CN109718363B - Peptide for preventing, relieving or treating Alzheimer disease and application thereof - Google Patents

Abstract

the present application provides the use of a peptide comprising amino acid sequence YEKLLDTEI (SEQ ID NO:1) or a functional variant thereof or a pharmaceutical composition comprising said peptide for the prevention, alleviation or treatment of Alzheimer's disease.

Description

Peptide for preventing, relieving or treating Alzheimer disease and application thereof

Technical Field

The present application relates generally to the field of medicine, and in particular, provides peptides and uses thereof for preventing, ameliorating or treating alzheimer's disease.

Background

Alzheimer's Disease (AD) is a common disease in the middle-aged and the elderly, and is a disease which is second only to cardiovascular and cerebrovascular diseases and tumors and seriously threatens the life and health of the elderly. The clinical manifestations are progressive cognitive dysfunction and behavioral impairment. The pathogenesis of alzheimer's disease is related to mutant genes, internal and external environments, and energy metabolism.

Under normal physiological conditions, tau protein levels in dendrites are much lower than that of axons. In some disease states, tau protein is abnormally enriched in dendrites, particularly in the early stages of a β amyloid-mediated alzheimer's disease. tau protein needs to act synergistically with Fyn, so dendritic targeting of Fyn is significantly reduced in Δ tau74 and tau-/-mice. In Δ tau74 mice, this was due to the fact that Δ tau competes with endogenous tau for binding sites for Fyn, acting as a competitive inhibitor. Similar phenomena occur with accumulation of Fyn in tau-/-bodies, suggesting that postsynaptic Fyn targeting requires endogenous tau mediation. Both delta tau74 mice and tau-/-mice have reduced postsynaptic levels of Fyn leading to a reduced degree of phosphorylation of Y1472 of Fyn-substrate NR2 b. Y1472 phosphorylation of NMDAR2B is a key condition to promote the interaction of NMDAR with PSD-95. Fyn and tau-mediated NMDAR/PSD-95 complex formation is the exact mechanism in the rat stroke model, targeted blocking of the NMDAR/PSD-95 interaction prevents excitotoxic injury and reduces lesion size. In agreement with this, the theory applies equally to the early stages of AD mediated by amyloid-a β. For Δ tau74 and tau-/-mice, there was no difference in NMDAR-mediated synaptic activity after treatment with Tat-NR2B9 c.

Excitotoxicity is increasingly recognized as one of the mechanisms by which amyloid-a β causes alzheimer's disease. Δ tau74 and tau-/-mice were not sensitive to excitotoxicity compared to wild type. Similarly, tau-deficient or primary neurons expressing Δ tau can also be immune to a β amyloid-induced toxicity. However, a β amyloid levels and plaque formation, as well as endogenous tau phosphorylation in APP23 mice (Δ tau74 in APP 23) indicate another mechanism of protection compared to APP mice. Y1472 phosphorylation and postsynaptic Fyn upregulation of NR2b was found to be fully restored in app23.Δ tau74 and app23. tau-/-mice in APP23 mice. Postsynaptic downregulation of Fyn in app23.Δ tau74 and app. tau-/-mice may lead to dendritic Fyn localization as well as to other tau mechanisms, i.e. partial Δ tau competitively inhibits Fyn localization. In addition to being involved in the pathogenesis of alzheimer's disease caused by amyloid a β, Fyn transgenic mice have premature seizure death. Neuronal apoptosis and microglia-induced inflammation are involved in the injury pathway, as are glutamate receptors and postsynaptic compact proteins in the neuronal apoptotic pathway. In addition, APP-related mortality is significantly reduced in fyn-/-individuals. The consistent behavior of app23.Δ tau74 and app23. tau-/-mice suggests that the deletion of conventional tau protein effectively inhibits the associated defects caused by a β amyloid.

The NMDAR/PSD-95 complex plays a key role in the a β amyloid toxicity mechanism in APP23 mice, as a downstream molecule of tau protein in alzheimer's disease pathology, it may be associated with both tau and non-tau mechanisms. When TatNR2B9c was administered to young APP23 mice, both tau and Fyn-induced amyloid A β toxicity could be effectively inhibited. The neuroprotective effect of TatNR2B9c in vitro and in vivo mainly depends on inhibiting the formation of NMDAR/PSD-95 complex, and can also improve the memory function and survival rate of APP23 mice. Notably, Tat-NR2B9 treated APP23 mice survived long term, indicating that short term treatment was sufficient to prevent a β amyloid toxicity.

In the field of alzheimer disease treatment, the research on pathogenesis is continuously advanced, and the field of alzheimer disease treatment still needs to develop treatment drugs according to new research to meet the requirements of the treatment market.

Summary of The Invention

In a first aspect, the present application provides the use of a peptide comprising amino acid sequence YEKLLDTEI (SEQ ID NO:1) or a functional variant thereof, or a pharmaceutical composition comprising said peptide, in the manufacture of a medicament for the prevention, alleviation or treatment of Alzheimer's disease in an individual.

In a second aspect, the present application provides a method of preventing, ameliorating or treating alzheimer's disease, the method comprising: administering to an individual in need thereof a peptide comprising amino acid sequence YEKLLDTEI or a functional variant thereof, or a pharmaceutical composition comprising said peptide.

In a third aspect, the present application provides a peptide comprising amino acid sequence YEKLLDTEI (SEQ ID NO:1) or a functional variant thereof or a pharmaceutical composition comprising said peptide for use in the treatment, amelioration or prevention of Alzheimer's disease in an individual.

In some embodiments of any of the above aspects, the functional variant is a variant resulting from one or more conservative substitutions in the LDTEI moiety in YEKLLDTEI, preferably the conservative substitutions are selected from the group consisting of a substitution between D and E, a substitution between L, V and I, and a substitution between T and S.

In some embodiments of any of the above aspects, the functional variant is a variant resulting from replacement of the LDTEI moiety in YEKLLDTEI with any of the following sequences: LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL, LDSEV, LDSDI, LDSDL, LDSDV, LETEI, LETEL, LETEV, LETDI, LETDL, LETDV, VDTEI, VDTEL, VDTEV, VDTDI, VDTDL, VDTDV, IDTEI, IDTEL, IDTEV, IDTDI, IDTDL, IDTDV, IETEI, IETEL, IETEV, IETDI, IETDL, IETDV.

In some embodiments of any of the above aspects, the peptide is a chimeric peptide comprising amino acid sequence YEKLLDTEI or a functional variant thereof and an internalization peptide, preferably the internalization peptide comprises amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2).

in some embodiments of any of the above aspects, the chimeric peptide comprises amino acid sequence YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 3).

In some embodiments of any of the above aspects, the peptide, pharmaceutical composition or medicament is capable of improving learning, memory (e.g., short-term memory) and/or spatial cognition in an individual with alzheimer's disease.

In some embodiments of any of the aspects above, the subject is a mammal.

In some embodiments of any of the aspects above, the subject is a human.

Drawings

FIG. 1 shows that the Pull-down experiment detects the interaction of P5 with the PDZ1/2 domain. M represents a protein molecular weight marker; lane 1 is His + PDZ1/2+ P5; lane 2 is P5 alone; lane 3 is His + P5; lane 4 is His + PDZ 1/2. The elution band shown in lane 1 contains both P5 and PDZ1/2, confirming that P5 is able to bind to the PDZ1/2 domain.

Fig. 2 shows the results of tests on the escape latency of rats in the alzheimer disease model according to different treatment regimens, wherein ″ "indicates p < 0.05.

Fig. 3 shows the results of tests on the number of crossing the platform in rats in alzheimer's disease model according to different treatment regimens, wherein ″) represents p <0.05 and ″) represents p < 0.01.

Detailed Description

The present inventors have conducted intensive studies on peptides capable of reducing the damaging effects of neurological disorders mediated at least in part by NMDAR excitotoxicity and have developed a new class of peptides with such effects. Without wishing to be bound by any theory, it is believed that such peptides act, at least in part, by inhibiting the interaction between NMDAR and post-synaptic density 95 protein (PSD-95) (i.e., PSD-95 inhibitors). On the basis, the inventor of the application tests the peptides by taking Alzheimer's disease as a target and obtains ideal results.

The pathogenesis of alzheimer's disease is continually explored in the art and theories have been established, such as the role of the neuronal inflammatory process in the pathology, and the cause and role of amyloid formation. However, whether amyloid is the cause or the consequence of the initiation of the pathogenesis of alzheimer's disease, the process of neuronal apoptosis is mediated from amyloid to the inflammatory response. Without wishing to be bound by any theory, applicants believe that the glutamate damage pathway is an important pathway, and NMDAR-PSD95 is an important complex of this pathway, inhibiting the formation of which inhibits neuronal apoptosis to some extent and may be a means of influencing the pathological progression of alzheimer's disease.

Therefore, the application provides a medicine and a method which can be effectively used for preventing, relieving and treating the Alzheimer disease, and provides a new choice for improving the symptoms of the Alzheimer disease patients and improving the quality of life.

Definition of

unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

The single-letter or three-letter abbreviations used herein for amino acids follow international conventions.

The term "chimeric peptide" means a peptide having two component peptides that are not naturally bound to each other, which can be bound to each other as a fusion protein or by a chemical bond.

The term "PDZ domain" refers to a modular protein domain of about 90 amino acids characterized by significant (e.g., at least 60%) sequence identity to brain synapsin PSD-95, Drosophila (Drosophila) separation junction protein Discs-Large (DLG), and epithelial tight junction protein Z01 (Z01). The PDZ domain is also known as the Discs-Large homology repeats ("DHRs") and GLGF repeats. PDZ domains are generally shown to retain the core consensus sequence (Doyle, D.A., 1996, Cell 85: 1067-76). Exemplary PDZ domain-containing proteins and PDZ domain sequences are disclosed in U.S. application Ser. No.10/714,537.

The term "NMDA receptor" or "NMDAR" refers to a membrane-associated protein known to interact with NMDA. These receptors may be human or non-human (e.g., mouse, rat, rabbit, monkey, etc.).

The term "specific binding" refers to the association between two molecules (e.g., a ligand and a receptor) characterized by the ability of one molecule (ligand) to bind to another specific molecule (receptor), even in the presence of many other different molecules, i.e., the ability to show preferential binding of one molecule to another in a heterogeneous mixture of molecules. Specific binding of the ligand to the receptor was also demonstrated as follows: in the presence of excess unlabeled ligand, the detectably labeled ligand has reduced binding to the receptor (i.e., a binding competition assay).

Statistically significant means a p-value <0.05, preferably <0.01, most preferably < 0.001.

The term "functional variant" refers to a variant having the same or similar biological function and properties as the parent. By way of non-limiting example, a "functional variant" may be obtained by making one or more conservative substitutions in the parent.

The term "internalization peptide", also known as a cell-penetrating peptide, is widely used in the field of protein pharmaceuticals and functions to facilitate the uptake and uptake by cells of the active peptide to which it is bound. As a non-limiting example, the internalization peptide can be a Tat peptide, wherein a non-limiting example of the Tat peptide is YGRKKRRQRRR (SEQ ID NO: 2).

In a first aspect, the present application provides the use of a peptide comprising amino acid sequence YEKLLDTEI (SEQ ID NO:1) or a functional variant thereof, or a pharmaceutical composition comprising said peptide, in the manufacture of a medicament for the prevention, alleviation or treatment of Alzheimer's disease in an individual.

In a second aspect, the present application provides a method of preventing, ameliorating or treating alzheimer's disease, the method comprising: administering to an individual in need thereof a peptide comprising amino acid sequence YEKLLDTEI or a functional variant thereof, or a pharmaceutical composition comprising said peptide.

In a third aspect, the present application provides a peptide comprising amino acid sequence YEKLLDTEI (SEQ ID NO:1) or a functional variant thereof or a pharmaceutical composition comprising said peptide for use in the treatment, amelioration or prevention of Alzheimer's disease in an individual.

the peptides, which are also referred to herein as "active peptides," are used as therapeutically active moieties in the chimeric peptides of the present application.

According to existing studies, some active peptides that inhibit the interaction between NMDAR and PSD-95 are based on the structure of NMDAR. For example, NMDAR2B has GenBank ID4099612, the C-terminal 20 amino acids FNGSSNGHVYEKLSSLESDV and PL motif ESDV. Some existing active peptides have selected a partial amino acid sequence from the C-terminus of NMDAR2B to produce competitive inhibition of PSD-95 with NMDAR 2B. It is thought that the ESDV or LESDV segments in the above peptides play an important role in inhibiting the interaction between NMDAR and PSD-95 proteins. Without being bound by any theory, the inventors of the present application surprisingly found that in the active peptide YEKLLDTEI disclosed herein (SEQ ID NO:1), which does not contain the SS two residues after KL relative to the C-terminal amino acid composition of NMDAR2B described above, while increasing the YEKL amino acid sequence in the N-terminal direction relative to the PL motif, the present application demonstrates that such changes can enhance the interaction of the active peptide with the PDZ1/2 domain. At the same time, the LDTEI at the C-terminus can be varied relative to the YEKL motif, which is not expected to affect the activity of the active peptide or may increase its activity. Thus, in some embodiments, the functional variants provided herein are those resulting from one or more conservative substitutions in the LDTEI moiety of SEQ ID No. 1.

In some embodiments, the conservative substitution is selected from the group consisting of a substitution between D and E, a substitution between L, V and I, and a substitution between T and S.

In more specific embodiments, the functional variant is a variant resulting from replacement of the LDTEI portion of SEQ ID NO:1 with any of the following sequences: LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL, LDSEV, LDSDI, LDSDL, LDSDV, LETEI, LETEL, LETEV, LETDI, LETDL, LETDV, VDTEI, VDTEL, VDTEV, VDTDI, VDTDL, VDTDV, IDTEI, IDTEL, IDTEV, IDTDI, IDTDL, IDTDV, IETEI, IETEL, IETEV, IETDI, IETDL, IETDV.

In some embodiments, functional variants disclosed herein also include amino acid sequences having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or even higher identity to the above-mentioned peptides. As is known in the art, "identity" between two proteins is determined by aligning the amino acid sequence of one protein with the sequence of a second protein substituted with its conserved amino acids. The degree of identity between two proteins is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm.

In some embodiments, functional variants disclosed herein include peptides having 1, 2, 3, 4, 5 or more substitutions, deletions, additions and/or insertions of amino acid residues as compared to the above-mentioned peptides that differ from the above-disclosed specific peptides.

As described above, the functional variants may be distinguished from the specific peptides disclosed above by one or more substitutions, deletions, additions and/or insertions. These variants may be naturally occurring or may be produced synthetically, for example, by modifying one or more of the above-described peptide sequences disclosed herein and assessing their biological activity as described herein using any of a variety of techniques well known in the art.

In some embodiments, the peptide is a chimeric peptide comprising amino acid sequence YEKLLDTEI (SEQ ID NO:1), or a functional variant thereof, and an internalization peptide that facilitates uptake of the chimeric peptide by a cell.

Those skilled in the art will appreciate that the purpose of chimerizing an active peptide and an internalization peptide is primarily to allow the active peptide to reach its target site of action better, and thus, an internalization peptide suitable for use in the present application is not limited to a particular class, as long as it achieves the purpose of membrane penetration and internalization. It will also be appreciated by those skilled in the art that since the target of action of an active peptide is primarily located inside a neuronal cell, internalization peptides that are specifically adapted to neuronal cells are preferred. In some embodiments, the internalization peptide can be a Tat peptide. In some embodiments, the amino acid sequence of the Tat peptide is YGRKKRRQRRR (SEQ ID NO: 2). In some embodiments, the chimeric peptide comprises amino acid sequence YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 3).

It will be appreciated that the internalization peptide may be linked to the active peptide via an amide bond as a fusion peptide, but may also be joined by other suitable means, such as chemical bonding. The coupling of the two components may be achieved by a coupling or conjugation agent. A large number of such reagents are commercially available and can be found in S.S. Wong, Chemistry of protein conjugation and Cross-Linking, CRC Press (1991). Some examples of crosslinking agents include J-succinimide-3- (2-pyridinedithio) propionate (SPOP) or N, N' - (1, 3-phenylene) bismaleimide; n, N' -ethylene-bis- (iodoacetamide) or other such reagents having 6 to 11 carbon methylene bridges (other thiol groups are relatively specific); and 1, 5-difluoro-2, 4-dinitrobenzene (which forms an irreversible linkage with the amino group and the tyrosine group). Other crosslinking agents include P, P '-difluoro-m, m' -dinitrodiphenyl sulfone (which forms irreversible crosslinks with amino and phenolic groups); dimethyl diethylaminohexanoate (which is specific for the amino group); phenol-1, 4-disulfonyl chloride (which reacts predominantly with amino groups); 1, 6-hexamethylene diisocyanate or diisothiocyanate, or phenylazo-p-diisocyanate (which reacts predominantly with amino groups); glutaraldehyde (which reacts with several different side chains) and bis-diazotized benzidine (which reacts primarily with tyrosine and histidine).

In addition, the aforementioned peptides can optionally be derivatized (e.g., acetylated, phosphorylated, and/or glycosylated) to promote affinity with the inhibitor, to promote the ability of the inhibitor to be transported across the cell membrane, or to promote stability.

The active peptides of the present application as well as fusion peptides fused to internalization peptides can be synthesized by solid phase synthesis or recombinant methods. Peptidomimetics can be synthesized using a variety of protocols and methods described in scientific and patent literature, such as Organic Syntheses Collective Volumes, Gilman et al (ed.) John Wiley & Sons, Inc., NY, al-Obeidi (1998) mol.Biotechnol.9: 205-223; hruby (1997) curr. opin. chem.biol.1: 114-119; 0stergaard (1997) mol. Divers.3: 17-27; 0stresh (1996) Methods enzymol.267: 220-234.

In some embodiments, the active peptides or chimeric peptides disclosed herein may be administered in the form of a pharmaceutical composition. The pharmaceutical compositions may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants which facilitate processing of the active peptide or chimeric peptide into a pharmaceutically acceptable formulation. Proper formulation depends on the chosen route of administration.

In some embodiments, administration may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Intravenous administration is preferred.

In some embodiments, the pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic. For injection, the active peptide or chimeric peptide may be formulated in aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulating agents such as suspending, stabilizing and/or dispersing agents.

Alternatively, the active peptide or chimeric peptide may be in powder form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.

for transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. This route of administration can be used to deliver the compound to the nasal cavity or for sublingual administration.

In some embodiments, for oral administration, the active peptide or chimeric peptide may be formulated with a pharmaceutically acceptable carrier as tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient to be treated. For oral solid formulations such as powders, capsules and tablets, suitable excipients include fillers such as sugars, for example lactose, sucrose, mannitol and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, carboxypropyl methyl cellulose, sodium carboxymethylcellulose, and/or povidone (PVP); granulating agent and adhesive. If desired, disintegrating agents can be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. If desired, the solid dosage form may be sugar coated or enteric coated using standard techniques. For oral liquid preparations such as suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycerol, oils, alcohols. In addition, a flavoring agent, a preservative, a coloring agent, and the like may be added.

in addition to the formulations previously described, active peptides or chimeric peptides may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example as a sparingly soluble salt.

Alternatively, other drug delivery systems may be used. Liposomes and emulsions can be used to deliver the chimeric peptides. Certain organic solvents such as dimethyl sulfoxide may also be used. In addition, compounds can be delivered using sustained release systems (e.g., semipermeable matrices of solid polymers containing the therapeutic agent).

Depending on its chemical nature, sustained release capsules may release the chimeric peptide for weeks up to over 100 days. Other strategies for protein stabilization may be used depending on the chemical nature and biological stability of the therapeutic agent.

In some embodiments, because the active peptides or chimeric peptides disclosed herein may contain charged side chains or termini, they may be included in any of the above formulations as free acids or bases or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are salts which substantially retain the biological activity of the free base and are prepared by reaction with a mineral acid. Drug salts tend to be more soluble in water and other protic solvents than the corresponding free base forms.

In some embodiments, the dosage range includes 0.001 to 20 μmol of active peptide or chimeric peptide per kg of patient body weight, optionally 0.03 to 3 μmol of active peptide or chimeric peptide per kg of patient body weight. In some methods, 0.1-20 μmol of active peptide or chimeric peptide per kg of patient body weight is administered. In some methods, 0.1 to 10 μmol of active peptide or chimeric peptide per kg of patient body weight is administered, more preferably about 0.3 μmol of active peptide or chimeric peptide per kg of patient body weight is administered. In other cases, the dosage range is 0.005 to 0.5 μmol of active peptide or chimeric peptide per kg of patient body weight. The different surface areas can be compensated by dividing by 6.2: mass ratio, the dose per kg body weight is converted from rat to human. Suitable doses of active peptide or chimeric peptide for use in humans may include 0.01 to 100mg/kg of patient body weight, or more preferably 0.1 to 30mg/kg of patient body weight or 1 to 10mg/kg (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg).

In some embodiments, the amount of active peptide or chimeric peptide administered depends on the subject being treated, the weight of the subject, the severity of the disease, the mode of administration, and the adjustment of the prescribing physician. Treatment may be repeated when symptoms are detectable or even undetectable. Treatment may be provided alone or in combination with other drugs.

In some embodiments, therapeutically effective doses of the active peptides or chimeric peptides disclosed herein are capable of providing therapeutic benefit without causing significant toxicity. Toxicity of the chimeric peptides can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example by determining LD50 (the dose lethal to 50% of the population) or LD100 (the dose lethal to 100% of the population). The dose ratio of toxic effect to therapeutic effect is the therapeutic index. Chimeric peptides or peptidomimetics that exhibit a high therapeutic index are preferred (see, e.g., Fingl et al, 1975, in The pharmaceutical Basis of Therapeutics, Chapter 1, page 1).

In some embodiments, the pharmaceutical composition is in the form of a pre-lyophilized formulation, or in the form of a reconstituted formulation obtained by combining the lyophilized formulation with an aqueous solution.

It should be understood that the above detailed description is only for the purpose of making the content of the present application more clearly understood by those skilled in the art, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will be apparent to those skilled in the art.

Examples

The following examples are provided merely to illustrate some embodiments of the present application and are not intended to be limiting in any way.

Example 1: screening of active peptide molecules

According to the reported research results, the Tat cell-penetrating peptide YGRKKRRQRRR (SEQ ID NO:2) is selected and linked with different numbers of amino acids to form a peptide library. The chimeric peptide molecules in the peptide library interact with PDZ1/2 structural domains expressed and purified in vitro respectively, and the polypeptides are primarily screened according to the strength of interaction force.

The immobilized molecule (ligand) is PDZ1/2 protein, molecular weight: 20kD, concentration: 2 mg/ml; molecules of the mobile phase (analytes): polypeptide to be screened, molecular weight: 2kD, concentration: 10 mg/ml. Immobilization was performed using a Biacore 3000 instrument, CM5 chip. The electrophoresis buffer solution is PBS + 0.005% Tween 20. Immobilization was performed using an amino coupling method. The concentration of ligand was 10. mu.g/ml. The fixation buffer was 10mM sodium acetate, pH 4.0. Fixed quantity: 1400RU, fixed to flow cell 2. The flow rate used was 10. mu.l/ml and the ligand was injected for 1 minute. Regeneration was performed at a flow rate of 30. mu.l/min using 10mM Gly at pH 2.0+2.5 as a regeneration solution. The sample injection time is 30 s.

Kinetic analysis was performed using the following conditions: control channel: a flow cell 1; the electrophoresis buffer solution is PBS; concentration gradients of 6.25n, 12.5n, 25n, 50n, 100n, 200n, 400nM using Kinetic Analysis Wizard model; the sample introduction time is 1 minute; the dissociation time is 2 min; the flow rate was 30. mu.l/min.

The data were fitted using the fitting software BIAevaluation 4.1 software. The fitted model is 1: 1 binding model. The dissociation constant KD value is inversely proportional to the applied force.

Through screening, a chimeric peptide with strong interaction capacity with the PDZ1/2 domain is obtained and named as P5, and the sequence is as follows:

P5:YGRKKRRQRRRYEKLLDTEI(SEQ ID NO:3)

For direct comparison with similar chimeric peptides in the reported studies, a control chimeric peptide NA-1 was introduced, the sequence of which is as follows:

NA-1：YGRKKRRQRRRKLSSIESDV

In addition, by comparing the structural difference between P5 and NA-1, a chimeric peptide YE-NA-1 in which two residues of YE were added to the N-terminus of the active peptide of the chimeric peptide NA-1 was additionally introduced, and the sequence was as follows:

YE-NA-1：YGRKKRRQRRRYEKLSSIESDV

The chimeric peptides NA-1, YE-NA-1 and P5 were simultaneously tested for interaction with the PDZ1/2 domain as described above, with the results shown in Table 1 below:

TABLE 1 detection of the interaction force of three chimeric peptides with the PDZ1/2 domain

Chimeric peptides	NA-1	YE-NA-1	P5
				KD(M)	7.53E-08	5.44E-08	2.99E-08

As shown in Table 1, chimeric peptides YE-NA-1 and P5 interacted more strongly with the PDZ1/2 domain and P5 was better acting than the control chimeric peptide NA-1. Therefore, it is speculated by the inventors that the two extra amino acid residues of YE at the N-terminal of the active peptide have certain enhancement effect on the interaction of the polypeptide and the PDZ1/2 domain. Furthermore, P5 reduced two less hydrophobic serines (SS) relative to the carboxy terminus of YE-NA-1, which, by the inventors' speculation, might therefore further increase the interaction of the polypeptide with the PDZ1/2 domain.

The chimeric peptide P5 was further tested in the following experiments, and NA-1 and YE-NA-1 were used as controls in some of the experiments.

Example 2: pull-down assay to detect the interaction of P5 with the PDZ1/2 domain

To demonstrate that P5 interacts with the PDZ1/2 domain, Pull-down experiments were performed.

The column was equilibrated for 5min with 100. mu.l of His beads and 1ml of MCAC-0 buffer. Shaking at 4 ℃. The mixture was centrifuged at 5000g for 1 min at 4 ℃ and the supernatant was discarded. To the mixture was added 1mg of PDZ1/2 protein and made up to 1ml with buffer. The mixture was spin-bonded for 1 hour at 4 ℃. The mixture was centrifuged at 5000g for 1 min at 4 ℃ and the supernatant was discarded. The cells were washed 3 times with 1ml of MCAC-0 buffer for 5 minutes each (shaking wash at 4 ℃). To the mixture was added 1mg of P5 protein and made up to 1ml with buffer. The mixture was spin-bonded for 2 hours at 4 ℃. The mixture was centrifuged at 5000g for 1 min at 4 ℃ and the supernatant was discarded. Washing was carried out 3 times with 1ml of lysis buffer for 5 minutes each time (washing with shaking at 4 ℃). After washing 20. mu.l MCAC-300 was added. Centrifuging, and taking the eluate for SDS-PAGE detection. The results of the experiment are shown in FIG. 1.

as confirmed in fig. 1, both the P5 and PDZ1/2 domains were contained in the elution band of chimeric peptide P5, thereby confirming that chimeric peptide P5 was able to bind to PDZ1/2 domain.

Example 3: acute toxicity test

Acute toxicity tests were performed in rats. The results show that: at the administration dose of 200mg/kg body weight, P5 has no lethal effect and other obvious toxic and side effects on rats.

Example 4: effect of P5 peptide on treating Alzheimer's disease

I. Instrument for measuring the position of a moving object

The apparatus used in this example was as follows:

1056189 double-head cold light source (Beijing Zhongdi Chuang science and technology development, LLC).

TJ-4A micro-injection pump (Beijing Zhongguo Tech development, Limited liability Co., Ltd.)

ZS-001Morris water maze video analysis system (Beijing Zhongdi Tech development, Limited liability company)

ZS-GSZ skull drilling instrument (Beijing Zhongguti Dichu scientific and technological development, Limited liability company)

II. Material

The materials used in this example were as follows:

Physiological saline (Shijiazhuang four drugs Co., Ltd., national drug Standard H13023200);

Polypeptide (P5, NA-1, manufactured by Hangzhou Zhongji peptide Co., Ltd.);

Chloral hydrate (national pharmaceutical group chemical agents limited, lot number 20150303);

Gentamicin (Hua Chinese medicine industry Co., Ltd., national drug Standard H42021503);

Amyloid a β (cat # a1075, available from sigma (china));

Dental cement (Beijing Zhongshidi scientific and technological development, LLC);

Memantine hydrochloride (Beijing Solaibao Biotech Co., Ltd.).

Experimental animals

the experimental animals used in this example were 240g to 280g SD rats purchased from Fukan Kangcheng Co., Ltd.

Method and procedure

1. Rat screening and grouping

80 male SD rats 240-280 g are selected as experimental mice and divided into 7 groups, wherein each group comprises 9-11 male SD rats, and the 7 groups respectively comprise: normal group, model group (AD group), P5 three dose groups (tail vein injection 1mg/kg, 3mg/kg, 10mg/kg), NA-1 (tail vein injection 3mg/kg, peptide drug control), memantine group (gavage 2mg/kg, chemical drug control).

2. Molding die

1) SD rats are anesthetized by intraperitoneal injection of 10% chloral hydrate solution, and the dosage is 4.5 ml/kg.

2) Fixing the head of a rat in a brain stereotaxic apparatus, conventionally disinfecting the surgical site by iodine tincture and alcohol cotton balls, and preparing skin.

3) The elbow scissors cut the epidermis, the blunt instrument separates the skin, the subcutaneous tissue and the periosteum, the skull is exposed, and the bregma position is found.

4) Marking 3mm behind bregma and one position at each of the left and right 2mm positions, polishing the marked skull to be thin enough and have two positions as much as possible by using a ZS-GSZ skull drilling instrument, pricking with a micro syringe of 10 μ l without puncture, and then continuing to insert a needle downwards by 3.5 μm.

5) Injecting amyloid A beta with the injection parameter of 2.5 mul/5 min, remaining the needle for 5 minutes after injection, and then slowly withdrawing the needle, wherein the amyloid A beta is 1 mg/ml.

6) The dental cement is used for sealing the skull drill hole, the skin needle and the 4.0 surgical suture are used for suturing the wound, the gentamicin is injected into the thigh muscle at 1mg/kg, and the commercial stock solution is diluted three times.

7) The day of surgery was the first day, and the 10 th day was the treatment of continuous administration.

3. Administration and testing

The Morris water maze (Morris water maze) test was used in this study. The test is an experiment for forcing the experimental animal to swim and learning and searching a platform hidden in water, is mainly used for testing the learning and memory capacity of the experimental animal to the spatial position sense and the direction sense (spatial positioning), and is widely applied to the drug evaluation research of the Alzheimer disease.

After each group of models is modeled for 10 days, the drug administration is started for 14 days, once a day, the water maze test is carried out immediately after the drug administration is finished, the waiting period is tested for 4 days continuously, the entering water quadrant is 4213, 1234, 2413 and 2143, the platform is removed on the 5 th day to carry out the platform penetrating experiment, and the entering water quadrant is 4321.

4. Incubation period test

Each rat in the different groups was immersed in water from the same location for each test, and the immersion quadrant was as described in "3. dosing and testing". When the rat enters water, the operator lifts the tail of the rat, the head of the rat is slowly placed into the water towards the wall, the rat finds the platform after entering the water and stays for 2 seconds, the experiment is stopped, the consumed time is the latency period, and the time is counted as 120 seconds when the platform is not found after exceeding 120 seconds. Periodically supplementing water, keeping the platform 2cm away from the water surface, and keeping the time point of the start of each test 1 minute away from the time point of the start of the next test.

5. Bench penetration test

After the rats are tested according to the quadrant continuous water maze latency period of 4 days shown in the '3. administration and test', the platform in the water maze is removed, the movement track of the rats entering the water is recorded, the platform intersects with the position once and is recorded as one-time platform passing, the total times of platform passing in 120 seconds of each rat is recorded, and the rats enter the water quadrant 4321.

Statistical analysis of V

Each rat was watered 4 times per day in 4 quadrants for 4 consecutive days, and the resulting 16 numbers were finally averaged for the final incubation period of that rat, and on day 5 each rat was watered 4 times in 4 quadrants for only one day, and the resulting 4 numbers were finally averaged for the number of landings of that rat. The table-up latency and the number of times of table-through of the model group and the normal group were compared respectively using the EXCEL self-contained T-test function (mantissa 2, type 3). Statistical differences were considered to be with p <0.05 and significant statistical differences were considered to be with p < 0.01. If the normal group and the model group (AD group) have statistical difference in latency and the number of times of platform crossing, the molding is determined to be successful.

And respectively comparing the model group with the upper station latency and the number of times of table crossing of each administration group. Statistical differences were considered to be with p <0.05 and significant statistical differences were considered to be with p < 0.01. Treatment groups were considered to have therapeutic effects if there was a statistical difference between the treatment group and the model group (AD group) in latency or number of landings.

VI. results of the experiment

1. Effect of different administration groups on escape latency of model rats

the escape latency statistics for each group of rats are shown in table 1 and fig. 2. The results show that: p <0.05 (not labeled in the figure) between the normal group and the AD group indicates successful molding. The three dose groups (1mg/kg, 3mg/kg, 10mg/kg) and NA-1 group of P5 compared to AD group, with P <0.05, demonstrated efficacy in this test, and the escape latencies of these four groups were substantially comparable to normal. The chemical control memantine group showed no efficacy in this test.

2. Effect of each treatment group on rat crossing platform test

Statistics of the number of platform crossings for each group of rats are shown in table 2 and fig. 3. The results show that: p <0.05 (not labeled in the figure) between the normal group and the AD group indicates successful molding. Two dose groups of P5 (3mg/kg, 10mg/kg) compared to the AD group, with P <0.05, indicated therapeutic efficacy in this test, and the number of plateau crossings in these two groups was substantially comparable to the normal group. The chemical control memantine group had a p <0.01 compared to the AD group, indicating that therapeutic efficacy was also produced. There was no statistical difference between the two effective dose groups of P5 (3mg/kg and 10mg/kg) and the memantine group. The peptide control NA-1 group showed no efficacy in this test.

VII. discussion

The two dose groups of P5 (3mg/kg and 10mg/kg) were statistically different from the AD group in both latency and number of landings (P <0.05), and the actual results were comparable to the normal group, showing therapeutic efficacy. However, the same 3mg/kg peptide control NA-1 group showed efficacy only in the latency test and no effect in the cross-hatch number test. Memantine hydrochloride is a commercially available drug for the treatment of alzheimer's disease, which only shows efficacy in the table-wearing number test, but no effect in the latency test. Combining the results of this study, the exemplary peptide P5 of the present application has good application prospects in the treatment of alzheimer's disease.

All publications and patent documents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the disclosure. Unless the context indicates otherwise, any feature, step, or embodiment of an embodiment of the present disclosure may be used in combination with any other feature, step, or embodiment.

Sequence listing

<110> Beijing Biotechnology Ltd

<120> peptide for preventing, alleviating or treating alzheimer's disease and use thereof

<130> 17C13031CN

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Tyr Glu Lys Leu Leu Asp Thr Glu Ile

1 5

<210> 2

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210> 3

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Tyr Glu Lys Leu Leu

1 5 10 15

Asp Thr Glu Ile

20

Claims (9)

1. Use of a peptide having the amino acid sequence YEKLLDTEI (SEQ ID NO:1) or a functional variant thereof, wherein the functional variant is a variant of YEKLLDTEI wherein one or more conservative substitutions in the LDTEI moiety are made, and wherein the conservative substitutions are selected from the group consisting of a substitution between D and E, a substitution between L, V and I, and a substitution between T and S, in the manufacture of a medicament for improving learning, memory and/or spatial cognition in an individual with Alzheimer' S disease.

2. The use of claim 1, wherein the functional variant is a variant resulting from replacement of the LDTEI moiety in YEKLLDTEI with any one of the following sequences: LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL, LDSEV, LDSDI, LDSDL, LDSDV, LETEI, LETEL, LETEV, LETDI, LETDL, LETDV, VDTEI, VDTEL, VDTEV, VDTDI, VDTDL, VDTDV, IDTEI, IDTEL, IDTEV, IDTDI, IDTDL, IDTDV, IETEI, IETEL, IETEV, IETDI, IETDL, IETDV.

3. Use of a chimeric peptide consisting of an active peptide having amino acid sequence YEKLLDTEI or a functional variant thereof, wherein the functional variant is a variant of YEKLLDTEI that results after one or more conservative substitutions in the LDTEI moiety, and wherein the conservative substitutions are selected from the group consisting of a substitution between D and E, a substitution between L, V and I, and a substitution between T and S, and an internalization peptide, in the manufacture of a medicament for improving learning, memory, and/or spatial cognition in an individual with alzheimer' S disease.

4. the use of claim 3, wherein the functional variant is a variant resulting from replacement of the LDTEI moiety in YEKLLDTEI with any one of the following sequences: LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL, LDSEV, LDSDI, LDSDL, LDSDV, LETEI, LETEL, LETEV, LETDI, LETDL, LETDV, VDTEI, VDTEL, VDTEV, VDTDI, VDTDL, VDTDV, IDTEI, IDTEL, IDTEV, IDTDI, IDTDL, IDTDV, IETEI, IETEL, IETEV, IETDI, IETDL, IETDV.

5. The use of claim 3, wherein the amino acid sequence of the internalization peptide is YGRKKRRQRRR (SEQ ID NO: 2).

6. The use of claim 3, wherein the amino acid sequence of the chimeric peptide is YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 3).

7. The use of any one of claims 1-6, wherein the memory capacity is short-term memory capacity.

8. The use of any one of claims 1-6, wherein the subject is a mammal.

9. The use of any one of claims 1-6, wherein the subject is a human.