WO2017041733A1 - Polypeptide qui inhibe l'agrégation des protéines aβ de la maladie d'alzheimer, et application de ce dernier - Google Patents

Polypeptide qui inhibe l'agrégation des protéines aβ de la maladie d'alzheimer, et application de ce dernier 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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PCT/CN2016/098501
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Chinese (zh)
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李继喜
黄静
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复旦大学
李继喜
黄静
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Priority claimed from CN201510579527.9A external-priority patent/CN105175494B/zh
Priority claimed from CN201510579526.4A external-priority patent/CN105175493B/zh
Application filed by 复旦大学, 李继喜, 黄静 filed Critical 复旦大学
Publication of WO2017041733A1 publication Critical patent/WO2017041733A1/fr

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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

L'invention concerne un polypeptide qui inhibe l'agrégation des protéines Aβ de la maladie d'Alzheimer, et une séquence d'acides aminés de ce dernier est Xaa1-Xaa2-Xaa3-Xaa4. Xaa1 et Xaa3 sont des acides aminés identiques et choisis parmi I, V ou L, Xaa2 est choisi parmi Q ou N, et Xaa4 est choisi parmi A, C, D, E, F, G, H, K, M, P, R, S, T, V, W ou Y. Le polypeptide inhibe l'agrégation de Aß40 et Aß42, et est utilisé pour préparer un médicament destiné au traitement de la maladie d'Alzheimer et d'autres maladies neurodégénératives.
PCT/CN2016/098501 2015-09-12 2016-09-09 Polypeptide qui inhibe l'agrégation des protéines aβ de la maladie d'alzheimer, et application de ce dernier WO2017041733A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201510579526.4 2015-09-12
CN201510579527.9A CN105175494B (zh) 2015-09-12 2015-09-12 一种具有抑制阿尔兹海默症Aβ蛋白聚集的多肽及其用途
CN201510579527.9 2015-09-12
CN201510579526.4A CN105175493B (zh) 2015-09-12 2015-09-12 一种具有抑制Aβ聚集活性的多肽及其用途

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