CN110522903A - Inhibit polypeptide/small molecule complexes, preparation method and the application of amyloid beta aggregation and toxicity - Google Patents
Inhibit polypeptide/small molecule complexes, preparation method and the application of amyloid beta aggregation and toxicity Download PDFInfo
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Abstract
The present invention provides polypeptide/small molecule complexes, preparation method and the applications of a kind of inhibition amyloid beta aggregation and toxicity, in the compound, polypeptide and small molecule are combined with each other, and, the amino acid sequence of the polypeptide is SEQ ID NO:2, and the small molecule is Epigallo-catechin gallate (EGCG).KLVFF/EGCG polypeptide/small molecule complexes binary inhibitor ingredients Biogenic the compatibility of the present invention for inhibiting amyloid beta aggregation is good, and aggregation inhibiting effect is obvious.In addition, the advantages of this polypeptide/small molecule complexes binary inhibitor combines different from the single inhibitor molecules of tradition and polypeptide drug, KLVFF/EGCG, has been provided simultaneously with multiple target point, high specificity and higher stability.
Description
Technical field
The invention belongs to protein structure detection and regulation and control fields, and in particular to a kind of inhibitions amyloid beta aggregation and malicious
Polypeptide/small molecule complexes, preparation method and the application of property.
Background technique
Alzheimer's disease (Alzheimer's disease, AD) is a kind of neurodegenerative disease, main clinical manifestation
For the memory disorders being constantly in progress, comprehensive hypophrenia, personality change and mental act are abnormal.Amyloid beta (β-
Amyloid protein, A β) aggregation be the important origin cause of formation on AD pathology, have found that A β is poly- in the brain tissue of AD patient
Collective deposits the patch to be formed.A β in human body by generating, from the monomer of solvable state to the oligomer with physiological-toxicity, original
The transformation of fiber, fiber.In accumulation process, aggregation is the important link of AD occurrence and development to the damage of nerve cell.Institute
To realize the regulation to A beta-aggregation process by the inhibitor of research and development amyloid protein, A β is reduced to the toxicity of nerve cell, tool
It is significant.Currently, being the drug of possible amyloidosis to the inhibitor of amyloid aggregation, can be divided by type has
Machine small molecule, polypeptide molecule and nano material etc..Small organic molecule such as Polyphenols inhibitor and A β have stronger combination
Ability, and polyphenol molecule stress have benign effect for cellular anti-oxidant, but specificity is poor, in conjunction with easy by dry
It disturbs.Polypeptide molecule such as KLVFF sequence can be specifically bound with A β, but be limited to that action site is single, and inhibitory effect is weaker.
Research before has shown that, if terpyridyl and the KLVFF compound assembled jointly can be effectively controlled the foldable structure of A β, regulates and controls A β
Assembling pattern.The compound of polyphenol small molecule blocking agent and peptide inhibitor can be obtained in conjunction with the two strong point, synergistic effect
The better effect of addition of opposite two kinds of independent roles is obtained, and A beta-aggregation, raising cellular anti-oxidant capacity can be inhibited by combining,
Slow down the carry out process of AD.
Summary of the invention
Therefore, the purpose of the present invention is to overcome the defects in the prior art, provides a kind of inhibition amyloid beta aggregation
With polypeptide/small molecule complexes, preparation method and the application of toxicity.
Before illustrating the content of present invention, it is as follows to define term used herein:
Term " A β " refers to: amyloid beta.
Term " EGCG " refers to: nutgall catechin gallic acid ester, Epigallocatechin gallate.
Term " HFIP " refers to: hexafluoroisopropanol.
Term " DMSO " refers to: dimethyl sulfoxide.
Term " ThT " refers to: thioflavine T, 2- [4- (dimethylamino) phenyl] -3,6-dimethyl
benzothiazolium chloride。
To achieve the above object, the first aspect of the present invention provides a kind of aggregation of inhibition amyloid beta and/or toxicity
Polypeptide/small molecule complexes, in the compound, polypeptide and small molecule are combined with each other, also, the amino acid sequence of the polypeptide
It is classified as SEQ ID NO:2, the small molecule is Epigallo-catechin gallate (EGCG).
Compound according to a first aspect of the present invention, wherein in the polypeptide/small molecule complexes, the polypeptide and small
The molar ratio of molecule is 10:1~1:10, preferably 5:1~1:5, most preferably 1:1.
The second aspect of the present invention provides polypeptide/small molecule complexes preparation method described in first aspect, institute
The method of stating includes: that the polypeptide and small molecule are mixed and sufficiently dissolved to form the polypeptide/small molecule complexes.
Preparation method according to a second aspect of the present invention, wherein the solvent is selected from one or more of: PBS buffering
Liquid, three times water, phosphate buffer；Preferably, the solvent is PBS buffer solution；Most preferably, the solvent is 1 × PBS buffering
Liquid.
Preparation method according to a second aspect of the present invention, wherein the described method comprises the following steps:
(1) it weighs the polypeptide and is dissolved in solvent, ultrasound makes dissolution completely, constant volume；
(2) it weighs the small molecule and is dissolved in solvent, ultrasound makes dissolution completely, constant volume；
(3) small molecule solution prepared by step (1) polypeptide solution and step (2) is mixed in proportion, ultrasound is allowed to abundant
Mixing, obtains the polypeptide/small molecule complexes.
The third aspect of the present invention provides a kind of method for inhibiting amyloid beta aggregation and/or toxicity, the side
Method includes: the sample containing amyloid beta to handle needed for polypeptide described in first aspect/small molecule complexes processing.
The fourth aspect of the present invention provides polypeptide/small molecule complexes described in a kind of first aspect in preparation for pressing down
Application in the drug of amyloid beta aggregation processed and toxicity.
The fifth aspect of the present invention provides a kind of for inhibiting the drug of amyloid beta aggregation and toxicity, the medicine
Object includes:
Polypeptide/small molecule complexes described in first aspect；And
Pharmaceutically acceptable carrier.
The sixth aspect of the present invention provides polypeptide/small molecule complexes described in first aspect in preparation for treating β
Application in the drug of diseases associated with amyloid protein.
Preferably, the disease is Alzheimer's disease.
The present invention relates to polypeptide molecules and more alkanol molecules in the application of regulation amyloid beta Assembling Behavior, is related to
Protein structure detection and regulation and control field.
The present invention devises a kind of novel polypeptide/small point for the physiological function regulation of regulation amyloid beta (A β)
Sub- composite regulator, the regulator derive from amyloid polypeptide critical sequences KLVFF and polyphenol epigallocatechin galla turcica
The compound that acid esters (EGCG) is formed.The sequence can for alzheimer's disease caused by A β (Alzheimer's disease,
AD treatment) provides feasible method, can be the discovery of the compound class lead compound for the treatment of diseases associated with amyloid protein
Thinking and reference standard are provided.
It is single the present invention be directed to be currently used in amyloid polypeptide inhibitor type, and there are problems that inherent defect, if
Polypeptide/small molecule class binary complex class inhibitor is counted, and a kind of regulation amyloid A beta-aggregation degree is provided, changes amyloid
Polypeptide A beta-aggregation pattern regulates and controls amyloid polypeptide A beta-aggregation body conformation, reduces the experimental method of the cytotoxicity of amyloid polypeptide.
" amyloid beta " in the present invention, sequence are as follows: DAEFR HDSGY EVHHQ KLVFF AEDVG SNKGA
IIGLM VGGVV IA(SEQ ID NO:1)。
The present invention provides a kind of polypeptide/small molecule complexes for inhibiting A beta-aggregation and toxicity, the amino acid of the polypeptide
Sequence is KLVFF (SEQ ID NO:2), and small molecule is Epigallo-catechin gallate (EGCG) (Epigallocatechin
gallate,EGCG)。
Preferably, the present invention provides polypeptide/small molecule complexes binary inhibitor preparation method, by KLVFF and EGCG
It is mixed according to molar ratio 10:1~1:10, and is sufficiently dissolved, form binary inhibitor.
It is further preferred that in polypeptide/small molecule complexes binary inhibitor KLVFF and EGCG molar ratio be 5:1~
1:5。
Most preferably, the molar ratio of KLVFF and EGCG is 1:1 in polypeptide/small molecule complexes binary inhibitor.
The present invention also provides a kind of aforementioned polypeptides/small molecule complexes binary inhibitor preparation for inhibit A beta-aggregation and
Application in the drug of toxicity.
The present invention provides a kind of aforementioned polypeptides/small molecule complexes binary inhibitor in preparation for treating A ss related diseases
Drug in application.
In conclusion it is an object of the invention to find that a kind of polypeptide/polyol molecule binary complex is inhibiting gathering for A β
New role in terms of collection and toxicity inhibits the drug of A β physiological-toxicity to have very important significance discovery and design.
One embodiment of the invention is the measurement polypeptide/small molecule complexes binary inhibitor and target β starch
Specific recognition effect between sample albumen, and the regulation of amyloid beta coherent condition and the inhibition of physiological-toxicity are made
With.Include the following steps:
1. by nuclear magnetic resonance spectroscopy (1H-Nuclear Magnetic Resonance, HNMR) technology, observe amyloid
The variation of polypeptide aggregation structure.
2. observing under solution environmental polypeptide/small molecule complexes binary inhibitor to A β to β-pleated sheet by ThT fluorescence spectrum
The influence of structure transition kinetics.
3. by polypeptide/small molecule complexes binary inhibitor under circular dichroism spectra observation solution environmental to A β secondary structure
It influences.
4. with atomic force microscopy observation polypeptide/small molecule complexes binary inhibitor to the shadow of A beta-aggregation bodily form looks
It rings.
5. with transmission electron microscopy observation polypeptide/small molecule complexes binary inhibitor to the shadow of A beta-aggregation state
It rings.
6. with neuroma cell line SH-SY5Y evaluation polypeptide/small molecule complexes binary inhibitor to A β cytotoxicity
Inhibit.
In short, the present invention is based on KLVFF/EGCG polypeptide/small molecule complexes binary inhibitor pair by largely testing
The aggregation for inhibiting A β and the exquisite ability in terms of toxicity, it was found that binary inhibitor is adjusting the new role in A beta-aggregation.We
It was found that KLVFF/EGCG can be combined with each other, and have extremely strong affinity to A β, stabilization is in the more a target spots of A β, Ke Yiyou
Effect inhibits the assembling and aggregation of amyloid beta, to inhibit its physiological-toxicity.
Polypeptide/small molecule complexes of the aggregation of inhibition amyloid beta and toxicity of the invention can have but be not limited to
Below the utility model has the advantages that
The KLVFF/EGCG polypeptide for inhibiting amyloid beta aggregation/small molecule complexes binary of the present invention inhibits
The ingredients Biogenic compatibility of agent is good, and aggregation inhibiting effect is obvious.In addition, this polypeptide/small molecule complexes binary inhibitor is different
It is combined in the single inhibitor molecules of tradition and polypeptide drug, KLVFF/EGCG, has been provided simultaneously with multiple target point, high specificity
And the advantages of higher stability.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows polypeptide of the present invention/small molecule complexes binary inhibitor and A β effect causes A beta structure
The core of change surveys resonance hydrogen spectrum；
Fig. 2 shows polypeptide of the present invention/small molecule complexes binary inhibitor to regulate and control the dynamic (dynamical) ThT of A beta-aggregation
Fluorescence spectrum；
Fig. 3 shows polypeptide of the present invention/small molecule complexes binary inhibitor and adjusts what A β secondary structure was formed
Circular dichroism spectra；
Fig. 4 shows regulation of the polypeptide of the present invention/small molecule complexes binary inhibitor to A beta-aggregation bodily form looks,
In, (a) sweeps subsurface imaging in atomic force microscope for A beta；One-component KLVFF (b) and one-component EGCG (c) are to A β
The atomic force microscope imaging of aggregation regulation, A β and one-component concentration ratio are 2:1；It (d) is the binary inhibitor and A β
Melting concn ratio is the atomic force microscope shape appearance figure of 1:2；(e) subsurface imaging is swept in transmission electron microscope for A beta；
The transmission electron microscope that A beta-aggregation regulate and control is imaged in one-component KLVFF (f) and one-component EGCG (g), A β and single group
Point concentration ratio is 2:1；(h) the transmission electron microscope pattern for being 1:2 for the binary inhibitor and A β melting concn ratio
Figure；
Fig. 5 shows the polypeptide/small molecule complexes binary inhibitor to the inhibiting effect of A β cytotoxicity, wherein
It (a) is influence of the various concentration A β to SH-SY5Y cell survival rate；It (b) is binary inhibitor described in various concentration to SH-SY5Y
The influence of cell survival rate；(c) A β is 42 μM, and when A β: KLVFF:EGCG:KLVFF/EGCG=2:1:1:1, SH-SY5Y is thin
Born of the same parents distinguish survival rate；(d) improvement of the binary inhibitor for A β cytotoxicity.
Specific embodiment
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only
It is used, is but should not be understood as present invention is limited in any form for specifically describing in more detail.
This part carries out general description to the material and test method that arrive used in present invention test.Although being
It realizes many materials used in the object of the invention and operating method is it is known in the art that still the present invention still uses up herein
It may detailed description.It will be apparent to those skilled in the art that within a context, if not specified, material therefor of the present invention and behaviour
It is well known in the art as method.
Reagent and instrument used in the following embodiment are as follows: reagent:
Amyloid beta, sequence D AEFRHDSGYEVHHQKLVFFAEDVG
SNKGAIIGLMVGGVVIA is purchased from Shanghai Ke Tai Co., Ltd (Shanghai Science Peptide
Biological Technology Co.,LTD)；
KLVFF, sequence KLVFF are purchased from Shanghai Ke Tai Co., Ltd (Shanghai Science Peptide
Biological Technology Co.,LTD)；
HFIP, DMSO, D2O, EGCG, ThT, Na2HPO4·12H2O, KH2PO4Reagent is purchased from from Sigma-Alder
Odd company (Sigma-Aldrich Co., LTD)；
RPMI-1640 culture medium, it is purchased from Corning company；
Neural oncocyte SH-SY5Y is purchased from Chinese Academy of Medical Sciences Beijing Union Medical College Hospital；
96 orifice plates are purchased from Corning company.
Instrument:
Magnetic nuclear resonance analyzer is purchased from Bruker, the U.S., model AVANCE III HD 400；
Circular dichroism absorption spectrometer is purchased from Jasco, Japan, model J-1500；
Scanning probe microscopy is purchased from Bruker, the U.S., model Fastscan；
Biological transmission electron microscope is purchased from Hitachi, Japan, model HT7700；
Continuous spectrum multi-function microplate reader is purchased from Tecan company, model Infinite M200.
Embodiment 1
The present embodiment is for illustrating1HNMR detects the polypeptide/small molecule complexes binary inhibitor to amyloid beta
Combination.
Amyloid beta dispersing method: first by A β solid powder 1mg, being dissolved in 1mLHFIP, ultrasound 30 seconds, vortex processing
1 minute.To after completely dissolution, place it on shaking table, 160rpm is shaken under homogeneous effect, is kept for 8 hours.Again by this A β's
HFIP solution is divided in Brown Glass Brown glass bottles and jars only by every bottle of 200 μ L, dries up HFIP solvent with nitrogen, this is contained to the vial of A β
It is placed in freeze drier and vacuumizes freeze-drying 60 minutes.The A beta monomers dispersed in every bottle containing 0.2mg at this time.
5 μ L DMSO are added into the vial containing the 0.2mg A beta monomers dispersed, ultrasound 5 minutes has dissolved A β
Entirely.With D2O is the A β solution that solvent configures final concentration of 0.4mg/mL.
KLVFF/EGCG binary inhibitor processing method: D is used2O is the KLVFF solution that solvent prepares 42 μM respectively, 42 μ
The EGCG solution of M, the two equal proportion are uniformly mixed and obtain 21 μM of binary inhibitor.
1H NMR spectra has reacted the migration of amyloid polypeptide structure.As shown in Fig. 1 (a), A β and EGCG/KLVFF mixing
After incubation, binary inhibitor regulates and controls amyloid beta structure and changes.Amyloid beta disappears at the peak 6.5-6.3ppm, and
Nearby there is new peak value in 0.2ppm.As shown in Fig. 1 .b, EGCG and KLVFF mixing after, the two formed binary complex, knot
Structure changes.New characteristic peak is formd at 3.0-2.8ppm.
Embodiment 2
The present embodiment is for illustrating that ThT dyeing detects the polypeptide/small molecule complexes binary inhibitor to beta-amyloyd egg
The adjustment effect of white ability of aggregation.
1 × PBS solution is prepared, for use.
It prepares ThT liquid storage: weighing 15.9mg ThT, 14.36g Na2HPO4·12H2O, 1.36gKH2PO4, measure 100mL
Ultrapure water makes it dissolve, and ultrasound makes sufficiently to dissolve for 10 minutes.
It prepares 10 μM of ThT test fluid: measuring 2mL ThT liquid storage, 718mg Na2HPO4·12H2O, 68mg KH2PO4, make
It is settled to 100mL with ultrapure water, uses 0.22 μm of water phase membrane filtration later, for use.
Amyloid beta dispersing method: as described in Example 1, final solvent is prepared with ultrapure water, and concentration is 42 μM.
Polypeptide/small molecule complexes binary inhibitor processing method: KLVFF takes 1mg, is dissolved using 5104 μ L1 × PBS,
Ultrasound makes dissolution for 1 minute completely, and the concentration of KLVFF liquid storage is 300 μM at this time.EGCG weighs 10mg, and 1000 μ L water three times is added
In, ultrasound makes dissolution for 1 minute completely, takes 40 μM of solution that ultrapure water is added and is settled to 2911 μ L, EGCG stock concentrations are 300 at this time
μM.1000 μ L equal proportions are respectively taken to mix KLVFF liquid storage and EGCG liquid storage, ultrasound makes sufficiently to dissolve for 10 minutes, and obtaining concentration is
150 μM of KLVFF/EGCG binary inhibitor.
Amyloid beta binary inhibitor solution preparation method: add in the vial for the 0.2mg A β that Xiang Hanyou has dispersed
Enter 5 μ L DMSO, ultrasound 5 minutes makes A β dissolution completely, and same procedure makes four times.The above-mentioned binary suppression of 106 μ L is added into bottle
Preparation liquid storage is settled to 1319 μ L with 1 × PBS, and it is 400 μm of glass marble (Sigma that a diameter is then added into bottle
Aldrich).At this point, the A β concentration in solution is 42 μM, binary inhibitor concentration is 21 μM.Similarly, control group is set: to
53 μ L KLVFF liquid storages are added in bottle, are settled to 1319 μ L with 1 × PBS, it is 400 μm of glass that a diameter is then added into bottle
Ball (Sigma Aldrich), this is A β+KLVFF control group.53 μ L EGCG liquid storages are added into bottle, are settled to 1 × PBS
1319 μ L, it is 400 μm of glass marble (Sigma Aldrich) that a diameter is then added into bottle, this is A β+EGCG control group.
1 × PBS is added into bottle and is settled to 1319 μ L, it is 400 μm of glass marble (Sigma that a diameter is then added into bottle
Aldrich), this is A β control group.Two clean vials are separately taken, 53 μ L KLVFF liquid storages are added in one, with 1 × PBS constant volume
To 1319 μ L, it is 400 μm of glass marble (Sigma Aldrich) that a diameter is then added into bottle, this is KLVFF control group,
Secondly 53 μ LEGCG liquid storages are added, 1319 μ L are settled to 1 × PBS, it is 400 μm of glass marble that a diameter is then added into bottle
(Sigma Aldrich), this is EGCG control group.
Sample is added drop-wise in 96 orifice plate (Corning) of black low adsorption amount according to this, 4 parallel control groups, every hole are set
200 μ L are added dropwise, add 20 μ LThT test fluids.In continuous spectrum multi-function microplate reader (Tecan Infinite M200),
Using 450nm as excitation wavelength, the transmitting optical signal of 482nm is collected, using continuous collecting signal mode, acquisition one in every 40 minutes
Secondary fluorescent value, yield value 100.
The amyloid accumulation process based on β-pleated sheet structure that ThT Coloration experiment reflects polypeptide changes with time.Knot
Fruit is as shown in Fig. 2, amyloid beta changed complete in the solution to the transformation of β-pleated sheet structure at the 14th hour.Binary suppression is added
After preparation, ThT fluorescence enhancement intensity is substantially reduced, i.e., the binary inhibitor can effectively prevent A β to β-pleated sheet structure at this time
Transformation.And the inhibiting effect of binary inhibitor is significantly stronger than KLVFF the or EGCG inhibitory effect of one-component.
Embodiment 3
The present embodiment is for illustrating that circular dichroism spectra detects the polypeptide/small molecule complexes binary inhibitor to beta-amyloyd
The regulation of Protein secondary structure
Amyloid beta dispersing method: with described in embodiment 1.
Polypeptide/small molecule complexes binary inhibitor processing method: with described in embodiment 2.
Amyloid beta+binary inhibitor solution preparation method: with described in 2.1.In 160rpm, the shaking table of 37 DEG C of constant temperature
After middle aging 48 hours, 300 μ L are taken out from sample solution, are added in the quartz colorimetric utensil that light path is 0.1cm.In circular dichroism light
In spectrometer (J-810), the circular dichroism value of 190nm-260nm is measured, slit width is set as 5.0nm, and measurement in triplicate, takes
Average value.
Circular dichroism spectrogram reflects the variation of polypeptide secondary structure.As a result as shown in figure 3, amyloid beta was through 48 hours
Afterwards, β-pleated sheet (negative peak at 217nm) and random coil structure (negative peak at 197nm) are its main space conformation.The binary is added
After inhibitor, amyloid beta is substantially reduced with beta structure folding in the mixed system of the polypeptide, at this time the binary inhibitor
It can effectively prevent A β to the transformation of β-pleated sheet structure.And the inhibiting effect of binary inhibitor is significantly stronger than one-component
KLVFF or EGCG inhibitory effect.
Embodiment 4
The present embodiment is for illustrating that atomic force microscopy detects the polypeptide/small molecule complexes binary inhibitor to β
The regulation of amyloid aggregation pattern.
Amyloid beta dispersing method: with described in embodiment 1.
Polypeptide/small molecule complexes binary inhibitor processing method and amyloid beta+binary inhibitor solution preparation side
Method: with described in 2.2.
Sample preparation methods: amyloid beta-binary inhibitor solution passes through in 160rpm, the shaking table of 37 DEG C of constant temperature
It is incubated for 48 hours.It takes 10 μ L to be added drop-wise to the mica surface of new cleavage, stands 10 minutes, inclination siphons away extra solution, stands dry
It is dry.Same method is incubated for amyloid beta, amyloid beta+KLVFF, amyloid beta+EGCG solution, and obtained pair
The scanning sample answered.
Utilize scanning probe microscopy (Bruker Fastscan, the U.S.), peak force tapping-mode, needle point selection
Scansyst Air (Bruker Fastscan, the U.S.), the pattern to test sample in mica surface is imaged.
AFM characterizes the variation for having reacted polypeptide aggregation morphology and size height.Shown in Fig. 4, A β self assemble forms diameter
For 10nm, 1 μm of length > of amyloid fiber.The binary inhibitor can obviously inhibit the fibrosis of A β, by binary inhibitor
Incubation is mixed with A β, forms the compound of indefiniteness on interface.The inhibition of binary inhibitor is significantly stronger than list at fiber ability
The KLVFF or EGCG of one component inhibit into fiber effects.
Embodiment 5
The present embodiment is for illustrating that transmission electron microscopy detects the polypeptide/small molecule complexes binary inhibitor pair
The regulation of amyloid beta aggregation pattern.
Amyloid beta dispersing method: with described in embodiment 1.
Polypeptide/small molecule complexes binary inhibitor processing method: with described in embodiment 2.
Amyloid beta-polypeptide mixed solution preparation method: with described in embodiment 2.
Sample preparation methods: amyloid beta-binary inhibitor solution passes through in 160rpm, the shaking table of 37 DEG C of constant temperature
It is incubated for 48 hours.It takes 50 μ L to be added drop-wise to ultra-thin plating carbon copper mesh surface, stands 10 minutes, remaining liq is siphoned away, in deposited samples
Copper mesh surface drip 20 μ L 1mg/mL ammonium phosphotungstate aqueous solution, deposit 5 minutes, remaining liq is siphoned away.The sample prepared
It is 8 hours dry in drier.Same method incubation amyloid beta, amyloid beta+KLVFF, amyloid beta+
EGCG solution, and corresponding TEM scanning sample is made.Using 120kV biology transmission electron microscope (Hitachi HT7700,
Japan) imaging, acceleration voltage is 80kV when imaging.
TEM characterizes the variation for having reacted polypeptide aggregation pattern.Shown in Fig. 4, it is 10nm, length that A β self assemble, which forms diameter,
The amyloid fiber of > 500nm.The binary inhibitor can obviously inhibit the fibrosis of A β, and binary inhibitor is mixed with A β and is incubated
It educates, is being formed within sweep of the eye without A beta.The inhibition of binary inhibitor is significantly stronger than the KLVFF of one-component at fiber ability
Or EGCG inhibits into fiber effects.
Embodiment 6
The present embodiment is for illustrating the neuroma that polypeptide/small molecule complexes binary inhibitor induces amyloid beta
The inhibition of cytotoxicity.
Amyloid beta dispersing method: with described in embodiment 2.
Polypeptide/small molecule complexes binary inhibitor processing method: with described in embodiment 2.
Amyloid beta-polypeptide mixed solution preparation method: with described in embodiment 2.
Using neural oncocyte SH-SY5Y as research model, (contain 15% North America tire ox blood using RPMI-1640 culture medium
Clearly, 1% mycillin) carry out cell culture.In 96 porocyte culture plates (Corning), 5000 cells, In are cultivated in every hole
In cell 12 hours after plate, the amyloid beta and the polypeptide that a series of concentration gradients are added into culture plate are incubated for.(tool
Bulk concentration is shown in Fig. 5 a) final volume of every hole culture solution is 200 μ L.After being incubated for 48 hours, into culture plate, 20 μ L are added in every hole
5mg/mL thiazolyl blue (MTT) 1 × PBS solution.It is reacted 4 hours at 37 DEG C.Solution in culture plate is siphoned away, 100 μ of every hole
The DMSO dissolution first a ceremonial jade-ladle, used in libation precipitating of L.In continuous spectrum multi-function microplate reader (Tecan Infinite M200), measure its
Absorbance value at 490nm obtains the data that amyloid beta cytotoxicity changes with concentration.Same method measures EGCG/
KLVFF binary inhibitor cytotoxicity changes with concentration.
Cytotoxicity experiment determines influence of the polypeptide to cell survival rate.As shown in Fig. 5 (a), amyloid beta pair
SH-SY5Y cell line has significant cytotoxicity.For testing the system, when the concentration of amyloid beta is 42 μM,
Cell survival rate is about 50%.As shown in Fig. 5 (b), binary inhibitor cytotoxicity is lower.As Fig. 5 (c) show A β (42 μ
M), KLVFF (21 μM), EGCG (21 μM), binary inhibitor (1:1 mixing, 21 μM) are added into 48 in SH-SY5Y cell suspending liquid
After hour, cell survival rate.When the concentration of the binary inhibitor is 21 μM, cell survival rate is 85% or more.Illustrate, In
Under experimental concentration, the preferable biological safety of binary inhibitor.Shown in Fig. 5 (d), amyloid beta and the binary inhibit
Agent is incubated for SH-SY5Y cell altogether, can significantly improve the survival rate of cell.When with 42 μM of A β incubated cell, SH-SY5Y is thin
Born of the same parents only have 50% survival rate, and when A β and binary inhibitor 2:1 addition, cell survival rate improves 29%, is significantly stronger than and is better than
The KLVFF (+8%) of one-component and the adduction of EGCG (+11%) detoxication.The i.e. described binary inhibitor can more effectively press down
Toxicity of the amyloid beta processed to SH-SY5Y cell.
Although present invention has been a degree of descriptions, it will be apparent that, do not departing from the spirit and scope of the present invention
Under the conditions of, the appropriate variation of each condition can be carried out.It is appreciated that the present invention is not limited to the embodiments, and it is attributed to right
It is required that range comprising the equivalent replacement of each factor.
Sequence table
<110>State Nanometer Science Center
<120>inhibit polypeptide/small molecule complexes, preparation method and the application of amyloid beta aggregation and toxicity
<130> YZDI-180040
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
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
<210> 2
<211> 5
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Lys Leu Val Phe Phe
1 5
Claims (10)
1. a kind of polypeptide/small molecule complexes for inhibiting amyloid beta aggregation and/or toxicity, which is characterized in that described compound
In object, polypeptide and small molecule are combined with each other, also, the amino acid sequence of the polypeptide is SEQ ID NO:2, and the small molecule is
Epigallo-catechin gallate (EGCG).
2. compound according to claim 1, which is characterized in that in the polypeptide/small molecule complexes, the polypeptide and
The molar ratio of small molecule is 10:1~1:10, preferably 5:1~1:5, most preferably 1:1.
3. the preparation method of polypeptide/small molecule complexes according to claim 1 or 2, which is characterized in that the method packet
It includes: the polypeptide and small molecule being mixed and sufficiently dissolution forms the polypeptide/small molecule complexes.
4. according to the method described in claim 3, it is characterized in that, the solvent is selected from one or more of: PBS buffering
Liquid, three times water, phosphate buffer；Preferably, the solvent is PBS buffer solution；Most preferably, the solvent is 1 × PBS buffering
Liquid.
5. the method according to claim 3 or 4, which is characterized in that the described method comprises the following steps:
(1) it weighs the polypeptide and is dissolved in solvent, ultrasound makes dissolution completely, constant volume；
(2) it weighs the small molecule and is dissolved in solvent, ultrasound makes dissolution completely, constant volume；
(3) small molecule solution prepared by step (1) polypeptide solution and step (2) being mixed in proportion, ultrasound is allowed to be sufficiently mixed,
Obtain the polypeptide/small molecule complexes.
6. a kind of method for inhibiting amyloid beta aggregation and/or toxicity, which is characterized in that the described method includes: with right
It is required that the sample containing amyloid beta handled needed for polypeptide described in 1 or 2/small molecule complexes processing.
7. polypeptide/small molecule complexes of any of claims 1 or 2 are in preparation for inhibiting amyloid beta aggregation and toxicity
Drug in application.
8. a kind of for inhibiting the drug of amyloid beta aggregation and toxicity, which is characterized in that the drug includes:
Polypeptide/small molecule complexes of any of claims 1 or 2；And
Pharmaceutically acceptable carrier.
9. polypeptide/small molecule complexes of any of claims 1 or 2 are in preparation for treating amyloid beta related disease
Application in drug.
10. application according to claim 9, which is characterized in that the disease is Alzheimer's disease.
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| CN201810516350.1A CN110522903A (en) | 2018-05-25 | 2018-05-25 | Inhibit polypeptide/small molecule complexes, preparation method and the application of amyloid beta aggregation and toxicity |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810516350.1A CN110522903A (en) | 2018-05-25 | 2018-05-25 | Inhibit polypeptide/small molecule complexes, preparation method and the application of amyloid beta aggregation and toxicity |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103536897A (en) * | 2012-07-16 | 2014-01-29 | 国家纳米科学中心 | Compound for inhibiting aggregation of amyloid polypeptide as well as preparation method and application thereof |
| CN104231053A (en) * | 2013-06-08 | 2014-12-24 | 国家纳米科学中心 | Polypeptide for regulating physiological toxicity of beta amyloid protein |
| CN104277105A (en) * | 2013-07-12 | 2015-01-14 | 国家纳米科学中心 | Polypeptide inhibitor for inhibiting aggregation and toxicity of beta amyloid protein and application of polypeptide inhibitor |
-
2018
- 2018-05-25 CN CN201810516350.1A patent/CN110522903A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103536897A (en) * | 2012-07-16 | 2014-01-29 | 国家纳米科学中心 | Compound for inhibiting aggregation of amyloid polypeptide as well as preparation method and application thereof |
| CN104231053A (en) * | 2013-06-08 | 2014-12-24 | 国家纳米科学中心 | Polypeptide for regulating physiological toxicity of beta amyloid protein |
| CN104277105A (en) * | 2013-07-12 | 2015-01-14 | 国家纳米科学中心 | Polypeptide inhibitor for inhibiting aggregation and toxicity of beta amyloid protein and application of polypeptide inhibitor |
Non-Patent Citations (3)
| Title |
|---|
| BALPREET等: "Development of retro-inverso peptides as anti-aggregation drugs for β-amyloid", 《PEPTIDES》 * |
| TEODOR等: "Amyloid Beta Peptide VHHQ, KLVFF, and IIGLMVGGVV Domains Involved in Fibrilization: AFM and Electrochemical Characterization", 《ANAL CHEM》 * |
| 董晓燕等: "多肽抑制剂抑制淀粉质多肽42构象转换的分子动力学模拟和结合自由能计算", 《物理化学学报》 * |
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