CN100999546B - Polypeptide compound with aza-containing heterocyclic modification and its application - Google Patents
Polypeptide compound with aza-containing heterocyclic modification and its application Download PDFInfo
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- CN100999546B CN100999546B CN2007100626284A CN200710062628A CN100999546B CN 100999546 B CN100999546 B CN 100999546B CN 2007100626284 A CN2007100626284 A CN 2007100626284A CN 200710062628 A CN200710062628 A CN 200710062628A CN 100999546 B CN100999546 B CN 100999546B
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Abstract
The present invention discloses one kind of nitrogen heterocycle modified polypeptide compound and its application in preparing medicine for preventing and treating Alzheimerdis, Parkinson's disease and other degenerative nerve system diseases and medicine for preventing and treating type II diabetes. Research shows that the nitrogen heterocycle modified polypeptide compound can inhibit Abeta aggregation, disaggregate Abeta, hydrolyze to eliminate Abeta protein and inhibit and eliminate Abeta toxicity, and possesses wide application foreground in pharmaceutical field.
Description
Technical field
The present invention relates to polypeptide compounds and application thereof, particularly relate to and have nitrogen heterocyclic ring modified polypeptides compounds and the application in nervous system degenerative disease medicines such as preparation treatment and prevention alzheimer's disease, Parkinson's disease, type-II diabetes thereof.
Background technology
Nervous system degenerative disease such as alzheimer's disease, Parkinson's disease and type-II diabetes are the protein conformation disease.Wherein, and alzheimer's disease (Alzheimer ' s disease, AD), claiming presenile dementia again, is a kind of central nervous system degenerative disease, and clinical manifestation is that cognitive disorder, memory are faded, final forfeiture elaborative faculty, dyskinesia be can't take care of oneself.Pathological characters mainly be senile plaque (senile plaque, SP), neurofibrillary tangle (neurofibrillary tangles, NFT) and selective neuronal and cynapse lose.Along with the aging of global population, alzheimer's disease has become human dead major reason.For many years, the main medicine for the treatment of this disease clinically is an anticholinesterase, and it acts on cholinergic nerve system, and illness is had mitigation, but can not stop the generation and the development of disease.At present, pathogenesis about alzheimer's disease is not fully aware of, but generally believe that crucial pathogenesis is excessive beta-amyloid polypeptide 1-(β-amyloid peptide, A β) make a mistake folding formed aggregate (main component of SP) cell membrane, cynapse and axon produces detrimental effect, and causes neurofibrillary tangle.Therefore, stop the gathering of A β or make A β degraded, just can suppress and the toxicity of removing A β, thereby reach treatment and the purpose of preventing alzheimer's disease.
Summary of the invention
It is inhibited to the purpose of this invention is to provide a kind of gathering to A β, simultaneously A beta peptide aggregation body is had unzipping and the removing A β that finally can degrade have a nitrogen heterocyclic ring modified polypeptides compounds.
Polypeptide compounds provided by the present invention has the general structure of formula I:
Wherein, R is any nitrogen heterocyclic ring alkane amino or the non-naphthenic hydrocarbon amino that contains 4 or 4 above nitrogen-atoms; N=1-5; X is for natural, alpha-non-natural amino acid residue arbitrarily or have the amino-acid residue of modification group accordingly, m=2-10; The C-terminal of described polypeptide compounds be carboxyl (-COOH) or amide group (-CONH
2) or for the hydroxyl after the reduction (-OH), amino (-NH
2) or alkyl.
R among the described formula I is preferably Cyclen (N connect-1,4,7,10-tetraazacyclododecanand) or Trpn (3,3 ', 3 "-triamino tripropylamine); N is preferably 1; X is preferably one of 20 kinds of natural amino acid residues or corresponding amido modified thing (be preferably and modify through methyl), and m is preferably 3-5 (more preferably 5).Formula II seen in the chemical structural formula of described Cyclen:
Formula III seen in the chemical structural formula of described Trpn:
X is lysine residue, leucine residue, Xie Ansuan residue, phenylalanine residue, tyrosine residues or aspartic acid residue more preferably.
X especially is preferably leucine residue, Xie Ansuan residue, aspartic acid residue or phenylalanine residue.
The amino-acid residue that constitutes described polypeptide compounds can be D type (dextrorotation), and L type (left-handed) conformation is preferably L type conformation.
The chemical synthesis process of available routine synthesizes polypeptide compounds of the present invention, as solid-phase synthesis, comprises uncle's fourth oxygen (acyl) carbonyl (t-Boc) chemical method and 9-fluorenylmethyloxycarbonyl (Fmoc) chemical method, is preferably the 9-fluorenylmethyloxycarbonyl chemical method.
The mixture that aforementioned polypeptides compounds and transition metal copper, cobalt, nickel, palladium, zinc or iron form also belongs to protection scope of the present invention.
Another object of the present invention provides a kind of medicine that is used for the treatment of and prevents nervous system degenerative diseases such as alzheimer's disease, Parkinson's disease, type-II diabetes.
The activeconstituents of medicine provided by the present invention is the aforementioned polypeptides compounds.
In addition, described polypeptide compounds also can be the form existence of pharmacy acceptable salt.Pharmacy acceptable salt comprises by organic or inorganic alkali, the salt that organic or inorganic acid obtains.The salt that is obtained by mineral alkali comprises aluminium salt, ammonium salt, calcium salt, mantoquita, molysite, ferrous salt, lithium salts, magnesium salts, manganese salt, manganous salt, sylvite, sodium salt and zinc salt etc., and effect is ammonium salt, sylvite, calcium salt, lithium salts, magnesium salts and sodium salt preferably; Acceptable nontoxic organic alkali salt comprises primary amine salt, secondary amine salt and tertiary ammonium salt on the pharmaceutics.Comprise hydrochloric acid, phosphoric acid, acetate, oxalic acid and tartrate etc. with the acid of polypeptide compounds reaction of the present invention.
When needing; in said medicine, can also add one or more pharmaceutically acceptable carriers, comprise thinner, vehicle, protective material, absorption enhancer, weighting agent, tamanori, wetting agent, disintegrating agent and the tensio-active agent etc. of pharmaceutical field routine.Described vehicle can be carboxymethyl starch, gelatin, Mierocrystalline cellulose, sodium bicarbonate, sodium-chlor or polyoxyethylene glycol; Described protective material can be EDTA or benzalkonium; Described short absorption agent can be Tween-80, azone, carboxymethyl cellulose or 9-lauryl ether etc.
Medicine of the present invention can also be made multiple medicament forms such as tablet, injection, solution, granule except that making capsule.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The oral consumption of the adult of this medicine is generally 0.05-50mg/kg, can use by one or many, and be 1 to 6 months the course of treatment.
The invention provides a kind of nitrogen heterocyclic ring modified polypeptides compounds that has.Studies show that this polypeptide compounds can not only suppress the gathering of A β, can also make accumulative A beta peptide aggregation body depolymerization, and finally A β is removed in hydrolysis, suppress and the toxicity of removing A β generation.With this polypeptide compounds is that the medicine of activeconstituents can be used for treatment and nervous system degenerative disease and type-II diabetes such as prevention alzheimer's disease, Parkinson's disease.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 detects polypeptide compounds of the present invention to the inhibiting result of A beta peptide aggregation for the ThT fluorescent method
Fig. 2 is the detected result of polypeptide compounds of the present invention to the unzipping of A beta peptide aggregation body
Fig. 3 is that polypeptide compounds is to the inhibiting electron microscopic observation result of A beta peptide aggregation
Fig. 4 is the electron microscopic observation result of polypeptide compounds to the unzipping of A beta peptide aggregation body
Fig. 5 is the MALDI-TOF-MS analytical results of polypeptide compounds to the hydrolysis scavenging(action) of A β
Fig. 6 is the A β generation H of polypeptide compounds to the copper mediation
2O
2Inhibiting analytical results
Fig. 7 passes through to suppress the analytical results of A β toxicity to the neuronic provide protection of rat hippocampus toe for polypeptide compounds
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The preparation of embodiment 1, polypeptide compounds
Synthetic polypeptide compounds of the present invention (molecular formula is seen formula IV) may further comprise the steps:
A. the synthetic monomer [4 that contains Cyclen, 7,10-three tertbutyloxycarbonyls-1,4,7,10-tetraazacyclododecane ten Second Academys-1-yl] acetate, method is: with Cyclen (available from U.S. Acros company) is raw material, earlier with Boc20 (tert-Butyl dicarbonate, available from Shanghai gill biochemical corp) reaction obtain 1,4,7-three tertbutyloxycarbonyls-1,4,7,10-tetraazacyclododecane ten Second Academys (3Boc-Cyclen) are (referring to Eiichi Kimura, Shin Aoki, Tohru Koike, and Motoo Shiro, A Tris (ZnII-1,4,7,10-tetraazacyclododecane) Complex as a New Receptor forPhosphate Dianions in Aqueous Solution, Journal of American Chemical Societv.1997, Vol 119,3068-3076), again product and ethyl bromoacetate are reacted (referring to Joong Won Jeon, Sang Jun Son, Chang Eun Yoo, In Seok Hong, Jung Bae Song, andJunghun Suh, Protein-Cleaving Catalyst Selective for ProteinSubstrate, Organic Letters, 2002, Vol 4,4155-4158) obtain 1,4,7-three tertbutyloxycarbonyls-10-(2-oxyethyl group-2-oxygen ethyl)-1,4,7,10-tetraazacyclododecane ten Second Academys, use the NaOH hydrolysis (referring to Joong Won Jeon at last, Sang Jun Son, Chang Eun Yoo, In Seok Hong, Jung Bae Song, and Junghun Suh, Protein-CleavingCatalyst Select ive for ProteinSubstrate, Organic Letters, 2002, Vol4 4155-4158) obtains containing the monomer [4,7 of Cyclen, 10-three tertbutyloxycarbonyls-1,4,7,10-tetraazacyclododecane ten Second Academys-1-yl] acetate.
B. amino acid (phenylalanine, phenylalanine, Xie Ansuan, leucine, Methionin) being connected Wang resin (available from Shanghai gill biochemical corp) one by one by Fmoc solid phase synthesis strategy (in general goes up, the C terminal of compound be carboxyl (-COOH) adopt the Wang resin, the C terminal is acid amides (-CONH
2) employing Rink resin).
The monomer [4 that c. will contain Cyclen, 7,10-three tertbutyloxycarbonyls-1,4,7,10-tetraazacyclododecane ten Second Academys-1-yl] acetate set by step among the b amino acid whose method of attachment link to each other with the little peptide that contains the Wang resin that step b obtains, condition of contact is that coupler HBTU (available from long-livingization of Shanghai Yan company) exists to descend (blending ratio: the coupler HBTU of the little peptide that contains the Wang resin of the monomer that contains Cyclen of 4 times of amounts, 1 times of amount, 4 times of amounts and the HOBt of 4 times of amounts) room temperature reaction to obtain the purpose product in 3 hours with HOBt (available from Shanghai gill biochemical corp).
D. used cutting reagent trifluoroacetic acid (TFA in synthesizing with polypeptide; purchase in Shanghai gill biochemical corp) step c is reacted polypeptide portion and resin cutting disconnection in the resin that is connected with polypeptide that obtains, remove blocking group simultaneously, filter; remove TFA, obtain the polypeptide compounds crude product.
E. with the polypeptide compounds crude product through HPLC method (chromatographic column: C18 filled column (available from U.S. Agilent company); Moving phase: acetonitrile/water mixed system; Flow velocity: 2.8mL/min; Detect wavelength: 215nm; Column temperature: room temperature) separate the pure product of polypeptide compounds of the present invention that obtain.
After testing, correct with the polypeptide compounds structure of method for preparing, molecular formula is seen formula IV, and purity reaches more than 95%.
The preparation of embodiment 2, polypeptide compounds
Synthetic polypeptide compounds of the present invention (molecular formula is seen formula V) may further comprise the steps:
A. use the method identical that amino acid (phenylalanine, phenylalanine, Xie Ansuan, leucine, Methionin, glycine) is connected on the Wang resin one by one with embodiment 1.
The monomer [4 that b. will contain Cyclen, 7,10-three tertbutyloxycarbonyls-1,4,7,10-tetraazacyclododecane ten Second Academys-1-yl] the little peptide that contains the Wang resin that obtains of acetate and step a links to each other, and condition of contact is that coupler HBTU obtained the purpose product in 3 hours with HOBt existence time (blending ratio: the coupler HBTU of the little peptide that contains the Wang resin of the monomer that contains Cyclen of 4 times of amounts, 1 times of amount, 4 times of amounts and the HOBt of 4 times of amounts) room temperature reaction.
C. used cutting reagent trifluoroacetic acid (TFA) reacts step b in the resin that is connected with polypeptide that obtains in synthesizing with polypeptide polypeptide portion and resin cutting disconnect, and remove blocking group simultaneously, obtain the polypeptide compounds crude product.
D. the polypeptide compounds crude product is separated the pure product of polypeptide compounds that obtain through HPLC method (separation condition is with embodiment 1).
After testing, correct with the polypeptide compounds structure of method for preparing, molecular formula is seen formula V, and purity reaches more than 95%.
The preparation of embodiment 3, polypeptide compounds
Synthetic polypeptide compounds of the present invention (molecular formula is seen formula VI) may further comprise the steps:
A. amino acid (aspartic acid, phenylalanine, Xie Ansuan, leucine) is connected on the Wang resin one by one.
The monomer [4 that b. will contain Cyclen, 7,10-three tertbutyloxycarbonyls-1,4,7,10-tetraazacyclododecane ten Second Academys-1-yl] the little peptide that contains the Wang resin that obtains of acetate and step a links to each other, and condition of contact is that coupler HBTU obtained the purpose product in 3 hours with HOBt existence time (blending ratio: the coupler HBTU of the little peptide that contains the Wang resin of the monomer that contains Cyclen of 4 times of amounts, 1 times of amount, 4 times of amounts and the HOBt of 4 times of amounts) room temperature reaction.
C. used cutting reagent trifluoroacetic acid (TFA) reacts step b polypeptide portion and the resin isolation in the resin that is connected with polypeptide that obtains in synthesizing with polypeptide, removes blocking group simultaneously, obtains the polypeptide compounds crude product.
D. crude product is separated the pure product of polypeptide compounds that obtain through HPLC method (separation condition is with embodiment 1).
After testing, correct with the polypeptide compounds structure of method for preparing, molecular formula is seen formula VI, and purity reaches more than 95%.
The preparation of embodiment 4, polypeptide compounds
Synthetic polypeptide compounds of the present invention (molecular formula is seen formula VII, and molecular formula please be provided) may further comprise the steps:
A. the monomer [3 (3 '; 3 that contains Trpn "-two tertbutyloxycarbonyls-3 '; 3 "-diamino) amino propyl amino] preparation of acetate: synthetic method is: with Trpn (available from U.S. Acros company) is raw material, earlier with Boc20 (tert-Butyl dicarbonate, available from Shanghai gill biochemical corp) reaction obtain 3 '; 3 "-two tertbutyloxycarbonyls-3,3 '; 3 "-triamino propyl group amine (2Boc-Trpn) is (referring to Eiichi Kimura, Shin Aoki, Tohru Koike, and Motoo Shiro, ATris (ZnII-1,4,7,10-tetraazacyclododecane) Complex as a New Receptor forPhosphate Dianions in Aqueous Solution, Journal of American Chemical Society, 1997, Vol 119,3068-3076), again product and ethyl bromoacetate are reacted (referring to Joong Won Jeon, Sang Jun Son, Chang Eun Yoo, In Seok Hong, Jung Bae Song, and Junghun Suh, Protein-Cleaving Catalyst Selective for ProteinSubstrate, Organic Letters, 2002, Vol 4,4155-4158) obtain [3 (3 ', 3 "-two tertbutyloxycarbonyls-3 '; 3 "-diamino) amino propyl amino] ethyl acetate, use at last the NaOH hydrolysis (referring to Joong Won Jeon, Sang Jun Son, Chang Eun Yoo, In Seok Hong, Jung Bae Song, and Junghun Suh, Protein-Cleaving CatalystSelective for ProteinSubstrate, Organic Letters, 2002, Vol 4,4155-4158) obtain containing the monomer [3 (3 '; 3 of Trpn "-two tertbutyloxycarbonyls-3 ', 3 "-diamino) amino propyl amino] acetate.
B. use the method identical that amino acid (phenylalanine, phenylalanine, Xie Ansuan, leucine, Methionin) is connected on the Wang resin one by one with embodiment 1.
The monomer [3 (3 '; 3 that c. will contain Trpn "-two tertbutyloxycarbonyls-3 '; 3 "-diamino) amino propyl amino] the little peptide that contains the Wang resin that obtains of acetate and step a links to each other, and condition of contact is that coupler HBTU obtained the purpose product in 3 hours with HOBt existence time (blending ratio: the coupler HBTU of the little peptide that contains the Wang resin of the monomer that contains Cyclen of 4 times of amounts, 1 times of amount, 4 times of amounts and the HOBt of 4 times of amounts) room temperature reaction.
D. used cutting reagent trifluoroacetic acid (TFA) reacts step c in the resin that is connected with polypeptide that obtains in synthesizing with polypeptide polypeptide portion and resin cutting disconnect, and remove blocking group simultaneously, obtain the polypeptide compounds crude product.
E. the polypeptide compounds crude product is separated the pure product of polypeptide compounds that obtain through HPLC method (separation condition is with embodiment 1).
After testing, correct with the polypeptide compounds structure of method for preparing, molecular formula is seen formula VII, and purity reaches more than 95%.
Embodiment 5, ThT fluorescent method detect polypeptide compounds of the present invention to the restraining effect of A beta peptide aggregation and unzipping to A beta peptide aggregation body
A β is gathered into fiber in external meeting under nearly physiological condition, fiber can with sulfo-yellow pigment T (ThioflavinT, ThT) combination, at excitation wavelength (excitation) 440nm, emission wavelength (emission) 485nm place shows specificity fluorescent, fluorescence intensity can go out the quantity of fiber by quantitative response, thereby can realize the aggregation extent of A β is carried out quantitative assay.Now detect polypeptide compounds of the present invention to the restraining effect of A beta peptide aggregation and to the unzipping of A beta peptide aggregation body with the ThT fluorescent method, experimental technique is as follows:
Preparation A β 42 storage liquid: in A β 42 (available from U.S. polypeptide company), add hexafluoroisopropanol (HFIP, available from U.S. Acros company), making peptide concentration is 1mg/mL, jog is 12 hours under the room temperature, dry up with nitrogen, add 200 μ M NaOH solution, finally be mixed with 100 μ M A β, 42 storage liquid, place-80 ℃ of refrigerators to preserve.
Prepare polypeptide compounds of the present invention storage liquid: with embodiment 1,2,3,4 synthetic polypeptide compounds respectively with the PBS (Na of pH 7.4
2HPO
412H
2O 3.73g, KH
2PO
40.43g NaCl 7.2g adds water to 1000mL, pH 7.4) be hybridly prepared into the storage liquid of 1.92mM polypeptide compounds, place-80 ℃ of refrigerators to preserve.
, detect the restraining effect of The compounds of this invention to the A beta peptide aggregation
Get A β 42 storage liquid, aforementioned polypeptides compounds storage liquid and cupric chloride are mixed with and contain 20uM A β 42,0 to 160uM aforementioned polypeptides compounds and 0.4 to 128 μ M Cu
2+Solution, transferring pH is 7.4,37 ℃ of following incubations 3 days, is contrast with the A β 42 storage liquid of incubation under the same conditions.The ThT measuring method is: get ThT (pH 7.4) solution that incubation sample liquid 20ul places 700ul 10uM, mix, measure at excitation wavelength (excitation) 440nm the fluorescent absorption at emission wavelength (emission) 485nm place.
Detected result as shown in Figure 1, with the blank sample aggregation extent of A β 42 be 100%, the gathering of A β is significantly suppressed (seeing curve I (polypeptide compound of embodiment 1 preparation), II (polypeptide compounds of embodiment 2 preparations), III (polypeptide compounds of embodiment 3 preparations), IV (polypeptide compounds of embodiment 4 preparations) among Fig. 1) behind adding embodiment 1,2,3, the 4 synthetic polypeptide compounds of the present invention, proves that polypeptide compounds of the present invention can significantly suppress the gathering of A β.
Two, detection compound is to the unzipping of A beta peptide aggregation body
Get A β 42 storage liquid, it is diluted to the solution that contains 20uM A β 42, transferring pH is 7.4, then at 37 ℃ of following incubations.After 2 days, the incubation sample is carried out ThT fluorescence and Electronic Speculum (TEM) detection, find that A β 42 assembles, the polypeptide compounds that adds embodiment 1 is then store liquid and cupric chloride and is mixed with and contains 20,40,80 μ M embodiment, 1,2,3,4 synthetic polypeptide compounds and 16,32,64 μ M Cu respectively
2+Solution (pH 7.4), under 37 ℃, continue to be incubated to 10 days, use the method identical to measure the ThT fluorescent absorption of A β 42 solution simultaneously, continuous detecting to 10 day with step 1.
Wherein, the detected result of the polypeptide compounds of embodiment 1 preparation of different concns such as the curve II among Fig. 2 (20 μ M polypeptide compounds), curve III (40 μ M polypeptide compounds), curve IV (80 μ M polypeptide compounds) show that polypeptide compounds of the present invention has good unzipping to accumulative A β 42 aggregates (curve I) take place.In addition, embodiment 2,3, the 4 synthetic polypeptide compounds of different concns also also have good unzipping to accumulative A β 42 aggregates take place, prove that polypeptide compounds of the present invention also has good unzipping to accumulative A β 42 aggregates take place.
A beta peptide aggregation body can be observed fiber form significantly under Electronic Speculum, now use the metamorphosis of Electronic Speculum direct viewing A β 42 samples on copper mesh, to the restraining effect of A beta peptide aggregation and unzipping to A beta peptide aggregation body, detection method is as follows with further detection polypeptide compounds of the present invention:
Polypeptide compounds storage liquid: the PBS (Na that embodiment 1,2,3,4 synthetic polypeptide compounds is added pH 7.4 respectively
2HPO
412H
2O 3.73g, KH
2PO
4O.43g, NaC1 7.2g adds water to 1000mL) in be mixed with the storage liquid of 1.92mM, place-80 ℃ of refrigerators to preserve.
One, the electron microscopic observation compound is to the restraining effect of A beta peptide aggregation
Get A β 42 storage liquid (prescription is seen embodiment 5), polypeptide compounds storage liquid, be mixed with cupric chloride respectively and contain 20 μ M A β 42,40 μ M polypeptide compounds and 32 μ M Cu
2+Solution, transferring pH is 7.4, as sample liquid, is contrast with the A β 42 storage liquid of incubation under the same conditions 37 ℃ of following incubations 7 days; Sample thief liquid 8 μ l place on the copper mesh that is coated with carbon film then, leave standstill 1 minute, inhale with filter paper and remove unnecessary liquid, wash copper mesh twice with water, treat that copper mesh is little dried, drips the dyeing of 10 μ l1-2% phospho-wolframic acids, left standstill 1 minute, and inhaled with filter paper and remove liquid, copper mesh was placed the dry 12-24 of moisture eliminator hour; Copper mesh with load sample places under the Electronic Speculum at last, at voltage 100kV, and observation sample form under magnification 50000 conditions.
Wherein, the electron microscopy observation of polypeptide compounds incubation after 3 days of A β 42 and embodiment 1 preparation the results are shown in Figure 3 (scale: the B figure 100nm), (the A figure among Fig. 3) compared with the control, this polypeptide compounds has significantly suppressed the gathering of A β.In addition, embodiment 2,3,4 synthetic polypeptide compounds also can significantly suppress the gathering of A β, show that polypeptide compounds of the present invention can significantly suppress the gathering of A β.
Two, the electron microscopic observation compound is to the unzipping of A beta peptide aggregation body
Get A β 42 storage liquid, it is diluted to the solution that contains 20 μ MA β 42, transferring pH is 7.4, then at 37 ℃ of following incubations.After 2 days, the incubation sample is carried out ThT fluorescence and Electronic Speculum detect, find that A β 42 assembles, add polypeptide compounds then respectively and store liquid and cupric chloride and be mixed with and contain 40 μ M polypeptide compounds and 32 μ M Cu
2+Solution (pH 7.4), down continued incubations 5 days at 37 ℃, carry out electron microscopic observation, be contrast with the A β 42 of incubation under the same conditions.
Wherein, A β 42 incubations the results are shown in Figure 4 (scales: the B figure 100nm) with the electron microscopy observation of polypeptide compounds incubation after 5 days of embodiment 1 preparation after 2 days again, contrast is the figure of the A among Fig. 4, show that this polypeptide compounds has good unzipping to accumulative A beta peptide aggregation body takes place, fundamental change has taken place than the form of A beta peptide aggregation body in the sample that polypeptide compounds plays a role on form.In addition, embodiment 2,3,4 synthetic polypeptide compounds also have good unzipping to accumulative A beta peptide aggregation body takes place, show that polypeptide compounds of the present invention has good unzipping to accumulative A beta peptide aggregation body takes place.
Embodiment 7, MALDI-TOF-MS analyze the hydrolysis scavenging(action) of polypeptide compounds of the present invention to A β
A β and aggregate thereof can be hydrolyzed into small segment under the effect of polypeptide compounds of the present invention, thereby reach the effect of removing A β.Now use ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MS) to analyze the situation that A β is hydrolyzed removing, concrete grammar is as follows:
Polypeptide compounds storage liquid preparation: the PBS (Na that embodiment 1,2,3,4 synthetic polypeptide compounds is added pH 7.4 respectively
2HPO
412H
2O 3.73g, KH
2PO
4O.43g, NaCl 7.2g adds water to 1000mL) in be mixed with the storage liquid of 1.92mM, place-80 ℃ of refrigerators to preserve.
Get A β 42 storage liquid (prescription is seen embodiment 5), polypeptide compounds storage liquid and cupric chloride are mixed with and contain 20 μ MA β, 42,160 μ M polypeptide compounds and 128 μ M Cu
2+Solution, transferring pH is 7.4,37 ℃ of following incubations 5 days as sample liquid.Sample thief liquid 20 μ L place little centrifuge tube, with ZiptipC18 purification column (Eppendorf company) desalination, analyze with carrying out MALDI-TOF-MS behind the 80% acetonitrile wash-out again.
Wherein, embodiment 2 synthetic polypeptide compounds are seen figure B among Fig. 5 to the MALDI-TOF-MS analytical results of A β hydrolysis scavenging(action), the molecular ion peak of A β 42 disappears in the sample of embodiment 2 synthetic polypeptide compounds effects after 5 days, the small pieces molecular ion peak occurs and strengthens, in addition, through embodiment 1,3, the molecular ion peak of A β 42 also disappears in the sample of 4 synthetic polypeptide compounds effects after 5 days, the small pieces molecular ion peak occurs and strengthens, and above-mentioned detected result shows that polypeptide compounds of the present invention has played the effect that hydrolysis is removed to A β really.There is not the A β 42 of polypeptide compounds that tangible molion cutting edge of a knife or a sword (seeing the figure A among Fig. 5) is then arranged in the control group.
A β can generate hydrogen peroxide (H under the mediation that copper is arranged
2O
2).Three-(2-carboxy ethyl) phosphonium salt hydrochlorates (tris (2-carboxyethyl) phosphine hydrochloride, TCEP, available from U.S. Alfa Aesar company) can with the hydrogen peroxide quantitative reaction, unreacted TCEP can with probe 5,5 '-two sulphur connection-(2-nitrobenzoic acids) (5,5 '-dithiobis (2-nitrobenzoic acid, DTNB, available from U.S. Sigma company) reaction generation 2-nitro-5-Thiosalicylic acid (2-nitro-5-thiobenzoate, NTB), NTB has UV, visible light to absorb near wavelength is the 410nm place.Therefore by detecting the concentration that uv-absorbing intensity can quantitative response goes out the hydrogen peroxide that A β produces, uv-absorbing is strong more, illustrate that DTNB and TCEP react manyly more, also just explanation is few more with the TCEP of hydroperoxidation, just illustrates that finally the hydrogen peroxide of A β generation is few more.Now detect the inhibition situation of polypeptide compounds of the present invention to A β generation hydrogen peroxide with the TCEP-DTNB method, experimental technique is as follows:
TCEP storage liquid preparation: the PBS (Na that TCEP is dissolved in pH 7.4
2HPO
412H
2O 3.73g, KH
2PO
40.43g NaCl 7.2g adds water to 1000mL) in obtain 10mM storage liquid.
DTNB storage liquid preparation: the PBS (Na that DTNB is dissolved in pH 7.4
2HPO
412H
2O 3.73g, KH
2PO
40.43g NaCl 7.2g adds water to 1000mL) in obtain 10mM storage liquid.
Gly-Cu storage liquid configuration: 10mMCuC12 and 60mMGly (available from gill biochemistry) are dissolved in the water room temperature vibration 24 hours.
Preparation contains 10 μ M A β, the embodiment 1,2,3 of 1 μ M Gly-Cu solution, 50 μ M TCEP and 50 μ M or 4 synthetic polypeptide compounds mixed solutions, at 37 ℃ of following incubation 60min, the DTNB that adds 50 μ M then, survey 412nm uv-absorbing, be contrast (catalase can decompose the hydrogen peroxide of generation, can think substantially that this system is the blank system of hydrogen peroxide) to add catalase (Catalase is available from Sigma company) mixed solution.
Detected result sees that (ordinate zou is represented the relative quantity of hydrogen peroxide to Fig. 6; X-coordinate is represented different experimental group, wherein I1 is the polypeptide compounds of embodiment 1 preparation, I2 is the polypeptide compounds of embodiment 2 preparations, I3 is the polypeptide compounds of embodiment 3 preparations, I4 is the polypeptide compounds of embodiment 4 preparations, and A β group is not for adding the control group of styrene compound of the present invention), compare with control group, show that polypeptide compounds of the present invention can significantly suppress A β catalysis and produce hydrogen peroxide, thereby weakened the toxic action of A β.
Embodiment 9MTT (Thiazolyl blue) method detects polypeptide compounds of the present invention to protecting neuron from acute
Mtt assay is a tetramethyl-azo azoles salt trace enzyme reaction colorimetry.MTT is a kind of thiazole salt, chemical name 3-(4,5-dimethyl-2-thiazole)-2, and 5-phenylbenzene bromination tetrazolium, the aqueous solution is yellowish-orange.Rat hippocampus toe neuronal cell is subjected to ConA effect back the propagation activation takes place, the corresponding rising of plastosome succinic dehydrogenase activity in its born of the same parents, MTT participates in reaction as its substrate, form blue De Jia Za (Formazan) particle deposition in cell or cell peripheral, after the DMSO dissolving is blue-violet solution, available enzyme mark determinator is measured the 0D value of cell culture, measures wavelength 570nm.Size according to the OD value is calculated cell proliferation degree in the reaction system.Now detect polypeptide compounds of the present invention to protecting neuron from acute with mtt assay, experimental technique is as follows:
The preparation of digitation of hippocamps neuronal cell: the rat neurone is referring to document (Isabella A.Graef, Paul G.Mermelstein, Kryn Stankunas, Joel R.Neilson, Karl Deisseroth, Richard W.Tsien, Gerald R.Crabtree, L-type calcium channels and GSK-3regulate theactivity of NF-ATc4in hippocampal neurons, Nature, 1999, Vol 401,703-708) the method preparation in.
Polypeptide compounds storage liquid preparation: the PBS (Na that embodiment 1,2,3 or 4 synthetic polypeptide compounds is added pH 7.4 respectively
2HPO
412H
2O 3.73g, KH
2PO
40.43g NaCl 7.2g adds water to 1000mL) in be mixed with the storage liquid of 1.92mM, place-80 ℃ of refrigerators to preserve.
Get A β 42 storage liquid (prescription is seen embodiment 5), polypeptide compounds storage liquid of the present invention and cupric chloride are mixed with and contain 40 μ MA β, 42,40 μ M polypeptide compounds storage liquid of the present invention and 32 μ M Cu
2+Solution, transferring pH is 7.4,37 ℃ of following incubations 3 days as sample liquid.The ratio of getting then in 1:4 is diluted to cell culture fluid in the neuronal cell of cultivation, and neuronal cell continues at 37 ℃, 5%CO
2Condition under cultivated two days, carry out the MTT experiment according to MTT product description (available from Sigma company) then.
Detected result sees that (ordinate zou is represented cell survival rate to Fig. 7; X-coordinate is represented different experimental group; wherein the I1 group is the polypeptide compounds of embodiment 1 preparation; the I2 group is the polypeptide compounds of embodiment 2 preparations; the I3 group is the polypeptide compounds of embodiment 3 preparations; the I4 group is the polypeptide compounds of embodiment 4 preparations; A β group is not for adding the control group of polypeptide compounds of the present invention; blank group is polypeptide compounds of the present invention and all un-added blank group of A β 42 storage liquid); compare with the control group that does not add polypeptide compounds of the present invention; polypeptide compounds of the present invention can obviously suppress the cytotoxicity of A β; thereby neurocyte is produced provide protection, make cell survival rate higher.
1) general toxicity test
Adopt cat and rat test model (available from Military Medical Science Institute), with (24 of cats, female, hero half and half, body weight 2.4-3.7kg, be divided into 4 groups, every group 6, dosage is respectively 0.05,50,120mg/kg) test, the polypeptide compounds of observing embodiment 1-5 preparation is to influences such as blood pressure, respiratory rate, hearts rate; With mouse (40, female, male half and half, body weight 18-20g is divided into 4 groups, 10 every group, dosage is respectively 0.05,30,50mg/kg) test, observe of the influence of the polypeptide compounds of embodiment 1-4 preparation to rat autonomic activities situation.Test-results shows that the polypeptide compounds of the present invention of three dosage does not all have obvious influence to blood pressure, respiratory rate and amplitude, heart rate, the rhythm of the heart of cat, and the autonomic activities number of times of rat is not had obvious influence yet.
2) acute toxicity test
Polypeptide compounds with embodiment 1 preparation is example detects polypeptide compounds of the present invention with following method a acute toxicity, concrete grammar is: adopt mouse test model (available from Military Medical Science Institute), (40 of Kunming mouses, be divided into blank group and the embodiment of the invention 1 preparation polypeptide group, every group each 20), the once maximum appetite clothes dosage 10.63g/kg that irritates, be equivalent to 2834 times of clinical plan consumption (3.75mg/kg), observed 28 days, comprise mouse body weight gradient, active situation and death condition, test-results is as shown in table 1:
Table 1 polypeptide compounds of the present invention is to the The acute toxicity tests of mouse
Test-results shows, once irritate appetite clothes maximum dosage-feeding 10.63g/kg, be equivalent to 2834 times of clinical administration amount, but acute toxic reaction does not take place all experimental animal, the rate of increase of rarely seen male mice body weight is higher than female mice, illustrates that the toxicity of polypeptide compounds of the present invention is very little.
3) long term toxicity test
Adopt the rat test model, SPF level Wistar rat is (available from Military Medical Science Institute, body weight 90-100g) is divided into three dosage groups (93.75 of polypeptide compounds of the present invention that blank group and embodiment 1-4 prepare, 187.5 and 375mg/kg), totally 13 groups, every group of 44 rats, the physiological saline that the blank group deturs talis dosis, every day gastric infusion once, serve on 6 months, observation index comprises the general physical signs and the peripheral blood phase of experimental animal, blood biochemical, liver function, the variation of main organs organ indexes such as kidney merit and histopathological examination inspection etc., test-results shows, the oral various dose (93.75 of rat, 187.5 polypeptide compounds of the present invention and 375mg/kg), be respectively 25 of clinical application amount, 50 and 100 times, successive administration 6 months does not see that every index is unusual, yet no significant difference (p〉0.05) between the various dose group, illustrate that it is safe taking polypeptide compounds of the present invention for a long time, avirulent storage effect.
Claims (4)
1. having nitrogen heterocyclic ring modified polypeptides compounds, is one of compound of following structural formula:
2. polypeptide compounds according to claim 1 is characterized in that: amino-acid residue described in the structural formula is the L configuration.
3. claim 1 or the 2 described polypeptide compounds application in the medicine of preparation inhibition A beta peptide aggregation.
4. claim 1 or the 2 described polypeptide compounds application in preparation A β depolymerizing agent.
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| CN2007100626284A CN100999546B (en) | 2007-01-11 | 2007-01-11 | Polypeptide compound with aza-containing heterocyclic modification and its application |
| PCT/CN2007/002985 WO2008083543A1 (en) | 2007-01-11 | 2007-10-18 | Nitrogen-containing heterocyclic modified compounds and uses thereof |
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| CN102816129B (en) * | 2011-06-07 | 2015-08-12 | 中国人民解放军军事医学科学院毒物药物研究所 | The benzene dicarboxylic acid derivative that isothiazoline (alkane) ketone replaces and the purposes as beta-secretase inhibitor thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1237978A (en) * | 1996-11-22 | 1999-12-08 | 伊兰药品公司 | N (aryl/heteroaryl) amino acid derivatives, pharmaceutical compositions comprising same, and method for inhibiting 'beta'-amyloid peptide release and/or its synthesis by use of such compounds |
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| CN1237978A (en) * | 1996-11-22 | 1999-12-08 | 伊兰药品公司 | N (aryl/heteroaryl) amino acid derivatives, pharmaceutical compositions comprising same, and method for inhibiting 'beta'-amyloid peptide release and/or its synthesis by use of such compounds |
Non-Patent Citations (4)
| Title |
|---|
| Michaelis K.Kalesse M.Selective Cleavage of the HIV-1 TAR-RNA With a Peptide-Cyclen-Conjugate.Angew Chem Int Ed Engl38 15.1999,38(15),2243-2245. |
| Michaelis K.Kalesse M.Selective Cleavage of the HIV-1 TAR-RNA With a Peptide-Cyclen-Conjugate.Angew Chem Int Ed Engl38 15.1999,38(15),2243-2245. * |
| 陈善勇.侧链含大环多胺的二肽的合成.大环化学和超分子化学研究进展——中国化学会全国第十二届大环第四届超分子化学学术讨论会论文集.2004,216. * |
| 陈善勇.大环多胺功能化二肽的合成与DNA裂解初探.2005,26-27. * |
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