CN102492022B

CN102492022B - Novel anti-senile dementia lead compound

Info

Publication number: CN102492022B
Application number: CN 201110405301
Authority: CN
Inventors: 梁桂兆; 张进; 苗霞; 温文轶; 吕凤林; 郑洁
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2011-12-08
Filing date: 2011-12-08
Publication date: 2013-05-29
Anticipated expiration: 2031-12-08
Also published as: CN102492022A

Abstract

The invention discloses a novel anti-senile dementia lead compound, which is characterized in that: an amino acid sequence of the lead compound is amino terminal-VTLWWG-carboxyl terminal. A peptide sequence is designed on the basis of a quantitative structural function relation model of a peptide, is detected through an atomic force microscope scanning technology, and is further obtained with cytotoxicity experimental evaluation. The lead compound has a good inhibiting effect on the aggregation of a toxic body-Abeta which leads to senile dementia, and can be further developed into a senile dementia resisting medicament.

Description

A kind of Novel anti-senile dementia lead compound

Technical field

The present invention relates to a kind of Novel anti-senile dementia lead compound, particularly a kind of anti-senile dementia disease toxicity body A β lead compound.

Background technology

At present, the whole world has at least 3,500 ten thousand people to suffer from senile dementia, and annual lethality rate rises.Total cost in the annual whole world is estimated to reach 2,000 hundred million dollars, studies show that, (Amyloid β-peptide) oligomer is the remarkable toxicity body in the senile dementia patient body to A β, and the generation that therefore suppresses the A beta oligomers is to stop senile dementia that the most effective strategy occurs.Yet, be used for the design of A beta inhibitor without effective ways at present, main serious challenge in the face of three aspects:: 1, lack effective high-throughput screening method: the experiment screening method need to be synthesized the β with purifying A, and this screening for a large amount of compounds is time-consuming beyond doubt, expensive and unrealistic.2, the high resolution structures that lacks the A beta oligomers: the A beta oligomers is metastable state, therefore utilizes X-ray diffraction and NMR technology to be difficult to obtain its structure, makes the Rational drug design based on structure be difficult to realize.3, lack understanding to A β self-assembly mechanism: comprise peptide which partly be formed in the amyloid fiber generative process and play keying action; Seed generates with fiber what relevant path and intermediate be; Whether A β is affine to specific acceptor; What the mechanism how A β generates toxicity body and toxicity is.Therefore, design new A beta inhibitor has important practice significance to senile dementia diagnosis and treatment.

Summary of the invention

In view of this, in order addressing the above problem, to the invention provides a kind of Novel anti-senile dementia lead compound, can further to be developed as anti-senile dementia disease medicine.

The object of the present invention is achieved like this: its aminoacid sequence is: aminoterminal-VTLWWG-carboxyl terminal.

A kind of Novel anti-senile dementia lead compound of the present invention, choose one group of 6 peptide sample with gathering behavior, design has the peptide molecule of gathering behavior through peptide quantitative structure-activity relation modeling technique, detect it to the inhibitory character of A β through atomic force microscope, estimate it to Cytotoxic restraining effect by cytotoxicity experiment, Using such method has found aminoacid sequence of the present invention, that is: aminoterminal-VTLWWG-carboxyl terminal.

Synthetic aminoacid sequence method of the present invention all is existing mature technologies, and it is made as follows:

Employing standard Fmoc scheme; the initial 0.0125mmol that selects; (ABI company produces the PSC resin; lot number A5F013); according to sequence signature claimed in claim 1; peptide chain is extended to the N end one by one from the C end; each amino acid whose consumption is 0.1mmol, and each seed amino acid blocking group is: the amino Pmoc protection of each amino acid whose alpha, all the other side chain protected groups; Arg (Mtr); Tyr (tBu), Thr (tBu), Asp (OtBu); for the modification of biotinyl and stearoyl group, Fmoc-Lys (biotin)-OH and stearic acid are connected respectively to C-terminal and the N-terminal of peptide.Per step condensation all adds the amino acid whose carboxyl of HoBT/Dcc activates relay.Per step condensation is removed the Fmoc protecting group with the nmp solution that contains 20% hexahydropyridine; after the peptide side chain is synthetic; step according to the recommendation of ABI company; resiniferous peptide chain is added in the mixed reaction solution that is under the condition of ice bath; the composition of reaction solution: crystallization benzoic acid 0.75g, ethylenediamine tartrate (EDT) 0.25ml, thioanisole 0.5ml; deionized water 0.5ml, trifluoroacetic acid 10ml.Continue to stir at ambient temperature, the reaction times is 4.5 hours, and peptide chain cracking from the branch is got off, and removes simultaneously the kinds of protect group.Mixed solution is filtered through the glass filter of 4G, with resin and the protection group that filters cutting-out, and with trifluoroacetic acid flushing reaction flask and filter; with filtrate at normal temperatures low pressure be evaporated to 1-2ml, the 50ml that adds diethyl ether, make polypeptide precipitation after; after the 6G filter filters, lyophilize, gained is peptide product.Above process all is to finish in ABI-431A solid phase automatic peptide synthesizer.Institute's synthetic peptide is through the RP-HPLC purifying, and purity reaches 95%, and identifies structure through TOF-MS.

With the restraining effect of the above-mentioned institute of afm scan technical measurement synthetic peptide to A β.

Estimate above-mentioned institute synthetic peptide to the restraining effect of A β toxicity with cytotoxicity experiment.

Other advantage of the present invention, target and feature will be set forth to a certain extent in the following description, and to a certain extent, based on being apparent to those skilled in the art to investigating hereinafter, perhaps can obtain from the practice of the present invention instruction.Target of the present invention and other advantages can be passed through following specification sheets, claims, and the specifically noted structure realizes and obtains in the accompanying drawing.

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:

Fig. 1 is that new designed peptide VTLWWG is to the inhibiting afm scan result of A β;

Fig. 2 is that new designed peptide is to the cytotoxic assay result of A β.

Embodiment

Below plant the design of main toxicity body-A beta inhibitor and be accredited as example and be described in detail adopting method of the present invention to be used for the senile dementia patient, may further comprise the steps:

A) the quantitative structure-activity relation modeling of peptide;

A1) peptide structural characterization: from document (Matsubara et al., Nat Methods, 2010,7 (3): 237; Thompson et al., PNAS, 2006,103 (11): 4074) select 278 peptide samples, but wherein the aggregatory peptides sample is 117, non-aggregatory peptides sample is 162, and each peptide contains 6 amino-acid residues.516 kinds of nature parameters of selected 20 kinds of natural amino acids by the factor analysis in the multiviate statistical analysis, through oblique rotation, and are extracted 6 factors with principal component analysis, and these 6 factors have been explained the information of original variable 83.47%.

6 factors are carried out loading analysis find that each factor relates separately to hydrophobicity, alpha-helix and corner tendency, bulk property, constitutive characteristic, local compliance and electrostatic property.Further calculate each factor score, see Table 1, these 6 factor score vectors combine 516 original amino acid nature parameters most information, can use it for peptide or protein structure and characterize.Each amino-acid residue in the peptide sequence characterizes with time 6 factor scores, and for each 6 peptide sequence, then available 6 * 6=36 variablees characterize.

6 factor scores of 516 property parameters of 20 kinds of natural amino acids of table 1

^a20 kinds of natural amino acids represent with conventional single English alphabet.

A2) set up the model of cognition of aggregatory peptides with linear discriminant analysis;

Select parameter with Step wise procedure, take F value corresponding to partial F test as foundation, when the F value greater than 3.84 the time, then this variable is stayed in the model, when the corresponding F value of this variable less than 2.71 the time, then reject this variable, the predictive ability through leaving-one method validation-cross model obtains 9 variable standardization models at last, model is 75.1% to the correct interest rate of identification of aggregatory peptides, sensitivity is 0.789, and specific degree is that 0.734, Ma Xiusi relation conefficient is 0.501, the recognition correct rate of leaving-one method validation-cross is 73.8%, sensitivity is 0.765, and specific degree is that 0.732, Ma Xiusi relation conefficient is 0.564.

B) design of peptide;

According to resulting linear model, with the peptide in 278 training sets, its sequence is that aminoterminal-GTVLFM-carboxyl terminal is template, designs 1 and may have and can assemble and more highly active peptide: aminoterminal-VTLWWG-carboxyl terminal.

C) peptide is synthetic;

Synthetic peptide in ABI-431A solid phase automatic peptide synthesizer.Detailed process is as follows: adopt standard Fmoc scheme; the initial 0.0125mmol that selects; (ABI company produces the PSC resin; lot number A5F013); according to sequence signature claimed in claim 1; peptide chain is extended to the N end one by one from the C end; each amino acid whose consumption is 0.1mmol, and each seed amino acid blocking group is: the amino Pmoc protection of each amino acid whose alpha, all the other side chain protected groups; Arg (Mtr); Tyr (tBu), Thr (tBu), Asp (OtBu); for the modification of biotinyl and stearoyl group, Fmoc-Lys (biotin)-OH and stearic acid are connected respectively to C-terminal and the N-terminal of peptide.Per step condensation all adds the amino acid whose carboxyl of HoBT/Dcc activates relay.Per step condensation is removed the Fmoc protecting group with the nmp solution that contains 20% hexahydropyridine; after the peptide side chain is synthetic; step according to the recommendation of ABI company; with resiniferous peptide chain add be under the condition of ice bath mixed reaction solution in; the composition of reaction solution: crystallization benzoic acid 0.75g, ethylenediamine tartrate (EDT) 0.25ml, thioanisole 0.5ml; deionized water 0.5ml, trifluoroacetic acid 10ml.Continue to stir at ambient temperature, the reaction times is 4.5 hours, and peptide chain cracking from the branch is got off, and removes simultaneously the kinds of protect group.Mixed solution is filtered through the glass filter of 4G, with resin and the protection group that filters cutting-out, and with trifluoroacetic acid flushing reaction flask and filter; with filtrate at normal temperatures low pressure be evaporated to 1-2ml, the 50ml that adds diethyl ether, make polypeptide precipitation after; after the 6G filter filters, lyophilize, gained is peptide product.Institute's synthetic peptide is through the RP-HPLC purifying, and purity reaches 95%, and identifies structure through TOF-MS.

D) peptide is to the inhibiting afm scan experiment of A β;

With single beam silicon cantilever probe, under the pattern of rapping (Tapping Mode) pattern, measure, scan at least 4 zones and correctly sample to guarantee structure.Fig. 1 be new designed peptide VTLWWG to the inhibiting afm scan result of A β, can find out that through 2 days, VTLWWG had obvious restraining effect to A β.This peptide can be used as A beta peptide aggregation inhibitor, and its sequence is: aminoterminal-VTLWWG-carboxyl terminal.

The new designed peptide VTLWWG of Fig. 1 to the inhibiting afm scan result of A β (the Tapping pattern, A is control experiment (unrestraint agent), the concentration of A β is 1 μ m, the concentration of A β is 20 μ M among the B, the concentration of six peptides is 50 μ M, deposits 37 ℃ 2 days)

E) cell toxicity test;

With Polyanionic dye calcein viable cell is carried out the green fluorescence mark, and measure its activity, ethidium-1 dyeing is carried out the red fluorescence mark to dead cell.With respect to the cell inactivation that is caused by A β, behind adding VTLWWG and freshly prepd A β and the cell co-culture, corresponding death is respectively 0.356 with becoming living cell rate, can reduce significantly apoptosis with respect to the control experiment that does not add peptide.When peptide with deposit 24 hours A β solution with after cell mixes, obtain after testing dead and the viable cell ratio is 0.877.Fig. 2 is that new designed peptide is to the toxicity inhibition result of A β.

The new designed peptide of Fig. 2 is to the cytotoxic assay result of A β (green expression viable cell, red expression dead cell, (A) without A β and peptide, (B) the A β of 20 μ M, (C) VTLWWG of the A β of 20 μ M and 50 μ M)

The above is the preferred embodiments of the present invention only, is not limited to the present invention, and obviously, those skilled in the art can carry out various changes and modification and not break away from the spirit and scope of the present invention the present invention.Like this, if of the present invention these are revised and modification belongs within the scope of claim of the present invention and equivalent technologies thereof, then the present invention also is intended to comprise these changes and modification interior.

Claims

1. an anti-senile dementia disease lead compound is characterized in that its aminoacid sequence is: aminoterminal-VTLWWG-carboxyl terminal.