WO2017041733A1 - 具有抑制阿尔兹海默症Aβ蛋白聚集的多肽及其用途 - Google Patents

具有抑制阿尔兹海默症Aβ蛋白聚集的多肽及其用途 Download PDF

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WO2017041733A1
WO2017041733A1 PCT/CN2016/098501 CN2016098501W WO2017041733A1 WO 2017041733 A1 WO2017041733 A1 WO 2017041733A1 CN 2016098501 W CN2016098501 W CN 2016098501W WO 2017041733 A1 WO2017041733 A1 WO 2017041733A1
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polypeptide
vqvg
iqig
crystal
aggregation
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李继喜
黄静
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Fudan University
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Fudan University
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Priority claimed from CN201510579526.4A external-priority patent/CN105175493B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides

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  • the invention belongs to the technical field of medicine, and in particular relates to a polypeptide and the use thereof for preparing a medicament for preventing or treating Alzheimer's disease.
  • AD Alzheimer's desease
  • a common chronic progressive degenerative disease that occurs in the elderly.
  • Common clinical manifestations include progressive memory and cognitive decline, speech disorders, and psychomotor abnormalities.
  • the number of Alzheimer's disease is increasing year by year, which not only jeopardizes the physical and mental health and quality of life of the elderly, but also imposes a heavy burden on families and society.
  • a ⁇ ⁇ -amyloid
  • APP ⁇ -amyloid precursor protein
  • a ⁇ As a core component of SPs, A ⁇ has two forms of A ⁇ 40 (human Amyloid B 1-40, amino acid sequence: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) and A ⁇ 42 (human Amyloid B 1-42, amino acid sequence: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) (in which A ⁇ 42 is more susceptible to amyloidosis), Both of them have toxic effects on neurons, and aggregation and abnormal deposition of A ⁇ are considered to be the primary and central links in the pathogenesis of AD.
  • a ⁇ 40 human Amyloid B 1-40, amino acid sequence: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
  • a ⁇ 42 human Amyloid B 1-42, amino acid sequence: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
  • drugs designed with A ⁇ as targets include: (1) degradation of A ⁇ , especially A ⁇ 42, such as ⁇ -secretase and ⁇ -secretase inhibitors; (2) promotion of A ⁇ clearance from the brain, inhibition of A ⁇ Accumulate and reduce the toxicity of its aggregates, such as A ⁇ aggregation inhibitors and degradation promoters; (3) Immunotherapy.
  • the existing drugs that interfere with A ⁇ aggregation can be divided into three major categories: small molecule organic compounds, antibodies, and peptides according to their chemical compositions.
  • a variety of short peptides have also proven to be effective molecules for the treatment of AD, such as glutathione as an antioxidant.
  • studies have shown that peptides and peptide molecules such as KLVFF and LPFFD have strong inhibitory properties on the pathogenesis of AD.
  • the short peptide is a dehydration condensation of amino acids, which has the advantages of easy absorption and no side effects, and is beneficial to the development of drugs, and thus has become a hot spot for the development of AD drugs.
  • the object of the present invention is to provide a polypeptide which can significantly inhibit the aggregation of A ⁇ (including A ⁇ 40 and A ⁇ 42). Drugs and their uses.
  • the test proves that the polypeptide can be used for preparing a medicament for preventing or treating Alzheimer's disease, in particular for the treatment or prevention of Alzheimer's disease, and has broad medical application prospects.
  • polypeptide for inhibiting A ⁇ aggregation comprises the following amino acid sequence (I), consisting of or consisting essentially of the following amino acid sequence (I):
  • Xaa1 and Xaa3 are the same amino acid selected from I, V or L;
  • Xaa2 is selected from Q or N;
  • Xaa4 is selected from the group consisting of A, C, D, E, F, G, H, K, M, N, P, Q, R, S, T, V, W or Y.
  • Xaa1, Xaa2, Xaa3, Xaa4 adopts the amino acid one-letter abbreviation code.
  • an amino acid refers to a carboxylic acid containing an amino group.
  • the various proteins in the organism are composed of 20 essential amino acids, and their structural formula is:
  • Xaa1 and Xaa3 are selected from V or L; Xaa2 is selected from Q or N; and Xaa4 is selected from C, G, N, Q, S, T.
  • Xaa1 and Xaa3 are selected from I or L; Xaa2 is selected from Q or N; and Xaa4 is selected from C, G, N, Q, S, T .
  • polypeptide of formula (I) is one of the following sequences:
  • polypeptide of formula (I) is VQVG or IQIG.
  • the polypeptide of formula (I) can be obtained by chemical synthesis, for example by standard polypeptides well known in the art.
  • Solid phase synthesis technology can be synthesized by using N-terminal protection strategies of tert-butoxycarbonyl (Boc) and fluorenylmethoxycarbonyl (Fmoc).
  • Boc tert-butoxycarbonyl
  • Fmoc fluorenylmethoxycarbonyl
  • the corresponding amino acids can be sequentially connected according to the solid phase synthesis method of the resin, and the Fmoc-protecting group is sequentially removed, and then the peptide is cut to obtain a crude product, and the crude product is separated and purified by a C18 column to obtain a formula (I). ) the polypeptide shown.
  • the preferred polypeptide VQVG or IQIG is synthesized by standard peptide solid phase synthesis technology, and the purity is greater than 95%, and the HPLC and MS detection analysis results are shown in FIG. 8 and FIG. 9 and FIG. 17 and FIG. 18
  • polypeptide of formula (I) is chemically modified at the N-terminus, C-terminus or both ends.
  • the N-terminus of the polypeptide of formula (I) is chemically modified by acetylation and the C-terminus is chemically modified by amidation.
  • the peptide form is Ac-VQVG-NH 2 or Ac-IQIG-NH 2 .
  • the inventors have found through exploratory experiments that the polypeptide represented by the formula (I) has an effect of significantly inhibiting the aggregation of A ⁇ (including A ⁇ 40 and A ⁇ 42).
  • the preferred polypeptide VQVG or IQIG surprisingly has a significant inhibitory effect on the aggregation of A ⁇ (including A ⁇ 40 and A ⁇ 42).
  • the polypeptide represented by the formula (I) and VQVG stabilize the A- ⁇ structure by inhibiting the ⁇ -helix structure of A ⁇ (including A ⁇ 40 and A ⁇ 42) by forming a complex with A ⁇ (including A ⁇ 40 and A ⁇ 42), thereby inhibiting the structure of ⁇ -sheet ( The conformation including A ⁇ 40 and A ⁇ 42) inhibits aggregation of A ⁇ (including A ⁇ 40 and A ⁇ 42).
  • a preferred crystalline form of the polypeptide VQVG is obtained.
  • the crystal of the polypeptide VQVG exhibits a long needle shape.
  • a crystallographic asymmetric unit contains one polypeptide chain, two water molecules and one sodium ion, and each chain consists of four amino acids, Val-Gln-Val-Gly.
  • the unit cell parameters of the polypeptide VQVG crystal are:
  • Crystal space group P2 1 2 1 2 1 ;
  • the invention also obtains a crystalline form of the polypeptide IQIG.
  • the crystal of the peptide IQIG exhibits a long needle shape, and a crystallographic asymmetric unit contains two polypeptide chains and two water molecules, each of which consists of four amino acids, Ile-Gln-Ile-Gly.
  • the two polypeptide chains are in an anti-parallel folded form.
  • the peptide cell parameters of the peptide IQIG crystal are:
  • Another object of the present invention is to provide a use of the above polypeptide for inhibiting A ⁇ aggregation for the preparation of a medicament for treating or preventing Alzheimer's disease.
  • the occurrence and progression of Alzheimer's disease is closely related to the aggregation of A ⁇ (including A ⁇ 40 and A ⁇ 42), so it is expected that the polypeptide of formula (I) (particularly preferably VQVG or IQIG) in the present invention is used in Alzheim.
  • the prevention or treatment of morbidity can play a positive role in improving the patient's condition or delaying the occurrence of the disease.
  • the fluorescence intensity of the experimental group added with VQVG or IQIG polypeptide was significantly lower than that of the A ⁇ 40 or A ⁇ 42 aggregation model of the model group, indicating that the polypeptide VQVG or IQIG can significantly inhibit A ⁇ 40 by the thiosulfinin T experiment. Or the aggregation activity of A ⁇ 42.
  • the gene for A[beta]40 or A[beta]42 is constructed into a suitable vector and transfected into HEK293T cells.
  • the results showed that the intensity of fluorescent spots formed in the cells was decreased compared with the control group, and the concentration of VQVG or IQIG was positively correlated with the concentration of the polypeptide VQVG or IQIG, indicating that the polypeptide VQVG Or IQIG can also significantly inhibit the aggregation activity of A ⁇ 40 or A ⁇ 42 at the cellular level.
  • the effect of the polypeptide VQVG or IQIG on the aggregation of ⁇ 40 or A ⁇ 42 was also examined by transmission electron microscopy. The results showed that the fiber density in the A ⁇ 40 or A ⁇ 42 samples was very high, linear, and crossed into a network.
  • the polypeptide drug of the present invention may be a free polypeptide or a pharmaceutically acceptable salt forming form of the polypeptide, such as a sulfate, a hydrochloride, a phosphate, a sulfonate, a citrate, an acetate, a lactate, a tartrate , mesylate, ethanesulfonate, besylate, and the like.
  • the polypeptide drug can be administered by, for example, oral, intravenous, intramuscular, subcutaneous or other routes.
  • the drug can be administered orally, for example, in the form of a tablet, gel, paste, ointment, liquid, powder or in other dosage forms.
  • compositions for oral administration include binders, flavoring agents, and wetting agents.
  • the drug may be contained in a toothpaste or mouthwash.
  • the oral agent can comprise milling and a blowing agent.
  • the drug can also be administered transdermally or as a suppository.
  • the invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of formula (1) and a pharmaceutically acceptable pharmaceutical excipient.
  • a suitable pharmaceutical composition of the invention typically comprises a therapeutically effective amount of a polypeptide of formula (1), a pharmaceutically acceptable diluent and an excipient (such as sterile water), according to a predetermined Use to adjust the final concentration range.
  • Preparation techniques are generally well known in the art, exemplifying Remington's Pharmaceutical Sciences (18th Edition, Mack Publishing Company, 1995).
  • the polypeptide of formula (1) may be used alone or in combination with other Alzheimer's therapeutic agents, including but not limited to somatostatin, norepinephrine, adrenaline, glutamic acid, phospholipids, physostigmine, vincristine, Vinblastine, vitamin E, dopamine, tacrine, clofibrate, naproxil, piracetam, nimodipine, aniracetam, citicoline, adenosine, cytochrome C, retinoic acid Ginkgo flavonoid glycosides, salvia miltiorrhiza, huperzine A, vasopressin, etc.
  • the human therapeutically administered dose ranges from 1 mg/kg to 4 g/kg by weight, preferably from 1 mg/kg to 100 mg/kg.
  • Figure 1 is a schematic diagram showing the results of the experimental results of the thiosulfurin T of the polypeptides VQVG and A ⁇ 40.
  • Figure 2 is a schematic diagram showing the results of the experimental results of the thiosulfurin T of the polypeptides VQVG and A?42.
  • Figure 3 is a schematic diagram showing the results of cell experiments of polypeptides VQVG and A?40.
  • Figure 4 is a schematic diagram showing the results of cell experiments of the polypeptides VQVG and A?42.
  • Figure 5 is a schematic diagram showing the results of transmission electron microscopy experiments of the polypeptides VQVG and A?40.
  • Figure 6 is a schematic diagram showing the results of transmission electron microscopy experiments of polypeptides VQVG and A?42.
  • Figure 7 is a schematic diagram showing the crystal structure of the polypeptide VQVG.
  • Figure 8 is a schematic diagram showing the results of HPLC analysis of the polypeptide VQVG.
  • Figure 9 is a schematic diagram showing the results of MS analysis of the polypeptide VQVG.
  • Figure 10 is a schematic diagram showing the results of the experimental results of the thiosulfurin T of the polypeptide IQIG and A?40.
  • Figure 11 is a schematic diagram showing the results of the thiosulfurin T experiment of the polypeptide IQIG and A?42.
  • Figure 12 is a schematic diagram showing the results of cell experiments of polypeptides IQIG and A?40.
  • Figure 13 is a schematic diagram showing the results of cell experiments of polypeptides IQIG and A?42.
  • Figure 14 is a schematic diagram showing the results of transmission electron microscopy experiments of polypeptides IQIG and A?40.
  • Figure 15 is a schematic diagram showing the results of transmission electron microscopy experiments of polypeptides IQIG and A?42.
  • Figure 16 is a schematic diagram showing the crystal structure of the polypeptide IQIG.
  • Figure 17 is a schematic diagram showing the results of HPLC analysis of the polypeptide IQIG.
  • Figure 18 is a schematic diagram showing the results of MS analysis of the polypeptide IQIG.
  • Protein A ⁇ 40 was synthesized by Shanghai Qiangyao Company with a purity greater than 95%.
  • the peptide VQVG is synthesized by Shanghai Qiangyao Co., Ltd., with a purity greater than 95% and a structural form of unmodified VQVG.
  • 1 mg of each of A ⁇ 40 and polypeptide VQVG was taken and completely dissolved with 100 ul of dimethyl sulfoxide.
  • Thioflavin T is purchased from sigma (Cat. No. T3516-5g) and has a molecular weight of 318.86.
  • a solution of 0.0319 g of ThT in 20 ml of a 20 mM Tris-HCl (pH 7.5) solution was weighed to obtain a 5 mM ThT mother liquor.
  • excitation was carried out at a wavelength of 430 nm, and the fluorescence intensity at a wavelength of 485 nm was detected, thereby determining the influence of the polypeptide VQVG on the formation of the ⁇ -starch fiber-like structure.
  • ThT 500uM
  • VQVG A ⁇ 1-40 A ⁇ 40 aggregation model group 5ul 0 5uM
  • VQVG experimental group 5ul 0.5uM 5uM
  • the peptide A ⁇ 42 is also synthesized by Shanghai Qiangyao Company, the purity is more than 95%, and the preparation method refers to the polypeptide A ⁇ 40.
  • the source and formulation of the polypeptides VQVG and thiosulfurin T were consistent with those described in Experimental Example 1. According to the experimental example 1, 100 ul of each experimental group solution was prepared as shown in Table 2. The prepared solution was added to a 96-well plate, placed in a microplate reader, incubated at 37 ° C, shaken once every 5 minutes, and the fluorescence intensity was measured after 48 hours. Each sample was repeated three times and averaged.
  • excitation was carried out at a wavelength of 430 nm, and the fluorescence intensity at a wavelength of 485 nm was detected, thereby determining the influence of the polypeptide VQVG on the formation of the ⁇ -starch fiber-like structure.
  • the gene corresponding to A ⁇ 40 was constructed into the vector of pEGFP-N1 (purchased from Clontech) through the restriction site XhoI/BamHI.
  • HEK293T cells were transfected with the correct sequencing plasmid, stimulated with 10 uM, 50 uM peptide VQVG, and cultured at 37 ° C.
  • a set of blank controls were set up and grown naturally without any stimulation.
  • 4% paraformaldehyde was fixed, 0.3% Triton X-100 was perforated, and DAPI was stained with nuclei.
  • the experimental results were observed with confocal (high-sensitivity laser scanning confocal microscope, model ZEISS LSM710), and the results are shown in Fig. 3.
  • the intensity of fluorescent spots formed in the cells was weakened by the addition of the polypeptide VQVG-stimulated cells, and was positively correlated with the concentration of the polypeptide VQVG.
  • the intensity of the fluorescent spots formed relative to the blank group was reduced to 25%; the intensity of the fluorescent spots formed by the 50 ⁇ M VQVG-stimulated experimental group was reduced to 10%.
  • the polypeptide VQVG can also significantly inhibit the aggregation activity of A ⁇ 40 at the cellular level and can be used for treating or preventing Alzheimer's disease.
  • the effect of the polypeptide VQVG on A ⁇ 42 protein was examined by the method of Example 3.
  • the results of the experiment were observed with confocal (high sensitivity laser scanning confocal microscope, model ZEISS LSM710), and the results are shown in Fig. 4.
  • the intensity of fluorescent spots formed in the cells was weakened by the addition of the polypeptide VQVG-stimulated cells, and was positively correlated with the concentration of the polypeptide VQVG.
  • Adding 10 uM VQVG-stimulated experimental group the intensity of fluorescent spots formed relative to the blank group was reduced to 30%; the intensity of fluorescent spots formed by the 50 ⁇ M VQVG-stimulated experimental group was reduced to 15%. It is indicated that the polypeptide VQVG can also significantly inhibit the aggregation activity of A ⁇ 42 at the cellular level, and can be used for treating or preventing Alzheimer's disease.
  • Example 5 Detection of the effect of polypeptide VQVG on A ⁇ 40 aggregation by transmission electron microscopy
  • the results are shown in Figure 5.
  • the fiber density in the A ⁇ 40 sample is very high, linear, intersecting, and the length is about 500 nm to 1 um; however, in the sample containing the peptide VQVG, it is difficult to observe the fiber distribution in the field of view. There are only a few short rods of fibers or round aggregates. It is indicated that the polypeptide VQVG can inhibit the aggregation of A ⁇ 40 and can be used for treating or preventing Alzheimer's disease.
  • Example 6 Detection of the effect of polypeptide VQVG on A ⁇ 42 aggregation by transmission electron microscopy
  • the present invention mixes an equal volume of polypeptide (VQVG, 5 mg/ml) with a crystallization reagent (0.2 M magnesium chloride, 0.1 M sodium cacodylate pH 6.5, 20% (v/v) PEG 200), and is placed at 20 degrees in a hanging drop method. Long needle-like crystals were obtained after 1 day. The prepared crystals were treated with 20% glycerol as an antifreeze and then frozen in liquid nitrogen. Crystal X-ray diffraction data were collected at the Shanghai Light Source (SSRF) Biomacromolecule Crystallography Beam Line Station (five-line six-station BL19U). The data processing uses HKL3000, and the structural analysis adopts the molecular replacement method.
  • SSRF Shanghai Light Source
  • BL19U Chinese Crystallography Beam Line Station
  • the Refmac program is supplemented with Coot software for further correction, and finally the crystal structure of the polypeptide VQVG is obtained.
  • the atoms in the three-dimensional structure of the crystal have at least 40% of the atomic coordinates listed in Table 3, or the backbone carbon skeleton of at least 40% of the amino acids in the three-dimensional structure of the 3D protein.
  • the average root variance of the atomic structure coordinates and the coordinates in Table 3 is less than or equal to
  • the atomic coordinates can be considered to have the same structure as the 3D protein.
  • the crystal three-dimensional structure of the polypeptide VQVG a crystallographic asymmetric unit contains a polypeptide chain, two water molecules, and one sodium ion, each chain consisting of four amino acids, namely Val-Gln-Val -Gly, as shown in Figure 7 (amino acids are represented by a ball stick model, red: oxygen atoms; green: carbon atoms; blue: nitrogen atoms; red stars: water molecules; purple: sodium ions).
  • the HPLC separation was carried out using an ODS column, eluting A solution containing 0.1% trifluoroacetic acid in 100% acetonitrile, and solution B as 0.1% trifluoroacetic acid in water.
  • the mass spectrometry report showed a charge-to-mass ratio of 401.85 [M+H] + , which is consistent with the theoretical value of 400.85.
  • Table 3 is a single molecule derived from the peptide VQVG, the coordinate group is as follows:
  • R work /R free are: 0.137 and 0.153 respectively
  • Protein A ⁇ 40 was synthesized by Shanghai Qiangyao Company with a purity greater than 95%.
  • the peptide IQIG is synthesized by Shanghai Qiangyao Co., Ltd., with a purity of more than 95%, and the structural form is unmodified IQIG.
  • 1 mg of each of A ⁇ 40 and polypeptide IQIG was taken and completely dissolved with 100 ul of dimethyl sulfoxide.
  • Thioflavin T is purchased from sigma (Cat. No. T3516-5g) and has a molecular weight of 318.86. Weigh 0.0319g of ThT dissolved in 20ml of 20mM Tris-HCl (pH 7.5) 5 mM ThT mother liquor was obtained in the solution.
  • 100 ul of the experimental group solution was prepared with pH 7.4, 20 mM Tris-HCl, and the final concentration of Thioflavin T was 25 uM, the final concentration of protein A ⁇ 40 was 5 uM, and the polypeptide IQIG was 0.5 uM.
  • the prepared solution was added to a 96-well plate, placed in a microplate reader, incubated at 37 ° C, shaken once every 5 minutes, and the fluorescence intensity was measured after 48 hours. Each sample was repeated three times and averaged.
  • excitation was carried out at a wavelength of 430 nm, and the fluorescence intensity at a wavelength of 485 nm was detected, thereby determining the influence of the polypeptide IQIG on the formation of the ⁇ -starch fiber-like structure.
  • ThT 500uM
  • IQIG A ⁇ 1-40 A ⁇ 40 aggregation model group 5ul 0 5uM
  • IQIG experimental group 5ul 0.5uM 5uM
  • the peptide A ⁇ 42 is also synthesized by Shanghai Qiangyao Company, the purity is more than 95%, and the preparation method refers to the polypeptide A ⁇ 40.
  • the source and formulation of the polypeptide IQIG and thiosulfurin T were all consistent with those described in Experimental Example 1. According to the experimental example 8, 100 ul of each experimental group solution was prepared as shown in Table 5. The prepared solution was added to a 96-well plate, placed in a microplate reader, incubated at 37 ° C, shaken once every 5 minutes, and the fluorescence intensity was measured after 48 hours. Each sample was repeated three times and averaged.
  • excitation was carried out at a wavelength of 430 nm, and the fluorescence intensity at a wavelength of 485 nm was detected, thereby determining the influence of the polypeptide IQIG on the formation of the ⁇ -starch fiber-like structure.
  • ThT 500uM
  • IQIG A ⁇ 1-42 A ⁇ 42 aggregation model group 5ul 0 5uM
  • IQIG experimental group 5ul 0.5uM 5uM
  • the gene corresponding to A ⁇ 40 was constructed into a vector of pEGFP-N1 (purchased from Clontech) through a restriction enzyme site XhoI/BamHI.
  • HEK293T cells were transfected with the correct sequencing plasmid, stimulated with 10 uM, 50 uM peptide IQIG, and cultured at 37 ° C.
  • a set of blank controls were set up and grown naturally without any stimulation. After 24 hours, 4% paraformaldehyde was fixed, 0.3% Triton X-100 was perforated, and DAPI was stained with nuclei.
  • the experimental results were observed with confocal (high-sensitivity laser scanning confocal microscope, model ZEISS LSM710), and the results are shown in Fig. 12.
  • the intensity of the fluorescent spots formed in the cells was weakened by the addition of the polypeptide IQIG-stimulated cells, and was positively correlated with the concentration of the polypeptide IQIG.
  • Adding 10 uM IQIG-stimulated experimental group the intensity of fluorescent spots formed relative to the blank group was reduced to 30%; the intensity of fluorescent spots formed with respect to the blank group was reduced to 15% by the 50 ⁇ M IQIG-stimulated experimental group. It is indicated that the polypeptide IQIG can also significantly inhibit the aggregation activity of A ⁇ 40 at the cellular level, and can be used for treating or preventing Alzheimer's disease.
  • the effect of the polypeptide IQIG on A ⁇ 42 protein was examined by the method of Experimental Example 10.
  • the results of the experiment were observed with confocal (high-sensitivity laser scanning confocal microscope, model ZEISS LSM710), and the results are shown in Fig. 13.
  • the intensity of the fluorescent spots formed in the cells was weakened by the addition of the polypeptide IQIG-stimulated cells, and was positively correlated with the concentration of the polypeptide IQIG.
  • Adding 10 uM IQIG-stimulated experimental group the intensity of fluorescent spots formed relative to the blank group was reduced to 35%; the intensity of fluorescent spots formed by the 50 ⁇ M IQIG-stimulated experimental group was reduced to 16%. It is indicated that the polypeptide IQIG can also significantly inhibit the aggregation activity of A ⁇ 42 at the cellular level and can be used for treating or preventing Alzheimer's disease.
  • Example 12 Detection of the effect of polypeptide IQIG on A ⁇ 40 aggregation by transmission electron microscopy
  • the results are shown in Figure 14.
  • the fiber density in the A ⁇ 40 sample is very high, linear, intersecting, and the length is about 500 nm to 1 um; however, in the sample containing the peptide IQIG, it is difficult to observe the fiber distribution in the field of view. There are only a few short rods of fibers or round aggregates. It is indicated that the polypeptide IQIG can inhibit the aggregation of A ⁇ 40 and can be used for treating or preventing Alzheimer's disease.
  • the fiber density in the A ⁇ 42 sample is very high, linear, intersecting into a network, and the length is more than 1 um; however, A sample of the peptide IQIG was added, and only a small amount of short fibers were observed in the field of view. It is indicated that the polypeptide IQIG can inhibit the aggregation of A ⁇ 42 and can be used for treating or preventing Alzheimer's disease.
  • the present inventors mixed an equal volume of polypeptide (IQIG, 5 mg/ml) with a crystallization reagent (0.2 M ammonium acetate, 0.1 M Bis-Tris pH 6.5, 45% (v/v) MPD), and suspended at 20 degrees. After 2 days of standing, long needle crystals were obtained. The prepared crystals were treated with 25% glycerol as an antifreeze and then frozen in liquid nitrogen. Crystal X-ray diffraction data were collected at the Shanghai Light Source (SSRF) Biomacromolecule Crystallography Beam Line Station (five-line six-station BL19U). Data processing was performed using HKL3000. The structural analysis was performed by molecular replacement method, and the Refmac program was used with Coot software for further correction.
  • SSRF Shanghai Light Source
  • SSRF Shanghai Light Source
  • Refmac program was used with Coot software for further correction.
  • the crystal structure of the peptide IQIG was obtained. It will be apparent to those skilled in the art that the atoms in the three-dimensional structure of the crystal have at least 40% of the atomic coordinates listed in Table 6, or at least 40% of the backbone carbon skeleton of the crystal three-dimensional structure of the 3D protein. The average root variance of the atomic structure coordinates and the coordinates in Table 6 is less than or equal to The atomic coordinates can be considered to have the same structure as the 3D protein.
  • the crystal three-dimensional structure of the polypeptide IQIG a crystallographic asymmetric unit contains two polypeptide chains and two water molecules, each chain consisting of four amino acids, namely Ile-Gln-Ile-Gly.
  • the two polypeptide chains are in an anti-parallel folded form, as shown in Figure 16 (amino acids are represented by a stick model, red: oxygen atoms; green: carbon atoms; blue: nitrogen atoms; red stars: water molecules).
  • the HPLC separation was carried out using a carbon 18 reverse phase column, eluting a solution of 100% acetonitrile containing 0.1% trifluoroacetic acid, and a liquid solution of 0.1% trifluoroacetic acid.
  • the mass spectrometry report showed a charge-to-mass ratio of 429.10 [M+H] + , which is consistent with the theoretical value of 429.52.
  • Table 6 is derived from the atomic coordinate group of the peptide IQIG as follows:
  • R work /R free are: 0.166 and 0.184 respectively

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Abstract

提供了一种具有抑制阿尔兹海默症Aβ蛋白聚集的多肽，其氨基酸序列为：Xaa1-Xaa2-Xaa3-Xaa4，其中，Xaa1和Xaa3为相同的氨基酸，选自I、V或L，Xaa2选自Q或N，Xaa4选自A、C、D、E、F、G、H、K、M、P、R、S、T、V、W或Y。所述多肽抑制Aβ40、Aβ42的聚集，并用于制备治疗阿尔茨海默症以及其他神经退行性疾病的药物。

Description

具有抑制阿尔兹海默症Aβ蛋白聚集的多肽及其用途 技术领域
本发明属于医药技术领域，具体涉及一种多肽及其在制备预防或治疗阿尔茨海默症药物中的用途。
背景技术
阿尔茨海默病(Alzheimer’s desease,AD)又称早老性痴呆，是一种常见的发生于老年人的慢性进行性精神功能衰退性疾病。常见的临床表现为进行性记忆力和认知力减退、言语障碍、精神运动异常等。随着人口老龄化日趋严重，阿尔茨海默病的发病数量呈逐年上升趋势，不仅危害着老年人的身心健康和生活质量，同时，也给家庭和社会带来沉重的负担。
关于AD的确切发病机制还不清楚，目前最为广泛接受的是以聚合和原纤维形成的β淀粉样蛋白(β-amyloid,Aβ)肽为生物标志物的Aβ学说。Aβ是机体的正常代谢产物，由β-淀粉样前体蛋白(β-amyloid precursor protein，APP)水解而来，在正常情况下它的产生和降解处于动态平衡，当某些原因导致APP代谢异常时，Aβ生成增多和(或)降解减少就会造成Aβ大量沉积，形成老年斑(senile plaques，SPs)。Aβ作为SPs的核心成分，存在Aβ40(human Amyloid B 1-40,氨基酸序列：DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV)和Aβ42(human Amyloid B 1-42，氨基酸序列：DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA)两种形式(其中Aβ42更易发生淀粉样变性)，均对神经元产生毒性作用，Aβ的聚集和异常沉积被认为是AD发病机制中的首要和中心环节。
目前临床上对AD的治疗尚没有任何特异性治疗的药物面世。现有的已进入临床使用的药物和极大部分处于研究阶段的治疗药物都属于治标的策略，只能减缓AD发病进程，改善临床症状。想要治本，根据AD发病机制来研究彻底治愈AD的有效方法则是攻克该疾病的关键。目前以Aβ为靶点设计的药物包括：(1)降解Aβ特别是Aβ42的生成，如β分泌酶和γ分泌酶抑制剂；(2)促进Aβ从脑内清除，抑制Aβ的 聚集并降低其聚集物的毒性，如Aβ聚集抑制剂和降解促进剂；(3)免疫治疗。现有的干扰Aβ聚集的药物按其化学成分的不同可分为小分子有机化合物、抗体、多肽三大类。
近来，针对Aβ的肽类抑制物的研究备受关注，一些多肽片段被认为可以结合到Aβ聚合体的中央疏水核心从而防止Aβ的沉积。多种短肽也被证明是治疗AD的有效分子，例如作为抗氧化剂的谷胱甘肽。此外，研究表明诸如KLVFF和LPFFD的多肽和肽类分子对AD的发病进程有较强的抑制特性。短肽为氨基酸脱水缩合而成，具有易吸收，无副作用等优点，利于药物发挥，因此成为研制AD药物的热点。
发明内容
为了解决现有技术中存在治疗阿尔茨海默症的药物不明确、效果不明显和药物毒副作用大的缺陷，本发明的目的在于提供一种能显著抑制Aβ(包括Aβ40及Aβ42)聚集的多肽药物及其用途。
试验证明该多肽可用于制备预防或治疗老年痴呆症药物，特别是可用于阿尔茨海默症的治疗或预防，具有广泛的医学应用前景。
本发明提供的抑制Aβ聚集的多肽，包含下述氨基酸序列(I)，由下列氨基酸序列(I)组成或基本上由下述氨基酸序列(I)组成：
Xaa1-Xaa2-Xaa3-Xaa4      (I)
其中，Xaa1和Xaa3为相同的氨基酸，选自I、V或L；
Xaa2选自Q或N；
Xaa4选自A,C,D,E,F,G,H,K,M,N,P,Q,R,S,T,V,W或Y。
其中，上述的Xaa1，Xaa2，Xaa3，Xaa4采用的是氨基酸单字母缩写代码。本领域技术人员所熟知，氨基酸是指含有氨基的羧酸。生物体内的各种蛋白质是由20种基本氨基酸构成的，其结构通式为：
Figure PCTCN2016098501-appb-000001
根据R基的不同，20种天然氨基酸的三字母和单字母缩写分别如下表所示：
Figure PCTCN2016098501-appb-000002
在一些优选的实施方案中，式(I)所示的多肽序列中，Xaa1和Xaa3选自V或L；Xaa2选自Q或N；Xaa4选自C,G,N,Q,S,T。
在另一优选的实施方案中，式(I)所示的多肽序列中，Xaa1和Xaa3选自I或L；Xaa2选自Q或N；Xaa4选自C,G,N,Q,S,T。
在优选的实施方案中，式(I)所示的多肽为如下序列之一:
VQVG；VNVG；LQLG；LNLG；LQVG；VQLG；VQVC；VQLQ；VNLG；VNVC；VNLC；VQVQ；VQVN；VNVQ；VNVN；IQIG；INIG；LQLG；LNLG；IQIS；IQIC；IQLG；LQIG；INLG；LNIG；INIC；INLC；IQIQ；IQIN；INIQ；ININ。
在特别优选的实施方案中，式(I)所示的多肽为VQVG或IQIG。
式(I)所示的多肽可以通过化学合成获得，例如可通过本领域公知的标准多肽 固相合成技术合成，可以采用叔丁氧羰基(Boc)和芴甲氧羰基(Fmoc)两种N端保护策略。例如采用Fmoc策略时，可按照树脂固相合成的方法依次连接相应氨基酸，期间依次脱去Fmoc-保护基团，然后切肽，获得粗品，粗品经C18柱分离纯化，即可制得式(I)所示的多肽。
本发明一个优选的实施方案中，优选的多肽VQVG或IQIG通过标准的多肽固相合成技术合成，纯度大于95％，其HPLC和MS检测分析结果分别如图8、图9以及如图17、图18所示
在一个实施方案中，式(I)所示的多肽在N-末端、C-末端或二端被化学修饰。
在另一实施方案中，式(I)所示的多肽的N-末端通过乙酰化化学修饰，C-末端通过酰胺化化学修饰。一个优选的实施方案中，所述的肽形式为Ac-VQVG-NH2或Ac-IQIG-NH2。
发明人经过探索性实验发现，式(I)所示的多肽具有显著地抑制Aβ(包括Aβ40及Aβ42)聚集的作用。特别是，发明人经实验发现，优选的多肽VQVG或IQIG令人惊讶的具有显著地抑制Aβ(包括Aβ40及Aβ42)聚集的作用。式(I)所示的多肽及VQVG是通过与Aβ(包括Aβ40及Aβ42)形成复合物来稳定Aβ(包括Aβ40及Aβ42)的α-螺旋结构，抑制β-折叠的结构形成，从而稳定Aβ(包括Aβ40及Aβ42)的构象，抑制Aβ(包括Aβ40及Aβ42)的聚集。
在本发明优选的一个实施方案中，获得了优选的多肽VQVG的结晶形式。多肽VQVG的晶体呈现长针状，一个晶体学不对称单位内含有1条多肽链、2个水分子以及1个钠离子，每条链由4个氨基酸组成即Val-Gln-Val-Gly。
多肽VQVG晶体晶胞参数为：
晶体空间群：P212121；
晶体参数：
Figure PCTCN2016098501-appb-000003
α＝90.0°,β＝90.0°,γ＝90.0°。
在本发明优选的另一实施方案中，本发明还获得了多肽IQIG的结晶形式。多肽IQIG的晶体呈现长针状，一个晶体学不对称单位内含有两条多肽链以及2个水分子，每条链由4个氨基酸组成即Ile-Gln-Ile-Gly。两条多肽链呈反平行折叠形式。
多肽IQIG晶体晶胞参数为：
晶体空间群：P1；
晶体参数：
Figure PCTCN2016098501-appb-000004
α＝100.97°,β＝89.98°, γ＝99.24°。
本发明的另一目的在于提供上述抑制Aβ聚集的多肽在用于制备治疗或预防阿尔茨海默症药物中的用途。阿尔茨海默症的发生和进展与Aβ(包括Aβ40及Aβ42)的聚集具有十分密切的关系，因此可以预见本发明中的式(I)的多肽(特别优选VQVG或IQIG)用于阿尔茨海默症的预防或治疗会对改善患者的病情或延缓疾病的发生起到积极的作用。
通过硫代硫磺素T实验，与未加上述多肽的模型组对比，加入VQVG或IQIG多肽的实验组的荧光强度明显低于模型组Aβ40或Aβ42聚集模型，表明多肽VQVG或IQIG可以显著的抑制Aβ40或Aβ42的聚集活性。在细胞水平上，将Aβ40或Aβ42的基因构建至合适的载体上，并转染HEK293T细胞。优选加入不同浓度的VQVG或IQIG多肽刺激，结果表明相比于对照组，加入多肽VQVG或IQIG刺激的细胞，细胞内形成的荧光斑点强度减弱，且与多肽VQVG或IQIG浓度呈正相关，表明多肽VQVG或IQIG在细胞水平也可以显著的抑制Aβ40或Aβ42的聚集活性。此外，还通过透射电子显微镜检测多肽VQVG或IQIG对β40或Aβ42聚集的作用。结果表明，Aβ40或Aβ42样品中的纤维密度非常很高，呈线状，交叉成网状。然而加入多肽VQVG或IQIG的样品，视野中仅能观察到少量短的纤维。这些结果均表明多肽VQVG或IQIG可以抑制Aβ40或Aβ42的聚集，因而可用于治疗或预防阿尔茨海默症。
本发明的多肽药物可以是游离多肽或多肽的药学上可接受的成盐形式，例如硫酸盐、盐酸盐、磷酸盐、磺酸盐、柠檬酸盐、醋酸盐、乳酸盐、酒石酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐等。多肽药物可通过例如口服、静脉注射、肌内注射、皮下注射或其他途径给药。药物可通过口服例如以片剂、凝胶、糊剂、膏剂、液体剂、粉剂或以其他剂型给药。口服给药的组合物包括，粘合剂、调味剂和润湿剂。药物可含在牙膏或漱口剂中。因此，口腔剂可包含磨粉和发泡剂。药物也可以经皮给药或栓剂给药。
本发明还提供了一种药物组合物，包括有效治疗量的式(1)所示多肽和药学上可接受的药用辅料。
一种优选的实施方案中，本发明的合适的药物组合物通常包含有效治疗量的式(1)所示多肽、可药用的稀释剂和赋形剂(如无菌水)，根据预定的用途以调节终浓度范围。制备技术通常是在这个领域熟知的，例示Remington′s Pharmaceutical Sciences(18th Edition，Mack Publishing Company，1995)。
式(1)的多肽可以单独或者和其他阿尔兹海默治疗药物联用，包括但不限于生长抑素、去甲肾上腺素、肾上腺素、谷氨酸、磷脂、毒扁豆碱、长春新碱、长春碱、维生素E、多巴胺、他克林、氯贝胆碱、萘呋胺、吡拉西坦、尼莫地平、茴拉西坦、胞磷胆碱、腺苷、细胞色素C、维A酸、银杏黄酮苷、丹参、石杉碱甲、加压素等。人体治疗给药剂量范围按体重计为1mg/kg-4g/kg，优选可为1mg/kg-100mg/kg。
附图说明
图1为多肽VQVG与Aβ40的硫代硫磺素T实验结果示意图。
图2为多肽VQVG与Aβ42的硫代硫磺素T实验结果示意图。
图3为多肽VQVG与Aβ40的细胞实验结果示意图。
图4为多肽VQVG与Aβ42的细胞实验结果示意图。
图5为多肽VQVG与Aβ40的透射电子显微镜实验结果示意图。
图6为多肽VQVG与Aβ42的透射电子显微镜实验结果示意图。
图7为多肽VQVG的晶体结构示意图。
图8为多肽VQVG的HPLC分析结果示意图。
图9为多肽VQVG的MS分析结果示意图。
图10为多肽IQIG与Aβ40的硫代硫磺素T实验结果示意图。
图11为多肽IQIG与Aβ42的硫代硫磺素T实验结果示意图。
图12为多肽IQIG与Aβ40的细胞实验结果示意图。
图13为多肽IQIG与Aβ42的细胞实验结果示意图。
图14为多肽IQIG与Aβ40的透射电子显微镜实验结果示意图。
图15为多肽IQIG与Aβ42的透射电子显微镜实验结果示意图。
图16为多肽IQIG的晶体结构示意图。
图17为多肽IQIG的HPLC分析结果示意图。
图18为多肽IQIG的MS分析结果示意图。
具体实施方式
下面采用具体实施例进一步说明本发明的内容。
以下内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定 本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。
实施例1.多肽VQVG与Aβ40的硫代硫磺素T实验
蛋白Aβ40由上海强耀公司合成提供，纯度大于95％。多肽VQVG由上海强耀公司合成提供，纯度大于95％，结构形式为未修饰的VQVG。取Aβ40与多肽VQVG各1mg,分别用100ul二甲基亚砜完全溶解。硫代硫磺素T(Thioflavin T)采购于sigma公司(货号T3516-5g)，分子量为318.86。称取0.0319g的ThT溶于20ml 20mM Tris-HCl(pH 7.5)溶液中即得5mM ThT母液。
按照表1所示，用pH 7.4，20mM Tris-HCl配制实验组溶液100ul，使硫代硫磺素T(Thioflavin T)终浓度为25uM，蛋白Aβ40终浓度为5uM，多肽VQVG为0.5uM。将配好的溶液加入96孔板中，放入酶标仪，37℃孵育，每5分钟进行一次摇匀，48h后检测其荧光强度。每个样品重复三次，取平均值。检测时，用430nm波长进行激发，检测485nm波长处的荧光强度，由此判定多肽VQVG对β淀粉纤维样结构形成的影响。
表1：多肽VQVG与Aβ40的硫代硫磺素T实验分组
  ThT(500uM) VQVG Aβ1-40
Aβ40聚集模型组 5ul 0 5uM
VQVG实验组 5ul 0.5uM 5uM
用上述方法，检测的各组的荧光强度结果见附图1。可以看出，与未加上述多肽的模型组对比，加入VQVG多肽的实验组的荧光强度明显低于模型组Aβ40聚集模型，表明多肽VQVG可以显著的抑制Aβ40的聚集活性，可用于治疗或预防阿尔茨海默症。
实施例2.多肽VQVG与Aβ42的硫代硫磺素T实验
多肽Aβ42同样由上海强耀公司合成提供，纯度大于95％，配制方法参考多肽Aβ40。多肽VQVG与硫代硫磺素T的来源与配制均与实验例1中所述一致。按照表2所示，参考实验例1配制各实验组溶液100ul。将配好的溶液加入96孔板中，放入酶标仪，37℃孵育，每5分钟进行一次摇匀，48h后检测其荧光强度。每个样品重复三次，取平均值。检测时，用430nm波长进行激发，检测485nm波长处的荧光强度，由此判定多肽VQVG对β淀粉纤维样结构形成的影响。
表2：多肽VQVG与Aβ42的硫代硫磺素T实验分组
  ThT(500uM) VQVG Aβ1-42
Aβ42聚集模型组 5ul 0 5uM
VQVG实验组 5ul 0.5uM 5uM
用上述方法，检测的各组的荧光强度结果见附图2。可以看出，与未加上述多肽的模型组对比，加入VQVG多肽的实验组的荧光强度明显低于模型组Aβ42聚集模型，表明多肽VQVG可以显著的抑制Aβ42的聚集活性，可用于治疗或预防阿尔茨海默症。
实施例3细胞水平研究多肽VQVG对蛋白Aβ40聚集的作用
为检测多肽VQVG在体内对Aβ1-40蛋白的作用，将Aβ40对应的基因通过酶切位点XhoI/BamHI构建在pEGFP-N1的载体(购自Clontech公司)。用测序正确的质粒转染HEK293T细胞，分别给与10uM，50uM多肽VQVG刺激，37℃培养，同时，设置一组空白对照，不加任何刺激，自然生长。24小时后，4％多聚甲醛固定，0.3％Triton X-100打孔，DAPI染核。用confocal(高感度激光扫描共聚焦显微镜，型号为蔡司LSM710)对实验结果进行观察，观察结果如图3。从图中可以看出，相比于对照组，加入多肽VQVG刺激的细胞，细胞内形成的荧光斑点强度减弱，且与多肽VQVG浓度呈正相关。加入10uM VQVG刺激的实验组，相对于空白组形成的荧光斑点强度减弱至25％；加入50uM VQVG刺激的实验组，相对于空白组形成的荧光斑点强度减弱至10％。说明多肽VQVG在细胞水平也可以显著的抑制Aβ40的聚集活性，可用于治疗或预防阿尔茨海默症。
实施例4细胞水平研究多肽VQVG对蛋白Aβ42聚集的作用
参照实施例3的方法检测多肽VQVG在体内对Aβ42蛋白的作用，用confocal(高感度激光扫描共聚焦显微镜，型号为蔡司LSM710)对实验结果进行观察，观察结果如图4。从图中可以看出，相比于对照组，加入多肽VQVG刺激的细胞，细胞内形成的荧光斑点强度减弱，且与多肽VQVG浓度呈正相关。加入10uM VQVG刺激的实验组，相对于空白组形成的荧光斑点强度减弱至30％；加入50uM VQVG刺激的实验组，相对于空白组形成的荧光斑点强度减弱至15％。说明多肽VQVG在细胞水平也可以显著的抑制Aβ42的聚集活性，可用于治疗或预防阿尔茨海默症。
实施例5采用透射电子显微镜检测多肽VQVG对Aβ40聚集的作用
用pH7.4的20Mm Tris-HCl将纯化好的蛋白Aβ40浓度稀释至100ug/ml,按蛋白：多肽＝10:1的摩尔比在体系中加入多肽VQVG，同时，设置空白组(只加入蛋白)，最后，用pH7.4的20Mm Tris-HCl将各实验组体积调整至100ul，蛋白终浓度为50ug/ml。37℃孵育48h后进行制样。
取5ul的样品加于铜网正面(购置于中镜科仪膜科技有限公司，规格400目)，1min后吸走，加5ul 2％醋酸釉保持1min，用滤纸吸走后放在玻璃皿中，用红外烘烤灯80°C烘烤20min，置于电镜(国家蛋白质科学中心，型号为Tecnai G2Spirit 120kV透射电镜)下进行观察，检测各样品的结构。
观察结果如图5所示：Aβ40样品中的纤维密度很高，呈线状，有交叉，长度约为500nm到1um不等；然而，加入多肽VQVG的样品，视野中很难观察到纤维分布，仅有少量很短的棒状纤维或圆形的聚集物。说明多肽VQVG可以抑制Aβ40的聚集，可用于治疗或预防阿尔茨海默症。
实施例6采用透射电子显微镜检测多肽VQVG对Aβ42聚集的作用
实验方法及过程参考实验例5中多肽VQVG对Aβ40的作用，观察结果如图6所示：Aβ42样品中的纤维密度非常很高，呈线状，交叉成网状，长度均超过1um；然而，加入多肽VQVG的样品，视野中仅能观察到少量短的纤维。说明多肽VQVG可以抑制Aβ42的聚集，可用于治疗或预防阿尔茨海默症。
实施例7多肽VQVG晶体的衍射收据收集及结构解析
本发明将等体积的多肽(VQVG,5mg/ml)与结晶试剂(0.2M magnesium chloride,0.1M sodium cacodylate pH6.5,20％(v/v)PEG 200)混合，20度以悬滴法放置1天后得到长针状晶体。制备的晶体以20％的甘油作为防冻剂处理后，冻存于液氮中。晶体X射线衍射的数据收集在上海光源(SSRF)生物大分子晶体学光束线站(五线六站BL19U)进行。数据处理使用HKL3000，结构解析采用分子置换法，利用Refmac程序并辅以Coot软件做进一步修正，最终解析得到多肽VQVG的晶体结构。本领域的普通技术人员可知，于其中所述晶体三维结构中的原子具有表3中所列的至少40％的原子坐标，或者3D蛋白的晶体三维结构中至少40％氨基酸的主链碳骨架的原子结构坐标 与表3中的坐标的平均根方差小于或等于
Figure PCTCN2016098501-appb-000005
的原子坐标，均可被认为与3D蛋白具有雷同结构。
具体而言，所述的多肽VQVG的晶体三维结构，一个晶体学不对称单位内含有一条多肽链、2个水分子以及1个钠离子，每条链由4各氨基酸组成即Val-Gln-Val-Gly，如图7所示(氨基酸以球棍模型表示，红色：氧原子；绿色：碳原子；蓝色：氮原子；红星：水分子；紫色：钠离子)。
多肽VQVG的HPLC和MS分析报告分别如图8和图9所示。
HPLC分离采用ODS色谱柱，洗脱A液含0.1％三氟乙酸的100％乙腈，B液为0.1％三氟乙酸的水。质谱报告显示荷质比为401.85[M+H]+，与理论值400.85一致。表3单分子的来源于多肽VQVG的原子，坐标群如下：
晶体空间群：P212121
晶体参数：
a,b,c
Figure PCTCN2016098501-appb-000006
4.81,17.15,29.56
α,β,γ(°)：90.0,90.0,90.0
I/σI：54.5
Rwork/Rfree分别为：0.137和0.153
分辨率范围
Figure PCTCN2016098501-appb-000007
50-1.27
数据完整性(％)：98.2
表3
Figure PCTCN2016098501-appb-000008
Figure PCTCN2016098501-appb-000009
实施例8.多肽IQIG与Aβ40的硫代硫磺素T实验
蛋白Aβ40由上海强耀公司合成提供，纯度大于95％。多肽IQIG由上海强耀公司合成提供，纯度大于95％，结构形式为未修饰的IQIG。取Aβ40与多肽IQIG各1mg,分别用100ul二甲基亚砜完全溶解。硫代硫磺素T(Thioflavin T)采购于sigma公司(货号T3516-5g)，分子量为318.86。称取0.0319g的ThT溶于20ml 20mM Tris-HCl (pH 7.5)溶液中即得5mM ThT母液。
按照表4所示，用pH 7.4，20mM Tris-HCl配制实验组溶液100ul，使硫代硫磺素T(Thioflavin T)终浓度为25uM，蛋白Aβ40终浓度为5uM，多肽IQIG为0.5uM。将配好的溶液加入96孔板中，放入酶标仪，37℃孵育，每5分钟进行一次摇匀，48h后检测其荧光强度。每个样品重复三次，取平均值。检测时，用430nm波长进行激发，检测485nm波长处的荧光强度，由此判定多肽IQIG对β淀粉纤维样结构形成的影响。
表4：多肽IQIG与Aβ40的硫代硫磺素T实验分组
  ThT(500uM) IQIG Aβ1-40
Aβ40聚集模型组 5ul 0 5uM
IQIG实验组 5ul 0.5uM 5uM
用上述方法，检测的各组的荧光强度结果见附图10。可以看出，与未加上述多肽的模型组对比，加入IQIG多肽的实验组的荧光强度明显低于模型组Aβ40聚集模型，表明多肽IQIG可以显著的抑制Aβ40的聚集活性，可用于治疗或预防阿尔茨海默症。
实施例9.多肽IQIG与Aβ42的硫代硫磺素T实验
多肽Aβ42同样由上海强耀公司合成提供，纯度大于95％，配制方法参考多肽Aβ40。多肽IQIG与硫代硫磺素T的来源与配制均与实验例1中所述一致。按照表5所示，参考实验例8配制各实验组溶液100ul。将配好的溶液加入96孔板中，放入酶标仪，37℃孵育，每5分钟进行一次摇匀，48h后检测其荧光强度。每个样品重复三次，取平均值。检测时，用430nm波长进行激发，检测485nm波长处的荧光强度，由此判定多肽IQIG对β淀粉纤维样结构形成的影响。
表5：多肽IQIG与Aβ42的硫代硫磺素T实验分组
  ThT(500uM) IQIG Aβ1-42
Aβ42聚集模型组 5ul 0 5uM
IQIG实验组 5ul 0.5uM 5uM
用上述方法，检测的各组的荧光强度结果见附图11。可以看出，与未加上述多肽的模型组对比，加入IQIG多肽的实验组的荧光强度明显低于模型组Aβ42聚集模型，表明多肽IQIG可以显著的抑制Aβ42的聚集活性，可用于治疗或预防阿尔茨海默症。
实施例10细胞水平研究多肽IQIG对蛋白Aβ40聚集的作用
为检测多肽IQIG在体内对Aβ1-40蛋白的作用，将Aβ40对应的基因通过酶切位点XhoI/BamHI构建在pEGFP-N1的载体(购自Clontech公司)。用测序正确的质粒转染HEK293T细胞，分别给与10uM，50uM多肽IQIG刺激，37℃培养，同时，设置一组空白对照，不加任何刺激，自然生长。24小时后，4％多聚甲醛固定，0.3％Triton X-100打孔，DAPI染核。用confocal(高感度激光扫描共聚焦显微镜，型号为蔡司LSM710)对实验结果进行观察，观察结果如图12。从图中可以看出，相比于对照组，加入多肽IQIG刺激的细胞，细胞内形成的荧光斑点强度减弱，且与多肽IQIG浓度呈正相关。加入10uM IQIG刺激的实验组，相对于空白组形成的荧光斑点强度减弱至30％；加入50uM IQIG刺激的实验组，相对于空白组形成的荧光斑点强度减弱至15％。说明多肽IQIG在细胞水平也可以显著的抑制Aβ40的聚集活性，可用于治疗或预防阿尔茨海默症。
实施例11细胞水平研究多肽IQIG对蛋白Aβ42聚集的作用
参照实验例10的方法检测多肽IQIG在体内对Aβ42蛋白的作用，用confocal(高感度激光扫描共聚焦显微镜，型号为蔡司LSM710)对实验结果进行观察，观察结果如图13。从图中可以看出，相比于对照组，加入多肽IQIG刺激的细胞，细胞内形成的荧光斑点强度减弱，且与多肽IQIG浓度呈正相关。加入10uM IQIG刺激的实验组，相对于空白组形成的荧光斑点强度减弱至35％；加入50uM IQIG刺激的实验组，相对于空白组形成的荧光斑点强度减弱至16％。说明多肽IQIG在细胞水平也可以显著的抑制Aβ42的聚集活性，可用于治疗或预防阿尔茨海默症。
实施例12采用透射电子显微镜检测多肽IQIG对Aβ40聚集的作用
用pH7.4的20Mm Tris-HCl将纯化好的蛋白Aβ40浓度稀释至100ug/ml,按蛋白：多肽＝10:1的摩尔比在体系中加入多肽IQIG，同时，设置空白组(只加入蛋白)，最后，用pH7.4的20Mm Tris-HCl将各实验组体积调整至100ul，蛋白终浓度为50ug/ml。37℃孵育48h后进行制样。
取5ul的样品加于铜网正面(购置于中镜科仪膜科技有限公司，规格400目)，1min后吸走，加5ul 2％醋酸釉保持1min，用滤纸吸走后放在玻璃皿中，用红外烘烤灯80° C烘烤20min，置于电镜(国家蛋白质科学中心，型号为Tecnai G2Spirit 120kV透射电镜)下进行观察，检测各样品的结构。
观察结果如图14所示：Aβ40样品中的纤维密度很高，呈线状，有交叉，长度约为500nm到1um不等；然而，加入多肽IQIG的样品，视野中很难观察到纤维分布，仅有少量很短的棒状纤维或圆形的聚集物。说明多肽IQIG可以抑制Aβ40的聚集，可用于治疗或预防阿尔茨海默症。
实施例13采用透射电子显微镜检测多肽IQIG对Aβ42聚集的作用
实验方法及过程参考实验例12中多肽IQIG对Aβ40的作用，观察结果如图15所示：Aβ42样品中的纤维密度非常很高，呈线状，交叉成网状，长度均超过1um；然而，加入多肽IQIG的样品，视野中仅能观察到少量短的纤维。说明多肽IQIG可以抑制Aβ42的聚集，可用于治疗或预防阿尔茨海默症。
实施例14多肽IQIG晶体的衍射收据收集及结构解析
本发明人将等体积的多肽(IQIG,5mg/ml)与结晶试剂(0.2M ammonium acetate,0.1M Bis-Tris pH6.5,45％(v/v)MPD)混合，20度以悬滴法放置2天后得到长针状晶体。制备的晶体以25％的甘油作为防冻剂处理后，冻存于液氮中。晶体X射线衍射的数据收集在上海光源(SSRF)生物大分子晶体学光束线站(五线六站BL19U)进行。数据处理使用HKL3000，结构解析采用分子置换法，利用Refmac程序并辅以Coot软件做进一步修正，最终解析得到多肽IQIG的晶体结构。本领域的普通技术人员可知，于其中所述晶体三维结构中的原子具有表6中所列的至少40％的原子坐标，或者3D蛋白的晶体三维结构中至少40％氨基酸的主链碳骨架的原子结构坐标与表6中的坐标的平均根方差小于或等于
Figure PCTCN2016098501-appb-000010
的原子坐标，均可被认为与3D蛋白具有雷同结构。
具体而言，所述的多肽IQIG的晶体三维结构，一个晶体学不对称单位内含有两条多肽链以及2个水分子，每条链由4个氨基酸组成即Ile-Gln-Ile-Gly。两条多肽链呈反平行折叠形式，如图16所示(氨基酸以球棍模型表示，红色：氧原子；绿色：碳原子；蓝色：氮原子；红星：水分子)。
多肽IQIG的HPLC和MS分析报告分别如图17和图18所示。
HPLC分离采用碳18反相色谱柱，洗脱A液含0.1％三氟乙酸的100％乙腈，B液为 0.1％三氟乙酸的水。质谱报告显示荷质比为429.10[M+H]+，与理论值429.52一致。
表6来源于多肽IQIG的原子坐标群如下：
晶体空间群：P1
晶体参数：
a,b,c
Figure PCTCN2016098501-appb-000011
4.79,15.05,17.76
α,β,γ(°)：100.97,89.98,99.24
I/σI：3.7
Rwork/Rfree分别为：0.166和0.184
分辨率范围
Figure PCTCN2016098501-appb-000012
50-1.60
数据完整性(％)：97.2
表6
Figure PCTCN2016098501-appb-000013
Figure PCTCN2016098501-appb-000014

Claims (15)

  1. 一种多肽，该多肽包含下述氨基酸序列(I)，由下述氨基酸序列(I)组成或基本上由下述氨基酸序列(I)组成：
    Xaa1-Xaa2-Xaa3-Xaa4         (I)
    其中，Xaa1和Xaa3为相同的氨基酸，选自I、V或L；
    Xaa2选自Q或N；
    Xaa4选自A,C,D,E,F,G,H,K,M,P,R,S,T,V,W或Y。
  2. 根据权利要求1所述的多肽，其特征在于：Xaa1和Xaa3选自I、V或L，Xaa2选自Q或N，Xaa4选自C,G,N,Q,S或T。
  3. 根据权利要求1所述的多肽，其特征在于：为下述序列之一:
    VQVG；VNVG；LQLG；LNLG；LQVG；VQLG；VQVC；VQLQ；VNLG；VNVC；VNLC；VQVQ；VQVN；VNVQ；VNVN；IQIG；INIG；LQLG；LNLG；IQIS；IQIC；IQLG；LQIG；INLG；LNIG；INIC；INLC；IQIQ；IQIN；INIQ；ININ。
  4. 根据权利要求1-3之一所述的多肽，其特征在于：多肽在N-末端、C-末端或二端被化学修饰。
  5. 根据权利要求4所述的多肽，其特征在于：多肽的N-末端通过乙酰化化学修饰，C-末端通过酰胺化化学修饰。
  6. 根据权利要求5所述的多肽，其特征在于：所述的肽形式为Ac-VQVG-NH2或Ac-IQIG-NH2。
  7. 根据权利要求1所述的多肽，其特征在于：是游离多肽或多肽的药学上可接受的成盐形式，包括硫酸盐、盐酸盐、磷酸盐、磺酸盐、柠檬酸盐、醋酸盐、乳酸盐、酒石酸盐、甲磺酸盐、乙磺酸盐或苯磺酸盐。
  8. 一种药物组合物，包括有效治疗量权利要求1-7任一项所述的多肽和药学上可接受的药用辅料。
  9. 如权利要求1-7任一项所述的多肽在制备治疗或预防阿尔茨海默症药物中的用途。
  10. 根据权利要求9所述的用途，其特征在于：所述多肽为单独使用，或者和其他阿尔兹海默治疗药物联用，所述其他阿尔兹海默治疗药物包括生长抑素、去甲肾上腺素、肾上腺素、谷氨酸、磷脂、毒扁豆碱、长春新碱、长春碱、维生 素E、多巴胺、他克林、氯贝胆碱、萘呋胺、吡拉西坦、尼莫地平、茴拉西坦、胞磷胆碱、腺苷、细胞色素C、维A酸、银杏黄酮苷、丹参、石杉碱甲、加压素中的一种或多种。
  11. 根据权利要求10所述的用途，其特征在于：多肽给药剂量范围按体重计为1mg/kg-4g/kg。
  12. 多肽VQVG的结晶体，其特征在于该晶体晶胞参数为：
    晶体空间群：P212121；
    晶体参数：
    Figure PCTCN2016098501-appb-100001
    α＝90.0°,β＝90.0°,γ＝90.0°；
  13. 根据权利要求12所述的晶体，其特征在于：多肽VQVG的晶体呈现长针状，一个晶体学不对称单位内含有1条多肽链、2个水分子以及1个钠离子，每条链由4个氨基酸组成即Val-Gln-Val-Gly。
  14. 多肽IQIG的结晶体，其特征在于：该晶体晶胞参数为：
    晶体空间群：P1；
    晶体参数：
    Figure PCTCN2016098501-appb-100002
    α＝100.97°,β＝89.98°,γ＝99.24°；
  15. 根据权利要求14所述的晶体，其特征在于：多肽IQIG的晶体呈现长针状，一个晶体学不对称单位内含有两条多肽链以及2个水分子，每条链由4个氨基酸组成即Ile-Gln-Ile-Gly，两条多肽链呈反平行折叠形式。
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