CN100543034C - A kind of integrase inhibiting peptide and the application in the AIDS-treating medicine preparation thereof - Google Patents
A kind of integrase inhibiting peptide and the application in the AIDS-treating medicine preparation thereof Download PDFInfo
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- CN100543034C CN100543034C CNB2007100675261A CN200710067526A CN100543034C CN 100543034 C CN100543034 C CN 100543034C CN B2007100675261 A CNB2007100675261 A CN B2007100675261A CN 200710067526 A CN200710067526 A CN 200710067526A CN 100543034 C CN100543034 C CN 100543034C
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- intergrase
- organic compound
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- polypeptide
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Abstract
The invention provides a kind of integrase inhibiting peptide, be selected from a kind of in a, b, c, d, e, f, the g seven peptide species organic compound, water-soluble, link to each other by peptide bond between each amino acid, can suppress HIV-1 intergrase activity, molecular weight is respectively: 974.3,770.8,822.9,830.9,849.0,789.8,788.8.Polypeptide organic compound of the present invention can suppress HIV-1 intergrase activity, interrupts increment and the life cycle thereof of viral DNA in host cell, can use in the medicine of preparation treatment acquired immune deficiency syndrome (AIDS).Also can be used for the further research work of intergrase structure, mechanism of action and treating AIDS medicine simultaneously.
Description
Technical field
The invention belongs to organic compound, relate generally to polypeptide class organic compound, especially about the polypeptide organic compound of synthetic and the application in the medicine of preparation treatment acquired immune deficiency syndrome (AIDS) thereof.
Technical background
The process that viral DNA is integrated into host cell chromosome DNA is a retrovirus in life cycle and an important link, in this link, play a crucial role be intergrase (integrase, IN).In the past, the medicine of classical acquired immune deficiency syndrome (AIDS) (AIDS) comprising: ucleosides reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and proteinase inhibitor; But the sudden change constantly of HIV virus produces tangible resistance to this three classes medicine, and this three classes medicine of life-time service also has the generation of side effects such as anaemia, neutrophilic granulocyte minimizing, myopathy, peripheral neuropathy.Therefore, in order to overcome the problem that exists in the AIDS treatment, people begin to seek to act on the medicine of new target molecule, and the medicine that suppresses the retrovirus integration has arisen at the historic moment.Owing to do not have and intergrase homologous albumen among the human cell, so the inhibitor of this enzyme may be very little to the human body toxic, integrase inhibitor becomes the medicine that reaches the tool potentiality of treatment AIDS.In more than ten years in the past, existing all kinds of integrase inhibitors are found and synthesize.
Summary of the invention
The purpose of this invention is to provide a kind of polypeptide organic compound, can suppress I type virus of AIDS (HIV-1) intergrase activity, interrupt the increment of viral DNA in host cell.The polypeptide organic compound that provides of invention is selected from a kind of, water-soluble among seven kinds of a, b, c, d, e, f, the g, links to each other by peptide bond between each amino acid, can suppress HIV-1 intergrase activity.
The aminoacid sequence of polypeptide organic compound a provided by the invention is: Lys-Lys-Met-Lys-Arg-Arg-Lys, molecular weight are 974.3; The aminoacid sequence of polypeptide organic compound b is: Thr-Pro-Ser-His-Ser-Ser-Arg, molecular weight are 770.8; The aminoacid sequence of polypeptide organic compound c is: His-Pro-Glu-Arg-Ala-Thr-Leu, molecular weight are 822.9; The aminoacid sequence of polypeptide organic compound d is: Ser-His-Leu-Gly-Phe-Arg-Asp, molecular weight are 830.9; The aminoacid sequence of polypeptide organic compound e is: Phe-Val-Arg-Pro-Phe-Ala-Leu, molecular weight are 849.0; The aminoacid sequence of polypeptide organic compound f is: Thr-Pro-Ser-Ala-Trp-Thr-Gln, molecular weight are 789.8; The aminoacid sequence of polypeptide organic compound g is: Ser-His-Ser-Gly-Trp-Ser-Glu, molecular weight are 788.8.
Second purpose of the present invention provides polypeptide organic compound a, b, c, d, e, f, the g application in the medicine of preparation treatment acquired immune deficiency syndrome (AIDS).Also be used for intergrase mechanism of action and research work thereof.
Advantage of the present invention has:
1. polypeptide organic compound of the present invention, because molecular composition has only 7 amino acid, thus can be synthetic in a large number with artificial chemical synthesis process, can obtain a large amount of purer little peptides.
The activity that 2. can suppress the HIV-1 intergrase, thus increment and the life cycle of viral DNA in host cell interrupted, can in the medicine of preparation treatment acquired immune deficiency syndrome (AIDS), use.
3. polypeptide organic compound of the present invention can suppress viral HIV-1 intergrase and human body cell albumen not had effect substantially.
4. polypeptide organic compound of the present invention can be used for HIV-1 intergrase structure, the research of mechanism of action etc.
5. because the composition amino acid quantity of polypeptide organic compound of the present invention is less, utilized and onset by body easily after being developed to medicine.
We utilize display technique of bacteriophage, have screened the small peptide of the intergrase specific combination that can produce with genetic expression in linear seven peptide storehouses.Phage that can specific combination is checked order, and synthetic corresponding seven peptides.The synthetic peptide sequence has and suppresses combining and suppressing the active effect of HIV-1 intergrase of corresponding positive colony phage and HIV-1 intergrase.
Description of drawings
Fig. 1 is that two kinds of synthetic seven peptides are competed phage combination rate graphic representation with showing the corresponding polypeptide phage.
Fig. 2 is that two kinds of synthetic polypeptide are integrated the maximum inhibition graphic representation to the HIV-1 intergrase.
Fig. 3 is the electrophoresis result of two kinds of synthetic polypeptide to HIV-1 intergrase desintegration activity inhibition.
Embodiment
The present invention reaches accompanying drawing with the following Examples and is described further.
Embodiment 1: as target protein, obtain positive phage clones with the display technique of bacteriophage screening with recombinant expressed HIV-1 intergrase.
Display technique of bacteriophage be utilize Protocols in Molecular Biology with the random sequence oligonucleotides fragment cloning of one group of certain-length of synthetic in the particular expression carrier, make its expression product be presented on phage surface with the form of fusion rotein.Because comprised all possible sequence of amino acid of the little peptide of this length in the peptide storehouse, each phage surface albumen presents a kind of peptide section wherein, be convenient to screening; The phage that screens can be increased by bacterium.Use the phage peptide library ehec infection, the oligonucleotide fragment at random that is recombined into phage is duplicated in intestinal bacteria, and in the coat protein of phage, express.Then, target protein is coated on the enzyme plate.With after target protein mixes, wash plate with this phage peptide library.If the coat protein on the phage can combine with target protein, just can not washed off.With acid or affinity elution liquid phage is eluted at last.Sieve 3-4 takes turns so continuously, just can sift out the phage stronger with the target protein bonding force.The dna sequence dna of surveying this phage obtain the recombinating sequence of oligonucleotide has also been known the sequence of corresponding polypeptide.Can obtain polypeptide by the method for artificial chemosynthesis then.
Polypeptide organic compound provided by the invention is selected from a kind of, water-soluble among seven kinds of a, b, c, d, e, f, the g, links to each other by peptide bond between each amino acid, can suppress HIV-1 intergrase activity.
The aminoacid sequence of polypeptide organic compound a provided by the invention is: Lys-Lys-Met-Lys-Arg-Arg-Lys, molecular weight are 974.3; The aminoacid sequence of polypeptide organic compound b is: Thr-Pro-Ser-His-Ser-Ser-Arg, molecular weight are 770.8; The aminoacid sequence of polypeptide organic compound c is: His-Pro-Glu-Arg-Ala-Thr-Leu, molecular weight are 822.9; The aminoacid sequence of polypeptide organic compound d is: Ser-His-Leu-Gly-Phe-Arg-Asp, molecular weight are 830.9; The aminoacid sequence of polypeptide organic compound e is: Phe-Val-Arg-Pro-Phe-Ala-Leu, molecular weight are 849.0; The aminoacid sequence of polypeptide organic compound f is: Thr-Pro-Ser-Ala-Trp-Thr-Gln, molecular weight are 789.8; The aminoacid sequence of polypeptide organic compound g is: Ser-His-Ser-Gly-Trp-Ser-Glu, molecular weight are 788.8.
Embodiment 2: the competition of synthetic seven peptides suppresses the ELISA experiment
Test method: the intergrase bag of using the 100ul/ hole is by 96 orifice plates, and 4 ℃ are spent the night, 5%BSA37 ℃ of sealing 2h.With 0,16.25, the TPSHSSR of 32.5,65,130,260,520 μ mol/L different concns; The HPERATL of 0,15,30,60,120,240,480 μ mol/L different concns mixes with the phage of corresponding seven peptides of displaying of certain titre, combines with the intergrase competition of bag quilt.Calculate phage combination rate: combination rate %=1-(A
450-A '
450)/A
450* 100%; A wherein
450Absorbancy under the 450nm wavelength during for inhibiting not, A '
450Be absorbancy under the 450nm wavelength behind the adding inhibitor.
Test-results: along with the increase of seven peptides that add, the combination rate of phage descends gradually, and just the competition inhibiting rate of corresponding polypeptide improves, and referring to Fig. 1, along with the increase of synthetic seven peptide concentrations, shows that accordingly the combination rate of phage and intergrase descends.These two peptides of this presentation of results are relative with the affinity of intergrase better.
Embodiment 3: the inhibition intergrase of synthetic seven peptides is integrated activity experiment
Test method: at first plasmid pBluescript SK (+) is cut with 30 ℃ of enzymes of Sma I and spend the night, product carries out 1% agarose gel electrophoresis, cut glue recovery enzyme and cut product, and be dissolved in coating buffer (20mmol/L Tris-HCl pH7.4,20mmol/L EDTA, 2mol/L NaCl) in, the DNA final concentration is 2ug/ml; By 96 orifice plates, 137 ℃ of bags of every hole 100 μ are by 2h, or 4 ℃ of bags are spent the night with its bag.Donor dna be by two single stranded oligonucleotide chains of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's synthetic (UV5BR:5 '-Biotin-GTGTGGAAAATCTCTAGCAGT-3 ', UV5:5 '-ACTGCTAGAGATTTTCCACAC-3 ') annealing form.VU5BR/VU5 is mixed in the TEN damping fluid (10mmol/L Tris-HCl pH8.0,1mmol/L EDTA pH8.0,0.1mol/L NaCl) by 1:1.2, and 80 ℃ of heating 3min slowly reduce to room temperature, leave 4 ℃ of refrigerators in.It is inferior that bag is given a baby a bath on the third day after its birth with distilled water by 96 good orifice plates, 37 ℃ of sealings of 2.5%BSA 3h, and 0.1% PBST gives a baby a bath on the third day after its birth inferior.At reaction mixture (20mmol/L HEPES pH7.5,10mmol/L MnCl
230mmol/L NaCl, 10mmol/L ddT, 0.05% NP-40,100 μ g/ml BSA, 1% DMSO, the 320nmol/L intergrase) the synthetic peptide that adds 0 μ mol/L-130 μ mol/L different concns in, 37 ℃ of first incubation 20min add 500pmol/LUV5/UV5BR again, the 37 ℃ of reactions in 100 μ l/ holes 1h.0.1% PBST gives a baby a bath on the third day after its birth inferior, and every hole adds horseradish peroxidase (1:3000, the 0.1% RSA+0.025% PBST dilution) solution of 100 μ l streptavidin marks, hatches 1h for 37 ℃.0.1%PBST gives a baby a bath on the third day after its birth inferior, adds TMB 100 μ l/ holes, places 20-30min, adds 50 μ l/ hole 2mol/L H for 37 ℃
2SO
4, the 450nm wavelength detects absorbancy down.
Test-results:, add 0,4 in the reaction system of 320nmol/L containing 500pmol/L UV5/UV5BR, 16.25,32.5,65, the TPSHSSR and 0,7.5 of 130 μ mol/L different concns, 15,30, the HPERATL of 60,120 μ mol/L different concns is along with the rising of peptide concentration, the activity of intergrase descends gradually, referring to Fig. 2.Above result proves that polypeptide is inhibited to the integration activity of HIV-1 intergrase.
Embodiment 4: the active experiment of inhibition intergrase desintegration of synthetic seven peptides
Test method: intergrase has the desintegration activity for a kind of " dumbbell shape " substrate.This substrate is formed by the oligonucleotide of 38-mer (5 '-TGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-3 ') self-annealing.Elder generation is with synthetic polypeptide and 320nM intergrase pre-reaction 20min in 37 ℃ of water-baths of different concns.Reaction system is: 20mM HEPES pH7.5,10mM MnCl
2, 10mM MgCl
2, 30mMNaCl, 10mM DTT, 0.05% NP-40,100 μ g/ml BSA.Add 1 μ M dumbbell shape substrate again, 37 ℃ of water-bath 1h.Run 20% native polyacrylamide gel electrophoresis.
Test-results: do not synthesizing in the reaction system of polypeptide, the dumbbell shape substrate is cut by HIV-1 intergrase enzyme, and along with the increase of HIV-1 intergrase concentration, amount of substrate reduces.In the system of synthetic polypeptide was arranged, along with the increase of peptide concentration, the dumbbell shape amount of substrate increased, referring to Fig. 3.Illustrate that synthetic polypeptide has restraining effect to HIV-1 intergrase activity.
The sequence that the present invention relates to
＜110〉Zhejiang University
＜120〉a kind of integrase inhibiting peptide and the application in the AIDS-treating medicine preparation thereof
<160>10
<210>1
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉according to the aminoacid sequence design, be used for the preparation of HIV-1 integrase inhibitor and treating AIDS medicine.
<400>1
Lys—Lys—Met—Lys—Arg—Arg—Lys
1 5
<210>2
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉according to the aminoacid sequence design, be used for the preparation of HIV-1 integrase inhibitor and treating AIDS medicine.
<400>2
Thr—Pro—Ser—His—Ser—Ser—Arg
1 5
<210>3
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉according to the aminoacid sequence design, be used for the preparation of HIV-1 integrase inhibitor and treating AIDS medicine.
<400>3
His—Pro—Glu—Arg—Ala—Thr—Leu
1 5
<210>4
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉according to the aminoacid sequence design, be used for the preparation of HIV-1 integrase inhibitor and treating AIDS medicine.
<400>4
Ser—His—Leu—Gly—Phe—Arg—Asp
1 5
<210>5
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉according to the aminoacid sequence design, be used for the preparation of HIV-1 integrase inhibitor and treating AIDS medicine.
<400>5
Phe—Val—Arg—Pro—Phe—Ala—Leu
1 5
<210>6
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉according to the aminoacid sequence design, be used for the preparation of HIV-1 integrase inhibitor and treating AIDS medicine.
<400>6
Thr—Pro—Ser—Ala—Trp—Thr—Gln
1 5
<210>7
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉according to the aminoacid sequence design, be used for the preparation of HIV-1 integrase inhibitor and treating AIDS medicine.
<400>7
Ser—His—Ser—Gly—Trp—Ser—Glu
1 5
<210>8
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223 〉, be used for the HIV-1 intergrase and integrate activity experiment according to the dna sequence dna design.
<400>8
gtgtggaaaa?tctctagcag?t
1 21
<210>9
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223 〉, be used for the HIV-1 intergrase and integrate activity experiment according to the dna sequence dna design.
<400>9
actgctagag?attttccaca?c
1 21
<210>10
<211>38
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the dna sequence dna design, be used for HIV-1 intergrase desintegration activity experiment.
<400>10
tgctagttct?agcaggccct?tgggccggcg?cttgcgcc
1 38
Claims (2)
1. integrase inhibiting peptide, be a kind of polypeptide organic compound, it is characterized in that: the polypeptide organic compound that provides is water-soluble, links to each other by peptide bond between each amino acid, its aminoacid sequence is: Thr-Pro-Ser-His-Ser-Ser-Arg, molecular weight are 770.8.
2. the application of a kind of integrase inhibiting peptide according to claim 1 in the medicine of preparation treatment acquired immune deficiency syndrome (AIDS).
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| CNB2007100675261A CN100543034C (en) | 2007-03-05 | 2007-03-05 | A kind of integrase inhibiting peptide and the application in the AIDS-treating medicine preparation thereof |
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| CNB2007100675261A CN100543034C (en) | 2007-03-05 | 2007-03-05 | A kind of integrase inhibiting peptide and the application in the AIDS-treating medicine preparation thereof |
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| CN100543034C true CN100543034C (en) | 2009-09-23 |
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| CN102695715A (en) * | 2009-03-19 | 2012-09-26 | 因特葛莱泰克蛋白质组学有限责任公司 | Inhibitors of viral integrase and methods of use |
| CN101845079B (en) * | 2009-03-27 | 2014-04-09 | 中国人民解放军军事医学科学院毒物药物研究所 | Hexapeptide or derivative thereof and medical application thereof |
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Non-Patent Citations (5)
| Title |
|---|
| AAB03055. EBI. 2000 |
| AAB03055. EBI. 2000;Anti-HIV and anti-tumor protein MAP30, a 30 kDasingle-strand type-I RIP, shares similar secondary structureand beta-sheet topology with the A chain of ricin, a type-IIRIP. YX Wang, et al.Protein Science,Vol.9 No.1. 2000 * |
| Anti-HIV and anti-tumor protein MAP30, a 30 kDasingle-strand type-I RIP, shares similar secondary structureand beta-sheet topology with the A chain of ricin, a type-IIRIP. YX Wang, et al.Protein Science,Vol.9 No.1. 2000 |
| Peptide Motifs That Recognize Differences inPolymer-FilmSurfaces. Takeshi Serizawa, et al.Angew. Chem.,Vol.46 No.5. 2006 |
| Peptide Motifs That Recognize Differences inPolymer-FilmSurfaces. Takeshi Serizawa, et al.Angew. Chem.,Vol.46 No.5. 2006 * |
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