CN102675419A - Abeta oligopeptide polymerization inhibitor and preparation and application thereof - Google Patents

Abstract

The invention relates to an Abeta oligopeptide polymerization inhibitor and a preparation and application thereof. The Abeta oligopeptide polymerization inhibitor is polypeptide with an amino acid sequence ALeuBProPheCLeu or a product obtained by modifying the polypeptide with a compound prolonging the half-life period of the polypeptide, wherein A is Ser or Thr, B is Ser or Thr, and C is Tyr, Ser or Thr. The Abeta oligopeptide polymerization inhibitor provided by the invention can be used for preparing a medicine for preventing or treating neurodegenerative diseases; compared with the known Abeta-precipitated small-molecular inhibitor RS-0406, the solubility is remarkably improved, and the effective drug concentration in in-vitro experiment is remarkably reduced; and meanwhile, since the compound comes from human protein alphaB, the possibility of initiating immunoreactions is greatly reduced.

Description

A kind of A β small peptide polymerization retarder and preparation thereof and application

Technical field

The present invention relates to biological technical field, be specifically related to a kind of A β small peptide polymerization retarder and preparation thereof and application.

Background technology

Alzheimer's disease (Alzheimer ' s Disease, AD) claim senile dementia again, be the nerve degenerative diseases that a kind of morbidity raises year by year.Present global AD patient is about 1700-2500 ten thousand, China 6,450,000 patients that have an appointment.Because the reduction of the significant self-care ability of patient AD has caused very big burden and pressure for patient family, society.For the world today that aging population increasingly sharpens, the novel method of exploring prevention and treatment AD is significant.

In recent years, molecular biology research has disclosed several gene relevant with AD and albumen, comprises amyloid beta protein precursor albumen (APP albumen), Tau albumen, apolipoprotein E (ApoE4), senilism albumen [1] such as (Presenilins, PS1 and PS2).Confirmed the dependency of these genes and AD for the research of natural mutation and gene knockout research, but the interaction relationship between these gene products is still very not clear and definite.Undeniable is that the polymerization of amyloid beta is the central step [2] of bringing out AD.In vivo, APP albumen by β-, after gamma-secretase was sheared, primary product was the A β small peptide [3] that contains 40 or 42 amino-acid residues, these A β small peptide major parts are secreted into the extracellular, polymerization forms beta-amyloyd deposition, wherein A β _1-42Form sedimentary fastest.In A β small peptide polymeric process, form a series of polymerisate, comprise dimer, tripolymer, the tetramer, solubility oligomer, fiber precursor, ripe amyloid fiber or the like [4].These polymerisates all are proved to be neurotoxicity in various degree, but and the solubility oligomer is pointed out in increasing in recent years experiment because its diffustivity and " seed " characteristic possibly have the strongest neurotoxicity [5,6]

NMR and x-ray crystal structure discover that amyloid beta precipitated fiber shape structure is based on the amino acid side chain residue and piles up the intersection β laminated structure [7,8] that (stacking) forms.Based on this discovery, scientists is being sought the method that can upset intersection β laminated structure always.In experiment in vitro, micromolecular inhibitor RS-0406 is proved to be the polymerization that can effectively suppress A β, but its low solubility in the aqueous solution might limit its application [9] in vivo.Found also that for the screening in pentapeptide and six peptide storehouses some have the little peptide that destroys β lamella characteristic; Like iA β 5 [10]; But the non-endogenous of these synthetic little peptides causes them to cause immunoreation easily and is prone to having been limited their application in vivo by System Cleaning.Therefore, start with from endogenous protein that to seek A β polymeric suppressor factor may be a more effective research direction.

Alpha-crystal albumen is a member of little heat-shock protein family, has chaperone activity, can combine multiple substrate protein, cohesion and the inactivation [11] of arrestin matter under multiple sex change condition.Alpha-crystal albumen is named owing to from people's lens, separating first.Found afterwards that alpha-crystal albumen had two hypotypes, α A and α B.Wherein α A is the lens specific protein, and α B has expression in multiple tissues such as brain, heart, Skelettmuskel.α B crystallin is the oligomer of many subunits, and each subunit is made up of 175 amino acid whose α B polypeptied chains.Discover; At some nerve degenerative diseases such as alzheimer's disease; In Parkinson's disease and the Crow Ci Feierte-cortico-striatal spinal degeneration, α B crystallin express to raise, and this is considered to body and avoids further a kind of of infringement for the neuroprotective cell and stress reflect [12].Experiment in vitro shows, people α B crystallin can with A β _1-42, α-synuclein, β 2-microglobulin, polypeptide or protein binding such as Regular Insulin, and suppress the sedimentary formation of its fiber [13].

[1]Selkoe，D.J.(2005).Defining?molecular?targets?to?prevent?Alzheimer?disease.Arch?Neurol62，192-5.

[2]Walsh，D.M.and?Selkoe，D.J.(2004).Deciphering?the?molecular?basis?of?memory?failurein?Alzheimer′s?disease.Neuron?44，181-93.

[3]Burdick，D.et?al.(1992).Assembly?and?aggregation?properties?of?synthetic?Alzheimer′sA4/beta?amyloid?peptide?analogs.J?Biol?Chem?267，546-54.

[4]Walsh，D.M.and?Selkoe，D.J.(2007).A?beta?oligomers?-?a?decade?of?discovery.JNeurochem?101，1172-84.

[5]Selkoe，D.J.and?Schenk，D.(2003).Alzheimer′s?disease：molecular?understanding?predictsamyloid-based?therapeutics.Annu?Rev?Pharmacol?Toxicol?43，545-84.

[6]Selkoe，D.J.(2008).Soluble?oligomers?of?the?amyloid?beta-protein?impair?synapticplasticity?and?behavior.Behav?Brain?Res?192，106-13.

[7]Barrow，C.J.and?Zagorski，M.G.(1991).Solution?structures?of?beta?peptide?and?itsconstituent?fragments：relation?to?amyloid?deposition.Science?253，179-82.

[8]Xu，M.，Shashilov，V.and?Lednev，I.K.(2007).Probing?the?cross-beta?core?structure?ofamyloid?fibrils?by?hydrogen-deuterium?exchange?deep?ultraviolet?resonance?Ramanspectroscopy.J?Am?Chem?Soc?129，11002-3.

[9]Nakagami，Y.et?al.(2002).A?novel?beta-sheet?breaker，RS-0406，reverses?amyloidbeta-induced?cytotoxicity?and?impairment?of?long-term?potentiation?in?vitro.Br?JPharmacol?137，676-82.

[10]Chacon，M.A.，Barria，M.I.，Soto，C.and?Inestrosa，N.C.(2004).Beta-sheet?breakerpeptide?prevents?Abeta-induced?spatial?memory?impairments?with?partial?reduction?ofamyloid?deposits.Mol?Psychiatry?9，953-61.

[11]Horwitz，J.(2003).Alpha-crystallin.Exp?Eye?Res?76，145-53.

[12]Link，C.D.，Taft，A.，Kapulkin，V.，Duke，K.，Kim，S.，Fei，Q.，Wood，D.E.and?Sahagan，B.G.(2003).Gene?expression?analysis?in?a?transgenic?Caenorhabditis?elegans?Alzheimer′sdisease?model.Neurobiol?Aging?24，397-413.

[13]Ghosh，J.G.，Houck，S.A.and?Clark，J.I.(2007).Interactive?sequences?in?the?stress?proteinand?molecular?chaperone?human?alphaB?crystallin?recognize?and?modulate?the?assembly?offilaments.Int?J?Biochem?Cell?Biol?39，1804-15.

Summary of the invention

The objective of the invention is to overcome defective of the prior art, a kind of new A β small peptide polymerization retarder and application thereof are provided.

The present invention at first discloses a kind of A β small peptide polymerization retarder; Be polypeptide or the said polypeptide product after prolonging polypeptide transformation period compound-modified of A Leu B Pro Phe C Leu for aminoacid sequence, wherein, A is Ser or Thr; B is Ser or Thr, and C is Tyr, Ser or Thr.

Further preferred, the aminoacid sequence of said A β small peptide polymerization retarder is Ser Leu Ser Pro Phe TyrLeu (SEQ ID NO:1).

Further, the said prolongation polypeptide transformation period is compound-modified in the carboxyl terminal of said aminoacid sequence.

The compound of said said prolongation polypeptide transformation period of prolongation polypeptide can be PEG.

The structural formula of said PEG is: HO (CH ₂CH ₂O) _nH, n are 4-40.

Preferably, said A β small peptide polymerization retarder meets structural formula (I):

The present invention also further discloses the preparation method of said A β small peptide polymerization retarder, comprises the following steps:

1) polypeptide peptide section is synthetic:

Adopt the synthetic polypeptide peptide section of standard solid state polypeptide synthesis;

2) with polyethyleneglycol modified polypeptide carboxyl terminal:

Specifically can comprise the following steps:

A). PEG (being dissolved in pyridine) is added BOC ₂Among the O, stirred 4-12 hour,, and use SiO the reaction mixture evaporation ₂Column purification is collected component and evaporation to obtain product P EG-BOC.

B). in the polypeptide (being dissolved in THF (THF)) of 0 ℃ of left and right sides N-end protection, add PPh3 (triphenylphosphine molecule); Stir after 5-20 minute; Adding PEG-BOC and DEAD (diethyl azodiformate) or DMAP (4-Dimethylamino pyridine) also at room temperature stirred 4-12 hour; Remove by filter OPPh3, evaporation is also crossed SiO ₂Post carries out purifying, collects component and evaporation to obtain product peptide-PEG-BOC.

C) .peptide-PEG-BOC (is dissolved in CH ₂Cl ₂) add TFA (trifluoroacetic acid), stir after 30 minutes, with dilution with toluene and evaporative crystallization or use SiO ₂Column purification obtains end product.

3) purifying and aftertreatment:

A β small peptide polymerization retarder after modification carbon 18 reverse chromatography columns capable of using are through the high pressure liquid chromatography (HPLC) purifying.

A β small peptide polymerization retarder of the present invention can be used for preparing the medicine of prevention or treatment nerve degenerative diseases.

The present invention further discloses the pharmaceutical composition of a kind of prevention or treatment nerve degenerative diseases, contain the A β small peptide polymerization retarder of the present invention of effective therapeutic dose.

A β small peptide polymerization retarder of the present invention can use through any known mode compounding pharmaceutical compsn in this area.This compsn comprises activeconstituents, adds one or more medicine acceptable carriers, thinner, weighting agent, wedding agent and other vehicle, the dosage form that this depends on administering mode and is designed.Inorganic or the organic carrier of the known treatment inert of this area branch art personnel includes, but is not limited to lactose, W-Gum or derivatives thereof, talcum, vegetables oil, wax, fat, polyol for example polyoxyethylene glycol, water, sucrose, ethanol, glycerine; Like that, various sanitass, lubricant, dispersion agent, correctives.Preserve moisture cut to pieces, inhibitor, sweeting agent, tinting material, stablizer, salt, damping fluid is like that also can add wherein; These materials are used to help the stability of filling a prescription as required or help to improve activity or its biological effectiveness or acceptable mouthfeel of generation or smell under oral situation; In this compsn, can use the form of its original chemical of suppressor factor itself; Or randomly use the form of the acceptable salt of its pharmacology; Protein of the present invention can be individually dosed, or with various combination medicine-feedings, and with other healing potion combining form administration.So composition prepared can select any suitable mode well known by persons skilled in the art to carry out administration to suppressor factor as required.When making pharmaceutical composition, be that the A β small peptide polymerization retarder of the present invention with safe and effective amount is applied to the people, wherein oral safe and effective amount is usually at least about 100 micrograms/kg body weight.Certainly, concrete dosage is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.

A β small peptide polymerization retarder of the present invention is compared solubleness with the sedimentary micromolecular inhibitor RS-0406 of known A β and is increased substantially, and effective administration concentration of its experiment in vitro reduces (can be low to moderate 2 μ M) significantly.Simultaneously, because this compound derives from people's source protein α B, it causes immunoreactive possibility and greatly reduces.Its 4 polyoxyethylene glycol unit not monoenergetic enough increases substantially its solvability, can also improve the RT of this compound in blood, improves holding time of effective blood drug concentration.

Description of drawings

Fig. 1: the Electronic Speculum picture of the external amyloid fiber of A β 1-42

A) the A β 1-42 of 10 μ M is at 37 ℃ of fiber pictures after hatching 24 hours

B) PG-105 and A β 1-42 with 1: 1 mol ratio at 37 ℃ of product pictures after hatching 24 hours

Fig. 2: thioflavin T fluorometric analysis experimental result

A. thioflavine T (ThT) and the external amyloid fiber of A β 1-42 be 0,5,20,60 hours mixed emitting fluorescence spectrum.

The external amyloid fiber of B.A β 1-42 mixes the back 0,5 with the PG-105 of different concns, mixed back thioflavin T and in the emitting fluorescence intensity of 485nm the polymerization incubation time is mapped in 20,60 hours.The fluorescence of 485nm is used for intersecting in the quantitative analysis amyloid deposition fiber content of β laminated structure.

Laser confocal microscope photo after Figure 31 0 μ M A β 1-42 and rat nerves unit cell are hatched altogether

A: hatched 1 hour; B: hatched 2 hours; C: hatched 24 hours; D: hatched 36 hours; E: hatch 55 hours (wherein, left side figure be the neurone of GFP green fluorescence mark, and right figure is that the phase microscope under the same visual field forms images) scheme in scale unification (seeing figure E) be 20 microns

Fig. 4: the laser confocal microscope photo after 10 μ M A β 1-42 and 10 μ M PG-105 and rat nerves unit cell are hatched altogether

A: hatched 1 hour; B: hatched 2 hours; C: hatched 24 hours; D: hatched 36 hours; E: hatch 55 hours (wherein, left side figure be the neurone of GFP green fluorescence mark, and right figure is that the phase microscope under the same visual field forms images) scheme in scale unification (seeing figure E) be 20 microns

Fig. 5: the laser confocal microscope photo after 10 μ M A β 1-42 and 10 μ M PG-105 and rat nerves unit cell is hatched altogether and with synaptophysin nerve synapse is dyeed after quantitative result.

A is that neurone and 10uM A β 1-42 cultivate 12,36,72 hours fluoroscopic image altogether: left side figure is the fluorescently-labeled neurone picture of GFP, and right figure is the fluorescence picture of the nerve synapse of synaptophysin mark under the same visual field

B is that neurone and 10uM A β 1-42 and 10uM PG-105 cultivate 12,36,72 hours fluoroscopic image altogether: left side figure is the neurone image under the phase microscope, and right side figure is the fluorescence picture of the nerve synapse of synaptophysin mark under the same visual field

C is the quantitative result of the nerve synapse of synaptophysin mark among A, B two figure

Embodiment

Further set forth the present invention below in conjunction with embodiment.Should be understood that these embodiment only are used to explain the present invention, and unrestricted scope of the present invention.

The preparation of embodiment 1 PG-105

The structural formula of PG-105:

Adopt following method to prepare PG-105:

1) polypeptide peptide section is synthetic: adopt the synthetic polypeptide peptide section of standard solid state polypeptide synthesis;

2) polyethyleneglycol modified:

A). 3 to 4 times of PEG4 (being dissolved in pyridine) are added one times of BOC ₂Among the O.Stirred 4-12 hour, and, and used SiO the reaction mixture evaporation ₂Column purification.Collect component and evaporation to obtain product P EG4-BOC.

B). in the polypeptide (being dissolved in THF) of 0 ℃ of one times of N-end protection, add one times of PPh3.Stir after 5-20 minute, add one times of PEG4-BOC and 1-1.5 times of DEAD or DMAP and at room temperature stirred 4-12 hour.Remove by filter OPPh3, evaporation is also crossed SiO ₂Post carries out purifying.Collect component and evaporation to obtain product peptide-PEG4-BOC.

C) .peptide-PEG4-BOC (is dissolved in CH ₂Cl ₂) add 25% volume TFA, stir after 30 minutes, with dilution with toluene and evaporative crystallization or use SiO ₂Column purification obtains end product.

3) purifying and aftertreatment: the A β small peptide polymerization retarder after the modification utilizes carbon 18 reverse chromatography columns through the high pressure liquid chromatography (HPLC) purifying.Experiment showed, that PG-105 in 0.1% trifluoroacetic acid, still can dissolve fully when concentration reaches 20 mg/ml.

Embodiment 2 PG-105 suppress A β 1-42 fiber deposition and form test

A β 1-42: (available from Biosource company)

PG-105: embodiment 1 preparation

Method:

The preparation of 10 μ M A β 1-42: A β lyophilized powder is dissolved as the solution of 20mM with the miliQ ultrapure water, uses to aimed concn (10 μ M) with the PBS gradient dilution then.(PBS damping fluid composition: 10mM sodium phosphate buffer, 150mM sodium-chlor, pH7.4) solution calcium ions not.

PG-105 and A β 1-421: the preparation of the mixing solutions of 1 mol ratio: PG-105 with the storing solution that PBS is mixed with 1mM, is diluted with PBS during use as required.

With 10 μ M A β 1-42 and PG-105 and A β 1-421: the mixing solutions of 1 mol ratio was directly hatched 24 hours at 37 ℃ respectively; Get 5 μ l deposit sample point samples respectively in being coated with on the carbon copper mesh through 400 order formvar films after the photoglow; After 1% acetate uranium negative staining, sample is carried out to picture with JEOL1200 XII projection ESEM.The parameters such as diameter of viewed polymerization of protein fiber are directly by measuring in the Electronic Speculum picture.

The result: it is as shown in Figure 1 that the projection ESEM detects the protein precipitation form; The pencil deposition that can observe diameter 10-40 nanometer during 10 μ M A β 1-42 are hatched after 24 hours forms; The deposition fiber length in submicron to micron dimension; PG-105 and A β 1-421: do not see the pencil deposition during the mixing solutions of 1 mol ratio is hatched after 24 hours; Diameter 5-10 nanometer is only arranged, and the oligomer of length 50-300 nanometer, this presentation of results PG105 can suppress polymerization and the sedimentary formation of pencil of A β 1-42.

Embodiment 3 thioflavin T fluorometric analyses experiment

Thioflavin T: available from Sigma company

PG-105: embodiment 1 preparation

Method:

Thioflavin T combines the beta-amyloyd post precipitation can send fluorescence, therefore can be used for the detection of amyloid deposition.

Prepare the thioflavin T of 25 μ M with the 50mM sodium phosphate buffer of pH7.

The thioflavin T solution of 492 microlitres, 25 μ M is moved in the cuvette; In 37 degree record emmission spectrum backgrounds, add 8 microlitre laboratory samples, build cuvette; Mixing sample several times overturns; Use the F-4500 of Hitachi spectrophotofluorometer, the 444nm wavelength excites, and record thioflavin T is at the fluorescence emission spectrum of 470-570nm.The emmission spectrum of solution that only contains thioflavin T is as contrast, and the generation fluorescence at 485nm after the amyloid deposition sample mix that thioflavin T and A β 1-42 experiment different time points produce significantly strengthens, and is used to indicate the generation of amyloid deposition.

The PG-105 combined experiments of external amyloid fiber of A β 1-42 and different concns: with 10 μ MA β 1-42 and PG-105 and A β 1-42 with 1: 5,1: 1,2; The mixing solutions of 1 mol ratio is hatched at 37 ℃ respectively; At take a sample respectively each 8 microlitre of 0,5,20,60 hour four time point of hatching; Behind the abundant mixing of thioflavin T solution of 492 microlitres, 25 μ M; Detect the fluorescence emission spectrum of thioflavin T, thioflavin T is mapped to the time in the fluorescence intensity of 485nm at 470-570nm.The result: the result is as shown in Figure 2; Visible by figure, under unrestraint agent situation, A β 1-42 has formed the amyloid deposition that typically is rich in intersection β laminated structure; When hatching altogether with PG-105; Polymerization is suppressed, and this restraining effect strengthens with the increase of PG-105 concentration, shows that PG-105 has significantly suppressed the polymerization of A β 1-42.

Embodiment 4 neurone culture experiments

Method:

Hippocampus neuronic cultivation of former generation: get pregnant 18 days SD rat embryo, separate hippocampus with surgical forceps after getting brain, the pancreatin with 0.125% was 37 ℃ of digestion 20 minutes.Remove pancreatin, with the DMEM nutrient solution suspension cell that contains 10% foetal calf serum and 10%F-12, with 65,000cells/cm ²Density cultivate in 35 millimeters Tissue Culture Dishs of poly-lysine embedding.Cultivated 24 hours, and partly changed liquid with the Neurobasal nutrient solution that contains 2% B27 and 0.25% Stimulina.Carry out twice half afterwards weekly and change liquid, vitro culture adds the 2mM cytosine arabinoside and suppresses the spongiocyte growth after 5 days.

Utilize the liposome transfection method GFP (green fluorescent protein) to be changed over to vitro culture 5-6 days neuronal cell.Transfection is handled cell with 10uM A β 1-42 and 10uM PG-105 after 24 hours simultaneously, and control group is only used 10uMA β.Processing 24-72 as a child pair cell checks.The green fluorescence that neurone is followed the tracks of GFP in the form of different time through laser confocal microscope is realized.The situation of cynapse is observed with laser confocal microscope after utilizing the synaptophysin immunofluorescence dyeing on the neurone.The data processing of Synaptophysin dyeing back fluorescence picture utilizes Image J software to carry out.

The result:

The laser confocal microscope photo is shown in Fig. 3-5; Visible by figure, when neurone was cultivated with 10uM A β 1-42 altogether, A β 1-42 had shown tangible neurotoxicity in 37 polymerizations of spending; The visible neuron axon atrophy of result under the fluorescent microscope, visible cell quantity reduces under the phase microscope; When neurone and 10uM A β 1-42 and 10uM PG-105 cultivate altogether; Phenomenons such as axonal atrophy and cell quantity minimizing receive obvious inhibition; Among Fig. 5 the quantitative result of nerve synapse is shown that PG-105 is hatched the reduction that has suppressed the nerve synapse quantity that the neurotoxicity by A β 1-42 polymkeric substance causes altogether.

Embodiment 5 preliminary toxicity research

The cultivation of hippocampus neuronal cell of former generation: cultural method as previously mentioned.

Under the normal neurons culture condition, the PG-105 that adds 100uM (being dissolved among the PBS) continues to cultivate after 5 days, observes cell growth condition and PBS contrast and does not see difference, and this explanation PG-105 does not have neurotoxicity in this concentration range.

Claims (9)

1. A β small peptide polymerization retarder; Be polypeptide or the said polypeptide product after prolonging polypeptide transformation period compound-modified of A Leu B Pro Phe C Leu for aminoacid sequence, wherein, A is Ser or Thr; B is Ser or Thr, and C is Tyr, Ser or Thr.

2. A β small peptide polymerization retarder according to claim 1 is characterized in that the aminoacid sequence of said A β small peptide polymerization retarder is Ser Leu Ser Pro Phe Tyr Leu.

3. A β small peptide polymerization retarder according to claim 1 is characterized in that, said prolongation polypeptide transformation period compound-modified in the carboxyl terminal of said aminoacid sequence.

4. A β small peptide polymerization retarder according to claim 1 is characterized in that the compound of said prolongation polypeptide transformation period is PEG.

5. like the said A β of claim 4 small peptide polymerization retarder, it is characterized in that the structural formula of said PEG is:

HO (CH ₂CH ₂O) _nH, n are 4-40.

6. A β small peptide polymerization retarder according to claim 1 is characterized in that said A β small peptide polymerization retarder meets structural formula (I):

7. like the preparation method of the said A β of the arbitrary claim of claim 1-6 small peptide polymerization retarder, comprise the following steps:

1) polypeptide peptide section is synthetic;

2) PEG modified polypeptide carboxyl terminal;

3) purifying and aftertreatment.

8. the purposes that is used to prepare prevention or treats the medicine of nerve degenerative diseases like the said A β of the arbitrary claim of claim 1-6 small peptide polymerization retarder.

9. the pharmaceutical composition of prevention or treatment nerve degenerative diseases contains the said A β of the arbitrary claim of the claim 1-6 small peptide polymerization retarder of effective therapeutic dose.

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