the above-mentioned targeted degradation of TMEM59 peptides include one or more selected from the following:

[0061] 1) The ability to specifically bind to TMEM59;

[0062] 2) Reduces the expression of TMEM59 protein;

[0063] 3) The ability to reduce TMEM59 protein expression in subjects (optionally, after conjugating the peptide to CPP);

[0064] 4) The ability to improve synaptic dysfunction and / or learning and memory dysfunction caused by excessive TMEM59 levels in subjects (optionally, after conjugating the peptide to CPP);

[0065] 5) The ability to treat diseases (e.g., neurological diseases) associated with abnormal TMEM59 expression in subjects (optionally, after conjugating the peptide to CPP).

[0066] The beneficial effects of this invention are: the targeted degradation TMEM59 peptide and fusion protein of this invention can significantly reduce the expression of TMEM59, and therefore can be used to treat diseases related to abnormal TMEM59 expression (such as neurological diseases such as Alzheimer's disease), which has significant clinical value. Attached Figure Description

[0067] Figure 1AF: Screening for the fragment in ATG16L1 that interacts most strongly with TMEM59-CTF. Among them: Figure 1 A and B: Co-IP analysis of the interaction between TMEM59-CTF and each WD of ATG16L1. Figure 1 CE: Co-IP analysis of the interaction between TMEM59-CTF and three fragments of WD7. Figure 1 F: Co-IP analysis of the interaction between mouse TMEM59 (m59) and the WD7-D3 fragment. n=3, one-way ANOVA with Tukey's post hoc test. Data represent mean ± standard error. **p<0.01, ***p<0.001, ****p<0.0001.

[0068] Figure 2 AC: Construction of LYTACs peptides targeting the degradation of TMEM59. Among them: Figure 2 A: The construction strategy of LYTACs peptides, wherein the PBD sequence is SEQ ID NO.07, the LTR sequence is SEQ ID NO.10, and the CPP sequence is SEQ ID NO.11. Figure 2 B and C: The efficiency of LYTACs peptides in crossing the blood-brain barrier was detected using two-photon microscopy. Data represent mean ± standard error.

[0069] Figure 3 AI: Utilizing LYTACs peptides to degrade TMEM59 significantly improved learning and memory abilities in 5xFAD mice. Specifically: Figure 3 AC: Immunoblot analysis of changes in TMEM59 protein expression levels after tail vein injection of LYTACs peptides in mice. n=6, one-way ANOVA with Tukey's post hoc test. Figure 3 D and E: Percentage of spontaneous alternation behavior (D) and distance traveled (E) in the Y maze test. Figure 3 F and G: In the Morris water maze test, the escape latency (F) of mice during training and the percentage of time mice spent in the target quadrant during the test (G). Figure 3H and I: Percentage of time mice exhibiting rigid behavior in the episodic memory test (H) and the cueed memory test (I) during conditioned fear tests. WT-Scr group n=12, 5xFAD-Scr group n=11, 5xFAD-CTM group n=11. D, E, and GI were evaluated using one-way ANOVA with Tukey's posthoc test, and F was evaluated using two-way ANOVA with Tukey's posthoc test. Data represent mean ± standard error. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0070] Figure 4 AI: Using LYTACs peptides to degrade TMEM59 to improve neurological dysfunction and motor-related behaviors in MCAO mice. Specifically: Figure 4 A and B: Immunoblot analysis of TMEM59 protein expression before and after MCAO modeling. n=3, unpaired t test. Figure 4 C and D: Immunoblot analysis of changes in TMEM59 protein expression levels after tail vein injection of LYTACs peptides in mice. n=3, one-way ANOVA with Tukey's post hoc test. Figure 4 E and F: TTC staining analysis of cerebral infarction area in MCAO mice. n=5, one-way ANOVA with Tukey's post hoc test. Figure 4 GI: mNSS score (G), rotarod test (H), adhesion removal test (I). Sham group n=14, MCAO group n=15, MCAO-Scr group n=14, MCAO-CTM group n=15, one-way ANOVA with Tukey's post hoc test. Data represent mean ± standard error. *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0071] The above technical solution will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0072] Sequence information

[0073] Information about the sequence involved in this invention is provided in Table 1 below.

[0074] Table 1: Sequence Description

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Usubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Reagents not otherwise specified in the examples are conventional reagents in the art or commercially available reagents.

[0081] I. Experimental Materials and Methods

[0082] A. Protein extraction and Western blotting (WB)

[0083] 1. Preparation of protein samples

[0084] (1) Cells: Discard the cell culture medium, wash 3 times with 1 mL of pre-cooled 1×PBS, then add an appropriate amount of 1% TNEN lysis buffer, scrape the cells with a cell scraper and place them in a 1.5 mL EP tube, lyse on ice for 30 min, and shake with a vortex mixer once every 10 min.

[0085] Tissue: Mouse brain tissue was removed using dissecting instruments and placed in pre-chilled 1×PBS buffer. The desired brain tissue sites were carefully dissected and placed in 1.5 mL EP tubes and weighed. An appropriate amount of RIPA lysis buffer was added according to the tissue weight, and the tissue was homogenized using a tissue homogenizer. Lysis was performed on ice for 1 h, with vortexing every 10 min.

[0086] (2) After lysis, centrifuge at 13000×g for 15 min at 4℃, and carefully aspirate the supernatant into a new 1.5mL EP tube.

[0087] (3) Protein concentration was determined using the BCA method (refer to the BCA Protein Assay Kit instructions). Based on the measured protein concentration, the required sample and loading buffer volumes were calculated, and the sample was prepared. 30–40 μg of protein sample was taken, and the corresponding volume of 5× loading buffer was added, with the remainder made up to 1× loading buffer. After mixing, the sample was placed in a sample cooker and boiled at 100°C for 5 minutes. The prepared sample was used for Western blot analysis.

[0088] 2. SDS-polyacrylamide gel (PAGE) electrophoresis

[0089] (1) Prepare SDS-polyacrylamide gels of different concentrations according to the molecular weight of the protein to be detected.

[0090] (2) Add electrophoresis buffer to the electrophoresis tank, slowly add the prepared sample into the gel well using a pipette, and adjust the voltage to 80V constant voltage; after the sample enters the separating gel, adjust the voltage to 120V.

[0091] (3) The location of the protein to be detected can be estimated based on the position of the protein marker electrophoresis, and electrophoresis can be stopped in time.

[0092] 3. Protein electrotransfer

[0093] (1) Pre-cool the transfer buffer at 4°C. Soak the PVDF membrane in methanol first and then put it into the pre-cooled transfer buffer. At the same time, soak the filter paper in the transfer buffer for a few minutes.

[0094] (2) Place the membrane clips in the order of 4 filter papers - protein gel - PDVF membrane - 4 filter papers from the negative electrode to the positive electrode. Avoid generating air bubbles during this process. Then place the membrane clips into the electrotransfer bath and perform electrotransfer at a constant current of 300mA for 90 minutes in an ice bath environment.

[0095] (3) After the protein electrotransfer is completed, the PVDF membrane is placed in a blocking solution containing 5% skim milk powder and blocked at room temperature for 1 hour.

[0096] 4. Antigen-antibody reaction

[0097] (1) Following the antibody instructions, dilute the primary antibody with 5% BSA (prepared with PBST) at an appropriate ratio, place it together with the PVDF membrane in the antibody incubation box, and incubate overnight at 4°C on a shaker.

[0098] (2) The primary antibody was recovered and then washed three times with PBST for 10 min each time.

[0099] (3) Dilute the secondary antibody with 5% skim milk (prepared with PBST) at a ratio of 1:3000 to 1:5000 and incubate it with the membrane at room temperature for 1 hour.

[0100] (4) Discard the secondary antibody and then wash with PBST 3 times, 10 min each time.

[0101] 5. Enhanced chemiluminescence (ECL) color development

[0102] (1) Place the PVDF film flat in the plastic sealant and then place it in the developing clip. Mix the A and B solutions of the ECL colorimetric solution evenly at a volume ratio of 1:1.

[0103] (2) In the darkroom, add ECL droplets to the film surface, cover with plastic film, press the film onto the plastic film, and expose it in the developing clamp. After a period of time, remove the film, develop and fix it.

[0104] B. Co-Immunoprecipitation (Co-IP)

[0105] 1. Protein sample preparation

[0106] (1) Discard the cell culture medium, add 1 mL of pre-cooled 1×PBS, and wash twice.

[0107] (2) Add 1 mL of pre-cooled 1×PBS, scrape the cells off with a cell scraper, transfer them to a 1.5 mL tube, centrifuge at 2000 rpm for 3 min at 4 °C, discard the supernatant and collect the cells.

[0108] (3) Discard the supernatant, add 800 μL of 1% TNEN lysis buffer (pre-added with protease inhibitor) to each tube, lyse on ice for 30 min, and vortex once every 10 min.

[0109] (4) Centrifuge at 13000×g for 15 min at 4℃, and carefully transfer the supernatant to a new 1.5mL EP tube.

[0110] 2. Immunoprecipitation

[0111] (1) Beads pretreatment: Recombinant Protein G beads were washed three times with 1×PBS and centrifuged at 3000 rpm for 2 min at 4 °C. Then, the beads were mixed with an equal volume of 1×PBS and set aside for later use.

[0112] (2) To determine the protein concentration, take 1 mg of the above protein lysis buffer and add 0.5% TNEN lysis buffer to 700 μL. Add 20 μL of the mixed Protein G beads and incubate at 4°C for 2 h by rotation. Take another 2% to 5% of the protein as input.

[0113] (3) Centrifuge at 3000 rpm for 3 min at 4℃. Transfer the supernatant to a new EP tube, add the antibody for the corresponding protein and 40 μL of the mixed Protein G beads. Incubate at 4℃ overnight by rotation.

[0114] (4) Centrifuge at 3000 rpm for 3 min at 4℃, discard the supernatant, wash 3 times with 1% TNEN lysis buffer, and incubate for 10 min each time.

[0115] (5) Use a microsyringe to aspirate the supernatant, add 2× loading buffer of the same volume as the beads, mix well and place in a sample cooker for 5 min for immunoblotting analysis.

[0116] C. Tail vein injection

[0117] The synthesized peptides were dissolved in physiological saline and kept on ice until ready for use. When injecting mice, ensure the mice were comfortably placed in the syringe and positioned using the tail vein syringe barrel, with the mouse's tail in the groove of the compression plate. Adjust the lighting to make the veins clearly visible, then begin injection from the base of the tail, slowly advancing the peptide solution while constantly monitoring the mouse's health. After injection, leave the needle in place to ensure proper drug absorption. After needle removal, apply pressure with a cotton swab for 1 minute to stop bleeding. Inject once every other day for a total of 10 injections.

[0118] D. Volume multiphoton microscopy

[0119] Mice were anesthetized with isoflurane. After deep anesthesia, part of the scalp was removed to expose the skull. A 4mm diameter cranial window was drilled into the dura mater using a skull drill. A glass coverslip was placed over the cerebral cortex instead of the skull and sealed with glue. A custom-made fixator was then fixed to the skull with dental cement. Imaging was performed using a multiphoton laser scanning microscope (FVMPE-RS) (Olympus). First, Texas red-dextran was injected intravenously to observe cortical vessels, followed by the injection of FITC-labeled peptides. During imaging, a 920nm wavelength laser was used to excite Texas red-dextran and FITC, and the emission light was detected using GaAsP PMTs with bandpass filters of 575-645 and 495-540, respectively.

[0120] E. Animal behavioral tests

[0121] All mouse behavioral experiments were conducted using TopScan Lite software (Clever Sys. Inc.) for data collection and analysis. Animal behavioral experiments were performed daily between 9:00 a.m. and 6:00 p.m., with a laboratory light intensity of 650 lux.

[0122] (1) Three days before the start of the experiment, touch the mice once a day, touch one mouse each time, gently grasp the mouse by the tail and pick it up, let the mouse stay in your hand for 30 seconds, then mark the mouse by drawing a line on the tail with a marker. After marking the mouse, keep it in your palm, grasp the tail, and then gently put it back into the cage.

[0123] (2) On the day of the experiment, the mice were transferred to the preparation room before the experiment and allowed to acclimatize for 60 minutes. Before the experiment began, the test chamber and maze were cleaned with 70% alcohol. After each experiment, the test chamber and maze were cleaned with 70% alcohol to remove the feces and urine excreted by the mice during the experiment and to eliminate the interference of residual mouse odor on the experiment.

[0124] 1. Y-maze Test: The Y-maze is a Y-shaped maze consisting of three arms measuring 30cm (L) × 6cm (W) × 15cm (H). The test mouse is placed in the center of the Y-maze and allowed to freely move between each arm for 5 minutes. The order in which each mouse enters each arm and the number of times it enters an arm are recorded. Successful movement between three different arms is counted as one correct spontaneous alternation behavior. The percentage of correct alternations is the ratio of the number of correct alternations to the total number of alternations.

[0125] 2. Morris Water Maze Test: The Morris water maze test is conducted in a large circular pool (120cm in diameter). An appropriate amount of water is added and the water temperature is maintained at 22°C. Titanium dioxide is added to the water to make it opaque, facilitating the concealment of platforms, which are hidden 1cm below the water surface. The maze walls are decorated with different shapes in four different directions to help mice develop spatial memory. The test specifically includes:

[0126] a. Training Phase: Mice were placed in the water maze from different starting positions, facing the pool wall. The time taken to reach and climb onto the platform was recorded (i.e., escape latency). If a mouse failed to find the platform within 60 seconds, it was guided to the platform and allowed to stay there for 10 seconds. Two different directions were selected for training each day for six consecutive days.

[0127] b. Testing Phase: On day 7, the platform beneath the water surface was removed, and the mice were placed in the water maze facing the pool wall from the position furthest from the platform, allowing them to swim freely for 60 seconds. The mice's swimming speed, distance traveled, time spent in each quadrant, and number of times they crossed the platform were recorded and analyzed.

[0128] 3. Fear conditioning tests, specifically including:

[0129] a. Training Phase: On Day 1, mice were placed in a testing chamber (square with an electric grid at the bottom to provide foot shocks) for 2 minutes to acclimatize. Then, an 80 dB noise stimulus was emitted for 30 seconds, followed by a 0.7 mA electric shock within the last 2 seconds of the noise stimulus. This process was repeated three times, with 30-second intervals between each shock. After the final electric shock, a 1-minute wait was observed before the mice were returned to their cages.

[0130] b. Contextual memory test: On the second day, place the mice in the same test box for 5 minutes and record the percentage of time the mice were in a state of rigidity.

[0131] c. Cue memory test: On day 3, the mice were placed in a test box that was completely different from the previous training environment (cylindrical, with walls and bottom of different colors and materials). After the mice adapted for 3 minutes, they were then given an 80dB noise stimulus for 3 minutes. The percentage of time the mice exhibited rigid behavior under the sound stimulus and without the sound stimulus was recorded.

[0132] 4. Modified Neurologic Severity Score (mNSS): The mNSS was used to comprehensively evaluate the neurological function of rats in each group after brain loss. The main observations included motor function (muscle status, abnormal behavior), sensory function (vision, touch, proprioception), balance, reflexes, and overall performance, to systematically evaluate the severity of neurological damage. The scale has a total score of 18 points, with higher scores indicating more severe neurological impairment: 1-6 points represent mild impairment, 7-12 points represent moderate impairment, and 13 points or higher represent severe impairment. Details are as follows:

[0133]

[0134]

[0135] 5. Rotary Bar Test: This test is used to evaluate the impairment of motor coordination and balance in mice after ischemic stroke modeling. Mice are trained continuously for 3 days prior to modeling, 3 times a day. The mouse is placed on the rotundum to adapt for 1 minute, then the rotundum is rotated at a constant speed of 20 rpm for 5 minutes. If the animal falls, it is placed back on the rotundum until the exercise time reaches 5 minutes. On the third day of training, a baseline test is performed after two training sessions. The rotundum is accelerated from 0 rpm to 40 rpm and then rotated at a constant speed, and the time it takes for the mouse to fall is recorded. The test is performed on mice after modeling, similar to the baseline test. The instrument is cleaned and dried with alcohol after training or testing.

[0136] 6. Adhesive Removal Test: This test is used to evaluate the loss of self-sensory perception in mice after ischemic stroke modeling. Before the experiment, mice should be familiarized with the test environment. Mice are trained 3 days prior to modeling. During the experiment, a small, round adhesive tag is placed on the palm of the mouse, ensuring consistent application method and pressure. The mouse is then placed in a transparent, sterile box and allowed to move freely. The mouse will touch and attempt to remove the tag with its teeth. The time taken for the mouse to touch and attempt removal is recorded.

[0137] 7. TTC (2,3,5-triphenyltetrazolium chloride) staining: After anesthetizing and euthanizing mice, the whole brain was quickly and carefully removed, rinsed in PBS, mounted on a brain mold, and flash-frozen at -20°C for 20 minutes. The brain was then cut into slices approximately 2 mm in size. The slices were immediately immersed in 0.5% TTC solution and heated in a 37°C water bath for 30 minutes, turning three times during the heating process to ensure even staining. After staining, the brain slices were stored in 4% paraformaldehyde in the dark for 24 hours. Images were then photographed and analyzed using ImageJ software. The infarct area ratio was calculated as: infarct area on the ischemic side / total brain tissue area × 100%.

[0138] II. Materials and Reagents

[0139]

[0140] Example 1: Determination of the ATG16L1 core sequence with strong binding affinity to TMEM59

[0141] Studies have reported interactions between the intracellular domain (CTF) of TMEM59 and the WD repeat region of ATG16L1. To further identify the binding regions, this study constructed GFP-modified human ATG16L1 WD1-WD7 plasmids (amino acid sequence information is shown in Table 2). These expression plasmids were co-transfected with myc-modified human TMEM59-CTF expression plasmids into HEK293T cells, and their interactions were analyzed using Co-IP assays. The results showed that the WD7 region of ATG16L1 (SEQ ID NO. 05) interacted most strongly with TMEM59. Figure 1 A, B). Further, this embodiment divides the WD7 region into three segments: D1 (aa575-590), D2 (aa582-598), and D3 (aa591-607, SEQ ID NO. 07). Figure 1 C) to determine the shortest sequence that binds to TMEM59, and found that D3 has the strongest binding affinity to TMEM59. Figure 1 CE). To determine whether D3 can bind to mouse TMEM59, this example co-transfected plasmids expressing D3 and mouse TMEM59 into HEK293T cells. Co-IP analysis showed that the D3 sequence could bind to mouse TMEM59 upon oxygen permeation. Figure 1 F).

[0142] The above results indicate that the 591-607 sequence region of ATG16L1 is a key region for interaction with TMEM59.

[0143] Table 2: Truncated human ATG16L1

[0144]

[0145]

[0146] Example 2: Detection of the efficiency of ATG16L1-based peptides in penetrating the blood-brain barrier

[0147] In this embodiment, a human ATG16L1 peptide containing D3 (CTM) was designed and synthesized using the principles of LYTACs. WD7-D3 (HVVSVDKGCKAVLWAQY; SEQ ID NO.07) was linked to the target lysosomal motif (KFERQKILDQRFFE; SEQ ID NO.10) and TAT (YGRKKRRQRRR; SEQ ID NO.11) via a linker (GSGS; SEQ ID NO.12), and modified with FITC. A peptide without the target lysosomal motif was used as a control (Scr). Figure 2 A). All of the above-mentioned peptides were synthesized by Sangon Biotech. To test whether the synthesized peptides could penetrate the blood-brain barrier, this embodiment used in vivo multiphoton microscopy to detect the efficiency of the two peptides in penetrating the blood-brain barrier. The imaging results showed that both Scr and CTM peptides could penetrate the blood-brain barrier and enter the brain parenchyma. Figure 2 B, C).

[0148] Example 3: Evaluation of the therapeutic effect of ATG16L1-based peptides in Alzheimer's disease (AD) model mice

[0149] 5xFAD mice are widely used as a typical AD model. Previous studies have shown that TMEM59 expression is abnormally increased in 5xFAD mice, and targeting the degradation of TMEM59 may be a potential treatment for AD. Therefore, this example further evaluates the therapeutic effect of an ATG16L1-based peptide on AD model mice.

[0150] In this embodiment, the synthesized Scr and CTM peptides were injected into 6-month-old 5xFAD mice or littermate control WT mice via tail vein injection. The injections were performed once every other day for 10 days. After the injections, the mice were subjected to behavioral tests and biochemical analysis.

[0151] Immunoblot analysis revealed that abnormally elevated TMEM59 levels in the hippocampus and cortical tissues of 5xFAD mice returned to normal levels after CTM peptide treatment. Figure 3 AC). Behavioral testing in mice showed that, in the Y-maze test, 5xFAD mice administered CTM peptides exhibited significantly improved working memory, but no impairment in motor function. Figure 3D, E); In the Morris water maze test, CTM peptide significantly improved the learning and memory abilities of 5xFAD mice compared to Scr peptide. Figure 3 F, G); In the conditioned fear behavior test, the episodic and cue memory abilities of 5xFAD mice given CTM peptides were significantly restored. Figure 3 The above results confirm that the ATG16L1-based peptide can significantly improve the disease-related phenotypes in AD model mice by targeting the degradation of TMEM59.

[0152] Example 4: Evaluation of the therapeutic effect of ATG16L1-based peptides in a mouse model of ischemic stroke.

[0153] The middle cerebral artery occlusion (MCAO) model in mice is a classic model of ischemic stroke. In this study, it was found that the level of TMEM59 protein in the brain tissue of MCAO mice was significantly reduced compared to the control group. Figure 4 (A, B). To investigate whether targeted degradation of TMEM59 can be used to treat ischemic stroke, this example further evaluated the therapeutic effect of an ATG16L1-based peptide on MCAO model mice.

[0154] In this embodiment, after MCAO modeling, the synthesized Scr and CTM peptides were injected into MCAO mice via tail vein injection once every 12 hours. After two injections, the mice were subjected to behavioral tests and biochemical analysis.

[0155] Immunoblot analysis revealed that CTM peptide treatment could further reduce the expression level of TMEM59 in MCAO model mice. Figure 4 C, D). Infarct volume following ischemic stroke in mice was assessed by TTC staining. Results showed that CTM peptide treatment reduced the infarct area in MCAO mice compared to the control group. Figure 4 E, F). Behavioral results showed that CTM peptide treatment improved the mNSS score in MCAO mice (E, F). Figure 4 G), which prolonged the time MCAO mice spent on the rotarod ( Figure 4 H), which reduced the time required for MCAO mice to remove adhesive tags (H), Figure 4 I). The above results confirm that the ATG16L1-based peptide can significantly improve neurological function and motor impairment in MCAO model mice by targeting the degradation of TMEM59.

[0156] The above description is merely a preferred embodiment and should not be construed as limiting the scope of the invention. All equivalent changes and modifications made in accordance with the scope of the patent and the description should still fall within the scope of the invention.

Claims

1. A polypeptide targeting TMEM59, characterized in that: the amino acid sequence of which is represented by SEQ ID NO. 05 or SEQ ID NO.

07.

2. A conjugate, characterized by: comprising the TMEM59-targeting polypeptide of claim 1 and a modification moiety selected from a detectable label or a protein tag.

3. A fusion protein, characterized in that: comprising the TMEM59-targeting polypeptide of claim 1 linked to a lysosomal targeting motif as represented by SEQ ID NO. 10 and TAT as represented by SEQ ID NO. 11 via a linker as represented by SEQ ID NO.

12.

4. An isolated nucleic acid molecule, characterized in that: encoding the TMEM59-targeting polypeptide of claim 1 or the fusion protein of claim 3.

5. A vector, characterized by: having the isolated nucleic acid molecule of claim 4.

6. A host cell, characterized in that: having the isolated nucleic acid molecule of claim 4 or the vector of claim 5, wherein the host cell is not a plant cell.

7. A method of producing a polypeptide targeting TMEM59 according to claim 1 or a fusion protein according to claim 3, characterized in that: comprising: culturing the host cell of claim 6 under suitable conditions and recovering the TMEM59-targeting polypeptide or the fusion protein from the culture of the host cell.

8. A pharmaceutical composition, characterized by: having the TMEM59-targeting polypeptide of claim 1, the conjugate of claim 2, the fusion protein of claim 3, the isolated nucleic acid molecule of claim 4, the vector of claim 5 or the host cell of claim 6, and a pharmaceutically acceptable carrier.

9. Use of the fusion protein of claim 3 for the manufacture of a pharmaceutical composition for the treatment of Alzheimer's disease and ischemic stroke.

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