HK1137765B - β -SHEET BLOCKING PEPTIDES FOR PREVENTING AND/OR TREATING ALZHEIMER’S DISEASE - Google Patents

Description

Beta-sheet blocking peptide for preventing and/or treating alzheimer's disease

Technical Field

The invention relates to polypeptides and application thereof, in particular to a beta sheet layer blocking peptide which can be used for preventing and/or treating Alzheimer disease, and application of the polypeptides in preventing and/or treating Alzheimer disease.

Background

Alzheimer's Disease (AD), also known as senile dementia, is a neurodegenerative disease with occult onset and progressive course, and is characterized by typical clinical features of comprehensive cognitive impairment and personality change, manifested as early-stage recent memory impairment, and then persistent intellectual decline, aphasia, judgment of reasoning ability loss and dyskinesia. This disease seriously affects the quality of life of the patient and his family members, and also imposes a heavy burden on the patient's family and society.

With the accelerated aging process of the population, the senile diseases become a prominent problem which obviously affects the health of human beings. Senile dementia, malignant tumor and cardiovascular and cerebrovascular accidents are listed as three diseases causing death of the elderly, and are the fourth enemy of human health in the 21 st century. The world health organization has listed AD as one of the five major diseases in the 21 st century. China entered the aging society in 1999. In the end of 2004, the population of the aged 60 years old and older in China reaches 1.43 hundred million, accounting for 10.97% of the total population. This number is predicted to reach 2 billion in 2014, 3 billion in 2026, more than 4 billion in 2037, and maximum in 2051, and thereafter is maintained on a scale of 3 to 4 billion. The increasing population of the elderly makes the incidence of alzheimer's disease relatively high. It is understood that more than 20% of the population in europe, japan and the united states over the age of 80 currently suffer from this disease. Over 5000 million people in the population over 65 years old worldwide suffer from different kinds of dementia.

The main features of pathological changes in alzheimer's disease are: a large number of Senile Plaques (SP) formed by deposition of beta amyloid peptide (beta amyloid peptide, abbreviated as Α β or β a) between nerve cells, Neurofibrillary Tangles (NT) caused by Tau protein that is hyperphosphorylated within nerve cells, and a large number of lost neurons are formed. There is a great deal of evidence that neurotoxicity of a β is a common junction of all etiologies. Prevention and/or treatment of targeted a β has therefore become one of the focus of AD research in recent years.

A β is a metabolite of β Amyloid Precursor Protein (APP). Under the action of alpha secretase, APP generates soluble sAPP alpha under normal conditions, and the sAPP alpha has the functions of reducing intracellular calcium concentration, regulating synaptic plasticity, promoting growth of synapses and protecting neurons, which is the main form of APP processing, and the pathway does not generate Abeta; another pathway is that APP produces sAPP β and C99 under the action of β secretase, and C99 further releases a β peptide under the action of γ secretase. Gamma secretase hydrolysis of C99 is heterogeneous, cleaving the site between alanine 713 and threonine 714 to produce A β_1-42(ii) a Cleavage of the site between valine 711 and isoleucine 712 to yield A.beta._1-40(Selkoe DJ.Alzheimer’s disease：genes，proteins，and therapy.Physiol Pev，2001，81(2)：741-766)。Aβ_1-42Accounts for about 10 percent of the total amount of the A beta protein, and the A beta_1-40Approximately 90%, but Abeta_1-42Then more readily aggregated, aggregated a β_1-42Is the basic component for senile plaque.

Aβ_1-42The primary structure of the peptide is shown below (SEQ ID NO: 4):

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-

1 5 10 15

Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-

16 20 25 30

Ile-Ile-Gly-Leu-Met-Val-Gty-Gly-Val-Val-Ile-Ala

31 35 40 42

Aβ_1-42c-terminal 10 amino acid residues 33-42 and 17-21 have high hydrophobicity, and form A beta_1-42A hydrophobic region; amino acid residues 28-42 are more likely to form a beta-sheet conformation, while amino acid residues 9-21 are also likely to form a beta-sheet conformation. Beta sheet conformation favours A beta_1-42Aggregation of the peptide. The experimental result shows that Val at the C terminal⁴⁰Ile⁴¹Ala⁴²The three amino acid residues play a stabilizing role in beta sheet conformation and are beneficial to the formation of beta sheet. Abeta peptide_1-42The N-terminal of (A) has hydrophilicity and is capable of forming an alpha-helical, random-helical or beta-sheet conformation depending on solution conditions. The results of the study indicate that the beta sheet conformation favors the aggregation of the A beta peptide, which is due to the interaction of its hydrophobic regions. Soto et al (Soto C, Kindy MS, Baumann M, et aL., Inhibition of Alzheimer's amyloidosis by peptides which is present in the region of the hydrophobic region of A.beta.peptide. biochem. Biophys. Res. Commun.1996, 226 (3): 672 and 680) replace amino acids in the vicinity of the hydrophobic region of A.beta.peptide with proline, and the resulting small peptide not only does not form a beta-sheet conformation, but also can bind to A.beta.peptide to maintain the conformation of the random helix and inhibit its aggregation. The research results show that one of the hydrophobic regions of the A beta peptide: abeta (beta)_16-20The pentapeptide fragment Lys-Leu-Val-Phe-Phe binds to the A.beta.peptide and thus prevents its aggregation. One by one substitution by alanine, indicating Lys¹⁶、Leu¹⁷And Phe²⁰Plays a key role therein. This illustrates A β^16-20Residue is AThe beta peptide is combined with the adjacent A beta peptide chain during the aggregation process. Research shows that the spatial conformation of A beta obviously influences the aggregation capability of A beta, and when the secondary structure of A beta is mainly alpha helix, the aggregation is slow; whereas with beta sheets as the main, aggregation is faster.

Under certain conditions, the hydrophobic region of the beta-sheet-rich structure is exposed, so that A beta can be promoted to aggregate to form oligomer, finally, insoluble substances are deposited in the gap of a neuron, neurotoxicity is generated, the activity of brain colloid cells is increased, inflammatory mediators and complement are generated, and amyloid plaques are formed together.

An increase in the number of hydrophobic charges of the molecule is one of the main causes of a β aggregation. And Abeta_1-40Comparison with Abeta_1-42The extended two amino acids not only increase the hydrophobicity of a β, making it more prone to aggregation, but also increase the stability of the aggregates, allowing selective deposition in amyloid plaques at an early stage. Abeta (beta)_1-42May be an initiating factor for the formation of oligomers, fibers and plaques by soluble A.beta. (Younkin, S.G.1995. infectious way A.beta.42 is the real cu lpipe in Alzheimer's disease. Ann.Neurol.37: 287-288.; Matsuok Y, Saito M, Lafrancois T, et al. Noval thermal approach for the treatment of Alzheimer's disease by the treatment of].J Neurosci，2003，23(1)：1-5)。

Jarrett et al propose: abeta (beta)_1-42Is first aggregated as a "seed" to initiate deposition of a β; other monomers gradually accumulate around the nucleus, and the extended (ligation) peptide chains form fibers that further accumulate and spread, eventually forming plaques (Jarrett JT, Lansbury PT Jr. feeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scientific. 1993, (6): 1055-.

The aggregation, precipitation and formation of senile plaques of a β and their accompanying neuronal damage are considered central links in the pathological mechanism of alzheimer's disease. Therefore, if the aggregation of a β peptide can be inhibited and the degradation and clearance of a β peptide can be accelerated, the purpose of preventing and/or treating AD can be fundamentally achieved.

The current international research on a β drugs aims to reduce a β production, increase a β clearance, prevent or reverse a β aggregation, inhibit a β toxicity, and the like. Researchers at the university of Washington, USA, have found that brain cells of Alzheimer's disease mice begin to recover function dramatically after clearing away amyloid plaques in the mouse brain. Indicating that drugs against a β have encouraging prospects. Among the various drugs directed against a β, β -sheet blockers are of increasing interest.

The current international drugs belonging to beta-sheet blockers are mainly of the following two types:

(1) the Neurochem company, canada, has designed and synthesized derivative compounds of the GAGs based on the discovery that low relative molecular mass amino glycoproteins (GAGs) act to stabilize plaques in amyloid beta plaques and hinder plaque degradation. Animal in vivo experiments show that the low relative molecular mass GAG analogues can significantly reduce the level of beta amyloid in blood plasma and brain, inhibit A beta aggregation, and can be used for treating AD. The small molecule compound Alzheimer is currently in phase III clinical trial.

(2) Chac pacific ya et al (Chac pacific MA, Barr i MI, Soto C, et aL., Beta-sheet breaker peptide precursors Beta-induced specific mechanisms with partial reduction of amyloid peptides. mol. major. 2004, 9 (10): 953-61) showed that iAbeta5p not only prevented the formation of a β fibers, but also had the ability to disintegrate a β fibers, which is currently in phase III clinical trials, by examining the neuronal protection by i.p. blocking peptide (i.a-amino-acid-peptide 5p) consisting of 5 amino acid residues.

There is a need in the art for monomers that are capable of binding to amyloid beta (A β)_1-42) Specific binding, stabilizing its normal space structure and inhibiting its formation of beta sheetA layer, a new active agent that prevents the formation of soluble beta amyloid oligomers and beta amyloid plaques. The agent should be capable of inhibiting aggregation of A beta peptide, accelerating degradation and clearance of A beta peptide, and thus be useful for prevention and/or treatment of AD.

Disclosure of Invention

An object of the present invention is to provide a monomer capable of reacting with beta-amyloid (A beta)_1-42) A polypeptide that specifically binds, stabilizes its normal spatial structure, inhibits its formation of beta sheets, prevents the formation of soluble beta amyloid oligomers and beta amyloid plaques, and has a lytic effect on a β fibers, which may be referred to as a beta sheet blocking peptide.

The present inventors have unexpectedly found that a polypeptide comprising the following amino acid sequence can achieve the above object:

His-X1-X2-Leu-X3-Phe-Phe-X4-Glu-Asp

wherein: x1 can be a lysine residue (Lys, K) or a glutamine residue (Gln, Q);

x2 can be a lysine residue (Lys, K) or a glutamine residue (Gln, Q);

x3 can be a valine residue (Val, V) or a proline residue (Pro, P);

x4 can be an alanine residue (Ala, a) or a glutamic acid residue (Glu, G).

Accordingly, in a first aspect of the invention there is provided a polypeptide comprising an amino acid sequence as described above.

The polypeptide can be combined with beta amyloid protein monomer (Abeta)_1-42) Specifically binds to, stabilizes the normal spatial structure thereof, inhibits the formation of beta sheets thereof, prevents the formation of soluble beta amyloid oligomers and beta amyloid plaques, and has a disintegrating effect on a β fibers, and thus can be used for the prevention and/or treatment of alzheimer's disease.

Accordingly, in a second aspect of the invention, there is provided the use of a polypeptide of the invention in the manufacture of a medicament for the prevention and/or treatment of alzheimer's disease.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising one or more polypeptides of the invention and a pharmaceutically acceptable carrier.

In a fourth aspect of the invention, there is provided a method of preventing and/or treating alzheimer's disease in a subject, said method comprising administering to said subject an effective amount of a polypeptide of the invention.

Drawings

FIG. 1 shows the steps of an exemplary method for preparing a polypeptide of the present invention and the results of testing the prepared product. FIG. 1A shows the main steps of an exemplary method for the synthesis and purification of the polypeptide H101 of the invention; FIG. 1B shows the results of chromatographic analysis of H101; FIG. 1C shows the results of mass spectrometric detection of H101.

FIG. 2 shows A.beta._1-42The results of the thioflavin T fluorescence analysis of peptide aggregation and fiber formation show the influence of the polypeptide of the present invention on the fluorescence intensity of A beta aggregation. A beta of given concentration_1-42Peptides were incubated with the polypeptides of the invention H101, H102 or H103 or Vitamin E (VE) or L5 for 24 hours at 37 ℃ and then ThT fluorescence intensity was measured. n is 5, and represents and Abeta_1-42Group phase P < 0.05.

FIG. 3 shows H102 and vitamin E inhibit A.beta._1-42Dose-dependent profile of fiber formation using concentrations of 10. mu. mol/L, 20. mu. mol/L, 44.30. mu. mol/L, 100. mu. mol/L H102 and vitamin E with 11.07. mu. mol/L A.beta._1-42After 24 hours of co-incubation at 37 ℃, ThT fluorescence intensity was measured. n is 5.

FIG. 4 shows H102 and vitamin E inhibit A.beta._1-42Time-dependent profile of fiber formation, H102 and vitamin at a concentration of 44.30. mu. mol/LElement E with 11.07. mu. mol/L of A.beta._1-42Co-incubation at 37 ℃ and ThT fluorescence intensity was measured at 12 hours, 1 day, 3 days, 5 days and 7 days, respectively. n is 5.

FIG. 5 shows the use of various polypeptides at a concentration of 44.30. mu. mol/L with 11.07. mu. mol/L of A.beta._1-42After incubation at 37 ℃ for 5 days, A.beta._1-42And (3) observing the result of the polypeptide fiber formation by using an electron microscope. Wherein: FIG. 5A shows A.beta._1-42Singly incubate electron microscope results for 5 days, magnification 35000 times; FIG. 5B shows A.beta._1-42Singly incubating the obtained product for 5 days to obtain an electron microscope result, wherein the magnification is 50000 times; FIG. 5C shows the conjugation of polypeptide L5 with A.beta._1-42Incubating the samples together for 5 days to obtain an electron microscope result, wherein the magnification is 35000 times; FIG. 5D shows the coupling of polypeptide H101 with A.beta._1-42Incubating the samples together for 5 days to obtain an electron microscope result, wherein the magnification is 35000 times; FIG. 5E shows the coupling of polypeptide H102 with A.beta._1-42Incubating the samples together for 5 days, wherein the magnification of the samples is 35000 times; FIG. 5F shows the coupling of polypeptide H103 with A.beta._1-42Incubating the samples together for 5 days to obtain an electron microscope result, wherein the magnification is 35000 times;

FIG. 6 shows different concentrations (10. mu. mol/L, 20. mu. mol/L and 40. mu. mol/L) of various polypeptides for A.beta.of 5. mu. mol/L_1-42Effect of survival of SH-SY5Y cells of human neuroblastoma cultured together for 72 hours. n-12 denotes a group represented by and a β_1-42Group phase P < 0.05.

FIG. 7 shows the results of immunohistochemical staining of neuronal sections from the CA1 region of the hippocampus of each group of mice with APPN terminal antibody and Abeta antibody at 400-fold magnification. FIG. 7A: the result of the immunohistochemical staining of the APP N-terminal antibody of the control group; FIG. 7B: a beta antibody immunohistochemical staining result of a control group; FIG. 7C: immunohistochemical staining results of APP N-terminal antibodies of the model group; FIG. 7D: immunohistochemical staining results of a β antibodies of the model group; FIG. 7E: performing immunohistochemical staining on APP N-terminal antibodies in the polypeptide injection group; FIG. 7F: and (3) performing immunohistochemical staining on the A beta antibody of the polypeptide injection group.

Fig. 8 shows the results of staining the temporal cortex of the brain and the hippocampus of each group of animals with congo red at 400-fold magnification. FIG. 8A: congo red staining of temporal cortex of brain of control group; FIG. 8B: congo red staining results of hippocampus of control group; FIG. 8C: congo red staining results of temporal cortex of brain of model group; FIG. 8D: congo red staining results of hippocampus of model group; FIG. 8E: congo red staining results of temporal cortex of brain of the polypeptide injection group; FIG. 8F: congo red staining of hippocampus of the polypeptide-injected group.

Detailed Description

The amino acid sequences of a series of polypeptides are disclosed herein, and it will be understood by those skilled in the art that when a sequence is represented by single or three letter amino acid residues, the sequence is represented from left to right as the sequence from the N-terminus (amino terminus) to the C-terminus (carboxyl terminus) of the polypeptide. For example, when "His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp" or "HQKLVFFAED" is used to indicate the sequence of a certain polypeptide, it means that the sequence of the polypeptide is "N-terminal-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-C-terminal", i.e., "N-terminal-HQKLVFFAED-C-terminal".

In a first aspect of the invention, there is provided a polypeptide comprising the amino acid sequence:

His-X1-X2-Leu-X3-Phe-Phe-X4-Glu-Asp

wherein: x1 can be a lysine residue (Lys, K) or a glutamine residue (Gln, Q);

x2 can be a lysine residue (Lys, K) or a glutamine residue (Gln, Q);

x3 can be a valine residue (Val, V) or a proline residue (Pro, P);

x4 can be an alanine residue (Ala, a) or a glutamic acid residue (Glu, G).

The polypeptide of the invention may comprise, consist essentially of, or consist of the amino acid sequence described above.

In one embodiment of the present invention, there is provided a polypeptide having the sequence designated H101 in the present invention:

H101：His-Lys-Gln-Leu-Val-Phe-Phe-Glu-Glu-Asp(HKQLVFFEED)(SEQID NO：1)。

in another embodiment of the present invention, there is provided a polypeptide having the sequence designated H102 in the present invention:

H102：His-Lys-Gln-Leu-Pro-Phe-Phe-Glu-Glu-Asp(HKQLPFFEED)(SEQID NO：2)。

in another embodiment of the present invention, there is provided a polypeptide having the sequence designated in the present invention as H103:

H103：His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp(HQKLVFFAED)(SEQID NO：3)。

the amino acid sequences of various polypeptides are disclosed herein. It will be apparent that various modifications may be made to the polypeptides of the invention. Such modifications include, but are not limited to: hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine and histidine side chains, acetylation of the N-terminal amino group and, in some cases, amidation of the C-terminal carboxyl group. It is understood that the various modifications described above will be readily apparent to those skilled in the art after knowing the amino acid sequence of the polypeptide of the invention, and polypeptides comprising the disclosed amino acid sequences containing such modifications are also within the scope of the invention.

The above-mentioned polypeptide of the present invention can be used as beta-sheet blocking peptide, and they can react with beta-amyloid monomer (Abeta)_1-42) Specifically binds to, stabilizes the normal spatial structure thereof, inhibits the formation of beta sheets thereof, prevents the formation of soluble beta amyloid oligomers and beta amyloid plaques, and has a decomposition effect on A beta fibers, and thus can be used as a medicament for preventing and/or treating Alzheimer's disease.

Therefore, in another aspect of the present invention, there is provided a use of the polypeptide of the present invention for the preparation of a medicament for the prevention and/or treatment of alzheimer's disease.

As used herein, "preventing" refers to reducing the risk of a subject developing a disease or delaying the time at which a patient develops a disease or symptoms. As used herein, "treatment" does not mean a complete cure. It refers to alleviation of the symptoms of the underlying disease and/or diminishment of one or more of the underlying cellular, physiological, or biochemical causes or mechanisms that cause the symptoms. It is understood that as used herein, alleviation is with respect to a disease condition, including the molecular state of the disease, and not just the physiological state of the disease.

The invention also provides a pharmaceutical composition comprising one or more polypeptides of the invention and a pharmaceutically acceptable carrier.

By "pharmaceutically acceptable carrier" is meant a substance that does not have a biologically or otherwise undesirable effect, i.e., the substance can be administered to a subject along with a polypeptide of the present invention without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. It will be apparent that the carrier will be selected to minimise any degradation of the active ingredient and to minimise any adverse side effects in the subject, as will be well known to those skilled in the art.

The pharmaceutical compositions of the invention generally comprise at least one polypeptide of the invention and one or more pharmaceutically acceptable carriers. Suitable vectors include, but are not limited to: antioxidants, preservatives, coloring agents, flavoring agents and diluents, emulsifying agents, suspending agents, solvents, fillers, extenders, buffers, carriers, diluents, excipients and/or pharmaceutically acceptable adjuvants. For example, suitable carriers may be physiological saline solution, citrate buffer or artificial CSF, and possibly other substances commonly used in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are also exemplary carriers. One skilled in the art can readily determine a variety of buffers that can be used in the compositions and dosage forms of the present invention. Typical buffering agents include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. Preferably, the buffer component is a water-soluble substance, such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof.

The primary solvent in the carrier may be aqueous or non-aqueous in nature. The carrier may additionally contain other pharmaceutically acceptable excipients for improving or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, dissolution rate or odor of the formulation. The pharmaceutical compositions of the present invention may also contain other pharmaceutically acceptable carriers for improving or maintaining the release rate of the polypeptide of the present invention. Such carriers are substances known to the skilled person for the formulation of sustained release formulations.

When the pharmaceutical composition of the present invention is formulated, it can be stored in sterile tubes in the form of a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. These formulations may also be stored in a ready-to-use form, as a lyophilized powder requiring reconstitution prior to use, or as a liquid requiring dilution prior to use. Preferably, the pharmaceutical compositions of the present invention are provided in a single use sterile tube form and stored at 2-8 ℃ until use. Immediately prior to administration, the pharmaceutical compositions of the present invention may be suitably diluted with a suitable sterile citrate buffer, such as any of those described above.

The present invention also provides a method for preventing and/or treating alzheimer's disease in a subject, said method comprising administering to said subject an effective amount of a polypeptide of the invention.

The term "effective amount" means an amount of a compound used that is sufficient to prevent the onset or symptoms of a disease or to ameliorate one or more causes or symptoms of a disease or disorder. The improvement need only be mitigated or altered and not necessarily eliminated. For the polypeptides of the invention, the prophylactically and/or therapeutically effective amount will vary depending on the subject to which it is directed and the polypeptide used. The prophylactically and/or therapeutically effective amount of the polypeptide used will also vary according to the age, size, weight, condition, etc. of the individual. It is within the ability of the person skilled in the art to determine a prophylactically and/or therapeutically effective amount of a polypeptide of the invention for a particular subject.

When the polypeptide of the present invention or the method for preventing and/or treating the same of the present invention is applied to a subject suffering from alzheimer's disease, the polypeptide of the present invention or the method for preventing and/or treating the same of the present invention has the effects of significantly inhibiting the aggregation of a β in the brain tissue of the subject, reducing the number and area of amyloid plaques, and improving the symptoms of alzheimer's disease. For example, activity and attention can be increased and reaction time can be reduced, and pronunciation, facial expression, body posture, smell, libido, sexual function and emotional condition can be improved and mental state can be enjoyed. In another embodiment of the invention, the polypeptides of the invention may suitably be administered as cognitive enhancers, for example to alzheimer's patients, to improve learning ability, especially impaired by dementia, or to inhibit cognitive decline and/or dementia.

Preparation of the polypeptide of the present invention

The polypeptides of the invention can be prepared by any method known to those skilled in the art for preparing polypeptides.

The polypeptides of the invention can be synthesized using chemical synthesis methods. The polypeptide can be synthesized in solution or by solid phase synthesis. The solid phase synthesis method of the polypeptide comprises Fmoc solid phase synthesis method and tBoc solid phase synthesis method. Methods for artificially synthesizing polypeptides are generally synthesized from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus).

In one embodiment of the invention, the polypeptides of the invention are synthesized using Fmoc solid phase synthesis and purified by HPLC. FIG. 1 shows, in an exemplary manner, the main steps for synthesizing and purifying a polypeptide H101 of the present invention and the results of detection of the prepared polypeptide in this embodiment. In this embodiment, the polypeptide is synthesized on the synthesis column using a polypeptide solid-state synthesizer, thereby greatly reducing the difficulty of product purification. In order to prevent side reactions, the side chains of the synthesis column and the added amino acids are protected. The carboxy terminus is free and must be activated prior to reaction. The polypeptides of the invention can be identified using Mass Spectrometry (MS) analysis after Fmoc synthesis and purification using preparative High Performance Liquid Chromatography (HPLC) columns. It will be appreciated that all polypeptides of the invention may be prepared, purified and identified using synthetic methods analogous to those described above.

The polypeptide of the present invention can also be produced by recombinant genetic engineering methods. Briefly, a polynucleotide encoding a polypeptide of the present invention can be synthesized, transformed into a suitable host cell by methods known in the art, expressed, and purified or treated to obtain the polypeptide of the present invention.

Examples

The present invention will be further described with reference to the following examples. The following examples are for the purpose of enabling those of ordinary skill in the art to better understand the present invention, are for illustrative purposes only, and are not intended to limit the scope of the present invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in degrees Celsius or is at ambient temperature, and pressure is near or at atmospheric pressure.

Example 1: preparation of the polypeptide of the present invention

In this example, a polypeptide of the invention, H101, was first synthesized using Fmoc/tBu solid phase polypeptide synthesis, having the sequence:

His-Lys-Gln-Leu-Val-Phe-Phe-Glu-Glu-Asp(SEQ ID NO：1)。

the raw materials used in the polypeptide synthesis method of this example were: Fmoc-Asp (OtBu) -Wang Resin (0.37mmol/g), Fmoc-Val-OH, Fmoc-Glu (OtBu) -OH, Fmoc-His (trt) -OH, Fmoc-Gln (trt) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH and Fmoc-Phe-OH.

The main steps of the polypeptide synthesis and purification method used in this example are shown in FIG. 1A.

The formulation of TFA reagent used in the experimental protocol shown in FIG. 1A was: [ TFA: H₂Mixing O, ethanedithiol and phenol at volume ratio of 92.5: 2.5]。

The crude peptide product obtained from the preparation was purified by HPLC to a purity of greater than 95% of the finally obtained peptide, the results of HPLC are shown in fig. 1B.

Polypeptide H101 prepared and purified by the above method was identified and sequenced using Mass Spectrometry (MS) analysis, the results of which are shown in FIG. 1C. The results of identification and sequencing show that the polypeptide sequence and molecular weight prepared by the above method are in agreement with expectations.

In a similar manner to this example, the following polypeptides of the invention were also subsequently prepared:

H102：His-Lys-Gln-Leu-Pro-Phe-Phe-Glu-Glu-Asp(SEQ ID NO：2)；

H103：His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp(SEQ ID NO：3)。

sequencing and characterization of several of the above synthetic polypeptides indicated that the synthesis and purification methods described in this example are applicable to a variety of polypeptides of the invention. The sequences and molecular weights of the various polypeptides prepared by the methods were consistent with expectations. And the polypeptide product with the purity of more than 95 percent can be obtained by the purification method.

Example 2: effect of the Polypeptides of the invention on amyloid beta

Materials and methods

1. Drugs and reagents: abeta (beta)_1-42(purity > 98%), vitamin E, Thioflayin T (ThT), Thiazol blue (MTT), dimethyl sulfoxide (DMSO), all from Sigma, USA; MEM medium, Fetal Bovine Serum (FBS) and trypsin (trypsin)Purchased from Gibco-BRL, USA; several polypeptides of the invention H101, H102 and H103 were prepared as described in example 1; also synthesized in a manner similar to that described in example 1 was a pentapeptide Leu-Pro-Phe-Phe-Asp, which has the same sequence as the beta sheet blocker peptide iAbeta5 of Soto-Jara et al and which is designated L5 in the present invention, and the L5 peptide will be used as a control in this and subsequent examples. The above polypeptides were synthesized by Shanghai Jier Biometrics, purified by High Performance Liquid Chromatography (HPLC), and analyzed by Mass Spectrometry (MS) to determine a purity of > 95%.

2. Thioflavin T (Thioflavin T, ThT) fluorescence analysis and electron microscopy: ThT fluorescence intensity reflects the degree of aggregation of A.beta.so ThT fluorescence was used to analyze the degree of aggregation of the protein. Vitamin E (ve) has been reported to be effective in inhibiting a β aggregation and fibril formation, and thus this experiment used vitamin E as a positive control. Mixing A beta_1-42The freeze-dried powder is prepared into 22.15 mu mol/L solution by 50mmol/L PBS (pH7.4), the four polypeptides (H101, H102, H103 and L5) are respectively prepared into 88.61 mu mol/L solution by the same PBS, and the vitamin E is prepared into 88.61 mu mol/L emulsion by 0.2 percent Tween 80-containing PBS. During the experiment, Abeta is added_1-42The solutions were mixed in equal volumes with each polypeptide solution to a final concentration of 11.07. mu. mol/L and 44.30. mu. mol/L, respectively. Mixing A beta_1-42Mixing with vitamin E emulsion at equal volume as positive control, and adding only Abeta_1-42And a solution not containing any of the above polypeptides or vitamin E as a negative control. After the solutions in each group are incubated for 24 hours at 37 ℃, 10 mu L of each group is added into 990 mu L of 3.0 mu mol/L ThT phosphate buffer solution, and the ThT fluorescence intensity is measured at the excitation wavelength of 453nm and the emission wavelength of 478-486 nm.

3. The polypeptide of the present invention and Abeta_1-42The dose-effect and the aging relation are as follows: selecting polypeptide H102, formulating with the above PBS to a series of concentrations, and mixing with 11.07. mu. mol/L A. beta_1-42The solution was incubated at 37 ℃ for 24 hours, and then a sample was taken for ThT fluorescence detection to confirm that the polypeptide of the present invention and A.beta._1-42Dose-effect relationship of. H102 and vitamin E with the concentration of 44.30 mu mol/L and Abeta with the concentration of 11.07 mu mol/L_1-42Co-incubation at 37 ℃ and ThT fluorescence detection at 12 hours (12h), 1 day (1d), 3 days (3d), 5 days (5d) and 7 days (7d) after the initiation of incubation, respectively, were performed to confirm the observation of the various polypeptides and A.beta._1-42While incubation of A β alone_1-42Peptides were used as negative controls.

4. And (3) observing by an electron microscope: to further observe the formation of fibers, various polypeptides were used at a concentration of 44.30. mu. mol/L with 11.07. mu. mol/L of A.beta._1-42After incubation at 37 ℃ for 5 days, 5. mu.l of each sample was dropped on a 300-mesh deionized copper mesh containing a carbon-supported membrane, and allowed to stand at room temperature for 15 min. And (3) carrying out light-shielding negative dyeing on 2% uranyl acetate for 2min, drying, and observing by using a transmission electron microscope. Simultaneously reacting Abeta_1-42Peptide incubation alone for 5 days served as a negative control.

5. Cytotoxicity experiments: human neuroblastoma SH-SY5Y cell line commonly used in the field, which was purchased from the Xuanwu Hospital of capital medical university and cultured in MEM medium according to a conventional method, to which 10% (v/v) of fetal bovine serum was added at 37 ℃ and 5% CO₂Cultured in an incubator and subcultured every 5 days. SH-SY5Y cells with stable cell growth state and logarithmic growth phase at density of 1.0 × 10⁴The cells were inoculated in 96-well plates (Costar products) at 200. mu.l/well, and 24 hours later, the medium was replaced with serum-free medium, and 5. mu. mol/L A. beta. was added_1-42And (4) solution and grouping. The four polypeptides were added to the culture medium of each group of cells at concentrations of 10. mu. mol/L, 20. mu. mol/L and 40. mu. mol/L, respectively. Adding serum-free culture medium only without adding A beta_1-42And polypeptide as positive control, adding only Abeta to the culture medium_1-42While the group of cells to which the above polypeptide was not added served as a negative control. 72 hours after the addition of the polypeptide, 20 μ l of 3- (4, 5-dimethyl-2-thiazole) -2, 5-diphenyl tetrazolium bromide (MTT) (5mg/ml) was added to each well, the incubation was performed at 37 ℃ for 4 hours, the stock culture was discarded, 200 μ l of DMSO was added to each well, the mixture was allowed to stand for 10min and shaken for 1min to completely dissolve the formazan particles, and the optical density at 492nm (Opti) was measured using a full-automatic enzyme-labeling machinecaldensity，OD)。

6. Statistical treatment: for data comparison between the two groups, a t-test method is adopted; for more than two data comparisons, the F-test method was used.

Results

1. Various polypeptide pairs tested for Abeta_1-42Inhibition of aggregation and fiber formation

Analysis of the various polypeptides tested and VE vs. A β by ThT fluorescence_1-42Effect of aggregation and fiber formation, Abeta_1-42Very high fluorescence intensity was formed in PBS solution, indicating that A.beta._1-42Can self-aggregate and form A beta fibers. Will contain only A beta_1-42Whereas the fluorescence intensity of the negative control without any test polypeptide or vitamin E was 100%. Calculation of the various polypeptide pairs tested for Abeta_1-42Inhibition (%) of aggregation and fiber formation. The results are shown in Table 1 below (note: background fluorescence generated by the instrument system itself has been subtracted from each data in Table 1).

Table 1: inhibition of A.beta.aggregation (%)

The polypeptide to be tested	L5	H101	H102	H103	Vitamin E
						Inhibition (% rate)	25.74	24.79	27.84	14.91	24.85

As can be seen from the results in Table 1 above, H102 is responsible for A.beta._1-42The inhibition rate of aggregation is highest, and the downward descending is sequentially as follows: l5, vitamin E, H101 and H103.

The results of the above table are plotted as a histogram, shown in fig. 2.

As shown in FIG. 2, H102 can significantly inhibit A.beta._1-42Aggregation and fiber formation; vitamin E and Abeta_1-42The incubation also obviously inhibits Abeta_1-42Aggregation and fiber formation, but the effect is weaker than that of H102.

2. The polypeptide of the present invention and Abeta_1-42The dose-effect relationship is as follows: FIG. 3 shows different concentrations of polypeptide H102 and vitamin E versus 11.07. mu. mol/L A. beta_1-42The results of ThT fluorescence detection were performed after incubating the solutions at 37 ℃ for 24 hours. As can be seen from fig. 3, H102 and vitamin E have a concentration-dependent effect on the inhibition of a β fibril formation, with the inhibition rate of H102 being greater at each concentration point than vitamin E. The highest inhibitory concentrations of H102 and vitamin E were around 20. mu. mol/L.

3. The polypeptide of the present invention and Abeta_1-42The aging relationship of (1): FIG. 4 shows the polypeptide of the invention H102 and vitamin E with 11.07. mu. mol/L A. beta_1-42The results of ThT fluorescence detection were performed after co-incubation of the solutions at 37 ℃ for various periods of time. As can be seen from FIG. 4, 11.07. mu. mol/L A. beta. is_1-42The fluorescence intensity was lower at 24H before incubation alone, increased sharply at day 3, followed by a plateau-type increase, and the fluorescence intensity at each time point of H102 and VE groups was significantly reduced, andthe effect of treating the group with H102 was most pronounced. Time T of occurrence of half peak of curve_1/2Representing the variation of the curve, T of the A β group_1/2On day 3. H102 and vitamin E groups compared to A β group_1/2Delay to day 4.

4. And (3) observing results by an electron microscope:

FIG. 5 shows the use of various polypeptides at a concentration of 44.30. mu. mol/L with 11.07. mu. mol/L of A.beta._1-42After incubation at 37 ℃ for 5 days, A.beta._1-42And (3) observing the result of the polypeptide fiber formation by using an electron microscope. As can be seen from the figures, A β_1-42After the single incubation for 5 days, a large amount of A beta fibers with the diameter of about 8-10 nm and the length of about 0.5-5 mu m appear, the fibers are in bur type crystal aggregation, have branches and even are interwoven into a net shape, and a small amount of amorphous structures in an aggregation state are sandwiched between the fibers (figures 5A and 5B, a control group); l5 and A beta are incubated for 5 days, the formed A beta fiber is obviously thin and is aggregated into a fiber with the diameter of about 4-gnm and the length of 0.3-5 mu m, and the fiber is interwoven into a net shape, and a small amount of amorphous structures in an aggregated state are sandwiched between the fibers (figure 5C); h101 and Abeta are incubated together for 5 days, Abeta fibers are formed, the diameter is thin and is about 5-8 nm, the length is 0.5-5 mu m, branches are interwoven into a net shape, and a small amount of amorphous structures in an aggregation state can be seen in the process (figure 5D); h102 and A beta are incubated for 5 days, the A beta fibers form obviously reduced and have thin diameters of about 3-6 nm and lengths of 0.3-3 mu m, and are interwoven into a net shape, and a small amount of amorphous structures in an aggregated state are sandwiched between the A beta fibers (figure 5E); h103 and A beta are incubated for 5 days, A beta fibers are formed and aggregated into barbed crystalline aggregates with the diameter of about 5-8 nm and the length of 0.5-5 mu m, the barbed crystalline aggregates are interwoven into a net shape, and a small amount of amorphous structures in an aggregated state are sandwiched between the barbed crystalline aggregates and the net shape (figure 5F).

5. Effect of the polypeptide of the present invention on the survival Rate of SH-SY5Y cells

FIG. 6 shows different concentrations (10. mu. mol/L, 20. mu. mol/L and 40. mu. mol/L) of various polypeptides for A.beta.of 5. mu. mol/L_1-42Effect of survival of SH-SY5Y cells of human neuroblastoma cultured together for 72 hours. n-12 denotes a group represented by and a β_1-42Group phase P < 0.05.

From the figureAs can be seen in FIG. 6, A.beta._1-42After the SH-SY5Y cells are acted for 72 hours, the cell survival rate is reduced, and 4 polypeptides (10 mu mol/L, 20 mu mol/L and 40 mu mol/L) with different concentrations and A beta are respectively used_1-42When the cells are incubated together, the survival rate of the cells is improved and a certain dose dependence relationship is presented. The above results indicate that H102 is most effective in improving cell viability and that the optimum concentration is 20. mu. mol/L.

Example 3: h102-pair of amyloid (Abeta) and pre-amyloid in brain of APP transgenic mice Effect of body protein (APP) expression

Materials and methods

Experimental animals: 30 APP 695 transgenic mice with 9 months of age, 10C 57BL/6J mice with the same genetic background and the same age, with unlimited sexes, were purchased from the research center of laboratory animals of Chinese medical academy of sciences, China coordination and medical university.

Drugs and reagents: abeta (beta)_1-42Antibodies were purchased from Chemicon, USA; APP antibody, instant SABC immunohistochemical staining kit, DBA color development kit, Congo red purchased from Dr. Wuhan, Dr. Co; other reagents are all domestic analytical purifiers. The H102 polypeptide was prepared, purified and characterized as described in example 1, and was prepared as a 40. mu. mol/L solution diluted with physiological saline at the time of the experiment.

Animal grouping and model preparation: 30 APP 695 transgenic mice are randomly divided into 15 model groups, and 15 polypeptide injection groups; c57BL/6J 10 was set as a normal control group only. Polypeptide injection group: each injection of 3. mu.l of 40. mu. mol/L H102 physiological saline solution into the lateral ventricle of the animal, control group and model group: the equal volume of normal saline is injected into the lateral ventricle of the animal every time, the injection is carried out once a day, and the injection is continuously carried out for 10 days. All animals were housed in the SPF grade clean rat house at the Experimental animals center of Tianjin medical university under the same conditions.

Performing abdominal anesthesia on animal with 10% chloral hydrate at a dosage of 400mg/Kg body weight, fixing on brain stereotaxic apparatus, cutting head hair, sterilizing with ammonia iodine, and placing alongThe head skin was cut at the midline to a length of about 1cm, and the periosteum was removed to reveal the bony landmarks. The mouse head plane was adjusted so that Bregma and Lambda were at the same height. Reference to George's mouse brain stereotaxic map, on the lateral ventricle (P) on the skull_0.58，R₁，H_-1.5) A small hole with the diameter of 1mm is arranged at the position of the needle inserting point. The next day after surgery, injections were started. Injecting 3 μ l polypeptide solution or normal saline into lateral ventricle slowly and uniformly via micro injector by means of animal brain stereotaxic apparatus, and slowly withdrawing the needle after injection is finished and the needle is retained for 1 min. Hemostasis by local compression, and continuous injection for 10 days.

Preparation of mouse hippocampal paraffin sections: after continuously injecting lateral ventricles of animals of each group for 10 days, feeding 0.1kg/L chloral hydrate into the stalks and carrying out intraperitoneal injection anesthesia according to the dose of 4mL/kg of body weight, quickly inserting tubes through the left ventricles, simultaneously cutting the right atrium, and quickly filling normal saline; after the liver becomes white, pouring 40g/L paraformaldehyde, wherein the speed is fast first and slow later until the limbs of the mouse are stiff and satisfied; taking mouse brain, quickly putting the mouse brain into paraformaldehyde solution containing 30% of sucrose, and fixing for 2-4 days. After the brain tissue sinks, the wax block can be embedded. The mice were sliced at sagittal brain surface, and after the hippocampal CA1 region appeared, the mice were serially sliced, 1 at 10 intervals, 3 for each antibody, and 5 μm thick.

Immunohistochemical staining: immunohistochemical staining of A.beta.and APP was performed according to the procedures of the immunohistochemical kit instructions, with first antibody dilutions of 1: 100 and 1: 200, respectively, and second antibody dilutions of 1: 100, respectively. The observation was done at 400 x magnification and photographed.

Congo red staining of mouse brain tissue sections: dewaxing the paraffin embedded section to water, immersing the paraffin embedded section in 10% formaldehyde solution for 15min, directly immersing the paraffin embedded section in Congo red staining solution for 15min, washing with water for 4min, immersing the section in hematoxylin solution for 2min, immersing the section in 0.5% hydrochloric acid ethanol after washing with water for differentiation, washing the section with water to bluish, dehydrating with ethanol, transparentizing with xylene, and sealing with neutral gum. The observation was done at 400 x magnification and photographed.

Results

APP N-terminal antibody immunohistochemical staining and Α β antibody immunohistochemical staining.

FIG. 7 shows the results of immunohistochemical staining of neuronal sections from the CA1 region of the hippocampus of each group of animals with the N-terminal antibody of APP and the antibody of A.beta..

As can be seen in fig. 7: (1) the results of immunohistochemical staining with APP N-terminal and Α β antibodies in the control group (fig. 7A and 7B, respectively) show: the cytoplasm staining of the neuron in the hippocampal CA1 area of the control group is negative or weakly positive; (2) the results of the immunodominant staining of the APP N-terminal antibody and the Α β antibody in the model group (fig. 7C and 7D, respectively) show: compared with a control group, the model group has the advantages that positive cells are increased, the expression is enhanced, and the cytoplasm is obviously deepened; (3) the results of immunohistochemical staining of APP N-terminal and Α β antibodies in the polypeptide-injected group (fig. 7E and 7F, respectively) show: compared with the model group, the APP in the polypeptide injection group has the advantages of reduction of positive cells, lightening of cytoplasmic staining and weakening of expression.

2. And (5) dyeing with Congo red.

After pathological sections of mouse brain tissue were stained with congo red, the expression of temporal cortex and hippocampal amyloid plaques in mouse brain were observed, and the staining results are shown in fig. 8.

As can be seen in fig. 8: (1) no positive amyloid plaques were seen in the temporal cortex of the brain (fig. 8A) and hippocampus (fig. 8B) of control mice; (2) the temporal cortex of brain (figure 8C) and hippocampus (figure 8D) of the model group mice have different sizes, most of light red chromophyte block-shaped amyloid plaque substances are round or similar to round, are scattered and distributed, have uneven distribution density, and can see a plurality of different neuron cells around; (3) the temporal cortex of the brain (fig. 8E) and hippocampus (fig. 8F) of the mice in the polypeptide-injected group also exhibited some lumpy amyloid plaque material, but the amyloid plaques in the polypeptide-injected group were significantly reduced compared to the model group.

Conclusion

Immunohistochemical staining results show: in the model group mice cerebrum CA1 region, A.beta._1-42Positive cells were significantly higher than the normal control. And the H102 polypeptide injection group has positive cells which are obviously reduced compared with the model group. It is presumed that H102 inhibits aggregation of a β in vivo and reduces a β toxicity. In addition, Congo red staining shows that amyloid plaques obviously appear in the brains of the mice in the transgenic group, plaques are not seen in the brains of the mice in the normal control group, and the number and the area of the amyloid plaques in the brains of the mice in the H102 polypeptide injection group are obviously reduced compared with those of the mice in the transgenic group. This also confirms the conclusion that H102 can inhibit the aggregation of a β, reducing its production.

Throughout this application, various publications are referenced. The publications mentioned are incorporated by reference in their entirety into this specification in order to describe more fully the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Sequence listing

<110> Tianjin medical university

Wang Wei

<120> beta-sheet blocking peptide for preventing and/or treating Alzheimer's disease

<130>CP1080054/CB

<160>4

<170>PatentIn version 3.3

<210>1

<211>10

<212>PRT

<213> Artificial sequence

<400>1

His Lys Gln Leu Val Phe Phe Glu Glu Asp

1 5 10

<210>2

<211>10

<212>PRT

<213> Artificial sequence

<400>2

His Lys Gln Leu Pro Phe Phe Glu Glu Asp

1 5 10

<210>3

<211>10

<212>PRT

<213> Artificial sequence

<400>3

His Gln Lys Leu Val Phe Phe Ala Glu Asp

1 5 10

<210>4

<211>42

<212>PRT

<213> human (Homo sapiens)

<400>4

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

Claims (3)

1.A polypeptide consisting of the amino acid sequence:

His-Lys-Gln-Leu-Pro-Phe-Phe-Glu-Glu-Asp(SEQ ID NO：2)。

2. use of a polypeptide according to claim 1 for the preparation of a medicament for the prevention and/or treatment of alzheimer's disease.

3. A pharmaceutical composition comprising a polypeptide according to claim 1 and a pharmaceutically acceptable carrier.