CN104311644A - Polypeptide, method for improving stability of polypeptide, pharmaceutical composition and application of polypeptide in preparation of medicaments - Google Patents
Polypeptide, method for improving stability of polypeptide, pharmaceutical composition and application of polypeptide in preparation of medicaments Download PDFInfo
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Abstract
The invention discloses an isolated polypeptide which has the nature of high enzyme stability. The polypeptide is obtained by at least one of the following modifications of an amino acid sequence of any one of (a) SEQ ID NO: 1-4: (1) compared with (a), the amino acid sequence has side chain modification; (2) compared with (1) and (a), the amino acid sequence inserts and/or substitutes and/or deletes one or more amino acids; (3) compared with (1) and (a), the amino acid sequence has non-natural amino acid shift; (4) compared with (1)-(3) and (a), the amino acid sequence has a modified N-terminal acetyl group; and (5) compared with (1)-(4) and (a), the amino acid sequence has C-terminal amide. The polypeptide disclosed by the invention can effectively inhibit the membrane fusion process of HIV (human immunodeficiency virus).
Description
Technical field
The present invention relates to field of medicaments, concrete, the present invention relates to isolated polypeptide and application thereof, more specifically, the present invention relates to isolated polypeptide, improve the method for polypeptide stability, pharmaceutical composition and isolated polypeptide are preparing the purposes in medicine.
Background technology
Acquired immune deficiency syndrome (AIDS) is (also referred to as " acquired immune deficiency syndrome (AIDS) ", Acquired Immune Deficiency Syndrome, AIDS) be a kind of lethal infectious diseases caused by human immunodeficiency virus (Human Immunodeficiency Virus, HIV).
HIV enters human cell can be divided into three processes.Adsorption process: HIV surface glycoprotein gp120 is combined with human cell surface receptors CD41, thus makes HIV be adsorbed onto human cell surface.Further process: HIV surface glycoprotein gp120 is combined with the auxiliary receptor CCR 5 of human cell or CXCR4 further, thus the distance of further HIV and human cell's film.Fusion process: HIV surface trans-membrane subunit gp41 conformation changes, the fusogenic peptide that then its N holds is inserted in host cell membrane.Finally start the fusion of peplos and human cell's film, complete cell entry host cell.
Acquired immune deficiency syndrome (AIDS) fusion inhibitor a kind ofly can cut off its medicine of prevention and therapy acquired immune deficiency syndrome (AIDS) of new generation infected at the HIV (human immunodeficiency virus) infection initial stage.It is by blocking the fusion process of HIV and human cell's film, and then blocking-up virus of AIDS enters human cell.Acquired immune deficiency syndrome (AIDS) fusion inhibitor is with HIV surface trans-membrane glycoprotein gp41 for target spot, by suppressing the formation of gp41 six spirochete (six-helixbundle, 6-HB), and then barrier film fusion process.
But existing HIV membrane fusion inhibitor still haves much room for improvement.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is that proposition can suppress the polypeptide of the film fusion process of HIV effectively.
According to an aspect of the present invention, the invention provides a kind of isolated polypeptide, there is the character that enzyme stability is high.According to embodiments of the invention, described polypeptide is that the aminoacid sequence described in any one of (a) SEQ ID NO:1 ~ 4 is modified through following at least one and obtains: (1) has the aminoacid sequence that side chain is modified compared with (a); (2) compare with (a) with (1), there is the aminoacid sequence of one or several amino acid whose insertion and/or replacement and/or disappearance; (3) compare with (a) with (1), there is the aminoacid sequence of alpha-non-natural amino acid displacement; (4) compare with (a) with (1) ~ (3), there is the aminoacid sequence that adorned N holds ethanoyl; And (5) are compared with (a) with (1) ~ (4), there is the aminoacid sequence that C end is acid amides.According to the embodiment of the present invention, described polypeptide can suppress the film fusion process of HIV effectively, has the activity suppressing HIV film and human cell surface fusion process.In addition, contriver is surprised to find, according to an embodiment of the invention polypeptidase good stability, and medicine is high for stability, effectively can suppress the film fusion of HIV, thus improves the efficiency suppressing HIV cell further.
According to a further aspect in the invention, the invention provides a kind of method improving polypeptidase stability.According to the embodiment of the present invention, the method can be included between described polypeptide two amino acid side chains and form covalent bonding, wherein, described covalent bonding be selected from carbon-carbon double bond, carbon-carbon single bond, carbon-sulfur bond, sulphur carbon bond, amido linkage or carbon-oxygen bond one of.According to a concrete example of the present invention, described bonding is carbon-carbon double bond.Contriver is surprised to find, and through the polypeptide that the method is modified, polypeptidase good stability, according to embodiments of the invention, can be applied to the medicine of raising polypeptide drugs for stability by the method.
According to another aspect of the invention, the invention provides a kind of pharmaceutical composition.According to the embodiment of the present invention, this pharmaceutical composition comprises: polypeptide of the present invention.Contriver is surprised to find, and this pharmaceutical composition can suppress the film fusion process of HIV effectively, has the activity suppressing HIV film and human cell surface fusion process.In addition, contriver is surprised to find, and according to the polypeptidase good stability of embodiment, medicine is high for stability, effectively can suppress the film fusion of HIV, thus improves the efficiency suppressing HIV cell further.
In accordance with a further aspect of the present invention, present invention also offers polypeptide of the present invention and preparing the purposes in medicine, described medicine is used for the treatment of or prevents AIDS, or described medicine merges for suppressing HIV film.Contriver is surprised to find, and effectively can suppress the film fusion process of HIV, has the activity suppressing HIV film and human cell surface fusion process.In addition, contriver is surprised to find, and according to the polypeptidase good stability of embodiment, medicine is high for stability, effectively can suppress the film fusion of HIV, thus improves the efficiency suppressing HIV cell further.According to embodiments of the invention, polypeptide of the present invention can be used for the treatment of or prevent AIDS, or described medicine merges for suppressing HIV film.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows the chromatogram picture of polypeptide circular dichroism spectrum according to an embodiment of the invention; And
Fig. 2 shows the Chymotrypsin degradation kinetics curve schematic diagram of polypeptide according to an embodiment of the invention.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.
According to an aspect of the present invention, the invention provides a kind of isolated polypeptide, there is the character that enzyme stability is high.According to embodiments of the invention,
Described polypeptide is the aminoacid sequence described in any one of (a) SEQ ID NO:1 ~ 4
Obtain through following at least one modification, wherein, SEQ ID NO:1 ~ 4 are specific as follows:
(1) there is the aminoacid sequence that side chain is modified compared with (a), and this polypeptide has the activity suppressing HIV film to merge.According to the embodiment of the present invention, described side chain modify comprise following one of at least: side chain increases, side chain shortens, heteroatoms is replaced.Thus, be easy between amino acid side chain form stable covalent linkage.
Wherein it should be noted that, the implication of the term " side chain modification " used in this article is not particularly limited, and suppresses HIV film fusion-activity as long as still have after modified.Preferably this side chain is the connecting arm side chain forming covalent linkage.Side chain growth refers to: the carbon atom number increasing side chain; Side chain shortening refers to: the carbon atom number reducing side chain; Heteroatoms is replaced and is referred to: what be selected from atom N, P atom and O atom one of at least replaces at least one carbon atom;
Term " polypeptide " used in this article should be interpreted broadly, and refers to and comprises at least two with the amino acid whose compound of peptide linkage, and it can containing the optional amino acid through modification or non-amino acid structure.In addition, in this article, as do not clearly not stated, " polypeptide ", " protein " and " peptide chain " can exchange use.
In the present invention, term used " separation " refers to as the material such as polypeptide or protein, and it is separated with its naturally occurring environmental facies.The material of described separation is optionally included in undiscovered material in its natural surroundings.
(2) compare with (a) with (1), there is the aminoacid sequence of one or several amino acid whose insertion and/or replacement and/or disappearance, and this polypeptide has the activity suppressing HIV film to merge.
Wherein it should be noted that, the implication of term " replacement " used in this article is not particularly limited, as long as the amino acid after replacing still has the activity suppressing HIV film to merge.According to embodiments of the invention, can be both common amino acid for the amino acid replaced, also can be the amino acid of conformation D-type, naturally occurring rare amino acid or manually modified amino acid.
(3) compare with (a) with (1), there is the aminoacid sequence of alpha-non-natural amino acid displacement, and this polypeptide has the activity suppressing HIV film to merge.Undertaken changing obtained polypeptide by alpha-non-natural amino acid on position in polypeptide, and this polypeptide still has the activity suppressing HIV film to merge.
Kind and the structure of term " alpha-non-natural amino acid " used in the text are not particularly limited, as long as the polypeptide obtained still has the activity suppressing HIV film to merge, are preferably easy to the alpha-non-natural amino acid forming covalent linkage.According to the embodiment of the present invention, described alpha-non-natural amino acid is (S)-2-(4 '-pentenyl)-L-Ala.Thus, amino acid side chain is easy to form covalent linkage, and covalent linkage good stability.
(4) compare with (a) with (1) ~ (3), there is the aminoacid sequence that adorned N holds ethanoyl, and this polypeptide has the activity suppressing HIV film to merge.Namely the N changing polypeptide in above-mentioned (1) ~ (3) and (a) holds ethanoyl to modify the polypeptide obtained, and this polypeptide still has the activity suppressing HIV film to merge.
(5) compare with (a) with (1) ~ (4), there is the aminoacid sequence that C end is acid amides, and described polypeptide has the activity suppressing HIV film to merge.Namely the C end changing polypeptide in above-mentioned (1) ~ (5) is acid amides and the polypeptide that obtains, and this polypeptide still has the activity suppressing HIV film to merge.
According to the embodiment of the present invention, the present invention have following one of a little at least: the activity suppressing HIV film to merge is strong, polypeptidase good stability, medicine are high for stability.Such as, the aminoacid sequence of the polypeptide after modified can be as follows:
The discovery that contriver is surprised, the polypeptidase good stability listed by upper table, medicine is high for stability, effectively can suppress the film fusion of HIV, thus suppresses HIV cell efficiently.
According to embodiments of the invention, described polypeptide comprises at least two alpha-non-natural amino acids further, and between the side chain of described alpha-non-natural amino acid, form covalent bonding (such as: carbon-carbon double bond, carbon-carbon single bond, carbon-sulfur bond, sulphur carbon bond, amido linkage, carbon-oxygen bond).Thus, the enzyme stability of polypeptide is good, and medicine is high for stability.
According to embodiments of the invention, between the side chain of described alpha-non-natural amino acid, be formed with the covalent bonding described in two.Thus, the stability of polypeptide is better, and polypeptide merges inhibit activities to HIV film and improves further.
According to embodiments of the invention, described covalent linkage be selected from carbon-carbon double bond, carbon-carbon single bond, carbon-sulfur bond, sulphur carbon bond, amido linkage or carbon-oxygen bond one of.According to a concrete example of the present invention, described covalent bonding is carbon-carbon double bond.Thus, covalent linkage good stability in peptide molecule.
According to embodiments of the invention, described polypeptide has alpha-helix conformation.The proteolytic enzyme identification that this feature makes polypeptide be not easy in body, thus be not easily easily degraded by proteases.Meanwhile, this constructional feature also make polypeptide and HIV binding ability stronger.Thus, the inhibit activities of polypeptide is strong, and enzyme stability is good, and medicine is high for stability.
According to a further aspect in the invention, present invention also offers a kind of method improving polypeptidase stability.According to the embodiment of the present invention, the method can be included between described polypeptide two amino acid side chains and form covalent bonding, wherein, described covalent bonding be selected from carbon-carbon double bond, carbon-carbon single bond, carbon-sulfur bond, sulphur carbon bond, amido linkage or carbon-oxygen bond one of.According to a concrete example of the present invention, described bonding is carbon-carbon double bond.Contriver is surprised to find, and through the polypeptide that the method is modified, polypeptidase good stability, medicine is high for stability.
According to another aspect of the invention, invention further provides a kind of pharmaceutical composition.According to embodiments of the invention, this pharmaceutical composition comprises: polypeptide of the present invention.Contriver is surprised to find, and this pharmaceutical composition is in vivo not easily by proteolytic degradation, and polypeptidase good stability, medicine is high for stability.
According to embodiments of the invention, described pharmaceutical composition comprises at least one of vehicle, additives and pharmaceutically acceptable carrier further.
Wherein, it should be noted that, " pharmaceutically acceptable carrier " refers to the bioactive mounting medium significantly not changing added active ingredient (such as, the polypeptide with suppression HIV film fusion-activity of the present invention) in the present invention.Pharmaceutically acceptable carrier includes but not limited to one or more of following material: water, damping fluid, physiological saline, 0.3% glycine, alcoholic solution, isotonic aqueous solution; One or more following material, such as glycerine can also be comprised; Oil; Salt, as sodium salt, sylvite, magnesium salts, ammonium salt and phosphoric acid salt; Carbonic ether; Lipid acid; Carbohydrate (such as N.F,USP MANNITOL); Polysaccharide; Polymkeric substance; Vehicle; With sanitas and/or stablizer etc.
According on the one hand of the present invention, the invention provides polypeptide of the present invention and preparing the purposes in medicine, described medicine is used for the treatment of or prevents AIDS, or described medicine merges for suppressing HIV film.
Pharmaceutical composition of the present invention or medicine comprise such as be suitable for oral, rectum, nose, locally, the composition that gives of peritonaeum and parenteral (comprising intramuscular, subcutaneous or intravenously).Or by sucking or be blown into the composition given.Can local or general give.Give preferably by oral or intravenous route.Pharmaceutical composition of the present invention comprises any pharmaceutical dosage forms that this area is determined; such as capsule, pill, tablet, powder, many granular preparations (such as pearl, particle or crystal), aerosol, sprays, foam, solution, dispersion agent, tincture, syrup, elixir, suspension, water-in-oil emulsion are as ointment, and water external emulsion is as emulsion, lotion and face cream.
Contriver is surprised to find, polypeptide of the present invention in vivo not easily by proteolytic degradation, polypeptidase good stability, medicine is high for stability, the restraining effect merged HIV film is strong, polypeptide of the present invention can be used for the treatment of or prevent AIDS, or described medicine merges for suppressing HIV film.
Below with reference to specific embodiment, the present invention will be described, it should be noted that, these embodiments are only illustrative, and can not be interpreted as limitation of the present invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.Experiment material used in following embodiment, if no special instructions, is purchased from routine biochemistry Reagent Company.
Be described subsequent embodiment for clearer, the symbol abbreviation providing in each embodiment each raw material used below represents, in embodiments, the place of using symbol abbreviation all refers to the content that the abbreviation of corresponding symbol is corresponding, specific as follows:
Embodiment 1
In an embodiment of the present invention, polypeptide provided by the present invention, all adopts standard Fmoc solid phase synthesis strategy to obtain.In order to shelter the negative charge of C end, Rink Aminde AM resin is adopted to be held by C last amino acid whose carboxyl to become acid amides.The amino acid adopted in solid phase synthesis process is standard Fmoc amino acid and Fmoc-S
5-OH.Treat that linear peptides synthesis is complete, utilize first-generation Grubbs catalyst to carry out olefin metathesis reaction on resin.Then TFA (trifluoroacetic acid)/TIPS (tri isopropyl silane base)/H is used
2polypeptide cuts down from resin by O.Finally utilize HPLC purified polypeptide, ESI-MS detects.Concrete synthesis step is as follows:
1. amino acid condensation: take 300mg substitution value be the Rink Amide AM resin of 0.33mmol/g in 80mL Peptide systhesis pipe, add 2mL DMF (dimethyl formamide) and 6mL DCM (methylene dichloride) mixed solvent is swelling.Solvent is drained with water pump after 0.5h.Use DMF (5mL*5) successively, DCM (5mL*5), DMF (5mL*5) washing resin.Then 5mL 20% Piperidine/DMF solution is used to slough Fmoc protecting group (5min+10min).Repeat above-mentioned washing resin, dose and method is described above.Finally add amino acid reaction solution (the Fmoc-AA-OH 4equiv. of preactivated a minute, HBTU (O-benzotriazole-tetramethyl-urea phosphofluoric acid ester) 3.9equiv. (equivalent, lower same), HOBt (I-hydroxybenzotriazole) 4equiv., DIEA (N, N-diisopropylethylamine) 8equiv., DMF5mL) react 1.5h.On resin, condensation natural amino acid step is all described above.Condensation alpha-non-natural amino acid Fmoc-S
5during-OH, reaction solution is combined as Fmoc-S
5-OH 2equiv., HATU (2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester) 1.9equiv., HOBt 2equiv., DIEA 4equiv., DMF 3ml, the reaction times is 2h.
2. olefin metathesis: polypeptide chain, after assembled on resin, carries out olefin metathesis reaction.1,2-dichloroethane solution (40mg/5mL) room temperature reaction of Grubs ' catalyzer is added under argon shield condition.1,2-dichloroethane solution of Grubs ' catalyzer is again added after 2h.Use DMF respectively after question response 2h, DCM washs.
3. acetylation modification: polypeptide N end all carries out acetylation modification, and concrete operations are as follows.After carrying out olefin metathesis, last amino acid whose Fmoc on polypeptide chain is removed, washs, then adds 5mL acetylation reagent (Ac
2o (diacetyl oxide): DIEA:DMF=1:1:8, v:v:v) reaction, again add acetylation reagent after 5min, finally re-use DMF, DCM washs.
4. protecting group excision: add 10mL cutting reagent (TFA:TIPS: water=95:2.5:2.5, v:v:v) in Peptide systhesis pipe, room temperature reaction.Cutting reagent is transferred in 50mL centrifuge tube after 2.5h, use Ar gas to blow the TFA in centrifuge tube to 2mL.Then in centrifuge tube, 40mL ice ether sedimentation, centrifugal is added.Most backward diethyl ether solution of pouring out stays precipitation.
5. peptide separation and qualification: utilize SHIMAZU high performance liquid chromatography, the performance liquid chromatographic column of GRACE Vydac C184.6 × 250mm carries out the analysis to thick peptide, moving phase is A: the acetonitrile containing 0.1%TFA, B: the water containing 0.1%TFA, carry out gradient elution, the B of 30min from the B of 10% to 80%, flow velocity is 1mL/min.Until polypeptide after ESI mass spectrum is determined, GRACE Vydac C18 high performance liquid phase semipreparative column is used to carry out separation and purification to thick peptide.
Solid-phase synthesis peptides route is as follows:
By aforesaid method, synthesize Four types altogether and amounted to 12 polypeptide (SFT-0, SFT-1, SFT-2; SC34EK-0, SC34EK-1, SC34EK-2; MT-SC22EK-0, MT-SC22EK-1A, MT-SC22EK-1B, MT-SC22EK-2, CP32M-0, CP32M-1, wherein 0,1,2 refer to the number forming stapling structure (i.e. covalent linkage) respectively).Wherein, SFT-0, SC34EK-0, MT-SC22EK-0, CP32M-0 are respectively the polypeptide shown in SEQ ID NO:1-4, and all the other polypeptide are the polypeptide derivative that above-mentioned four peptide species obtain after modifying.The concrete aminoacid sequence of aforementioned polypeptides is as follows:
Embodiment 2
Circular dichroism detector is adopted to analyze 12 polypeptide (SFT-0, SFT-1, SFT-2 obtaining in embodiment 1; SC34EK-0, SC34EK-1, SC34EK-2; MT-SC22EK-0, MT-SC22EK-1A, MT-SC22EK-1B, MT-SC22EK-2, CP32M-0, CP32M-1) physicochemical property.
1, experimental technique
12 polypeptide are dissolved in the phosphate buffered saline buffer of pH=6.8 respectively, finally obtain concentration and be 50 μMs of test solns.At 25 DEG C, test soln is transferred in the quartz colorimetric utensil of 0.1mm, uses the circular dichroism spectrum of Jasco-715 spectropolarimeter test polypeptide.Spectropolarimeter measuring parameter: wavelength 190-260nm, step-length 0.5nm, accumulative 3 times.
2, experimental result
Typical alpha-helix has positive absorption peak at the 195nm place of circular dichroism spectrum, has negative absorption peak at 208nm and 222nm two place.There is two negative peak and occur positive absorption peak at 195nm place in 12 polypeptide as can be seen from Figure 1 at 208nm and 222nm place, show that synthesized polypeptide all has alpha-helix conformation.The helix degree of polypeptide is by its CD absorption peak [θ] at 222nm
222determine.After the CD absorption peak [θ] 222 at 222nm place is normalized, use polypeptide helix degree calculation formula [θ]
222/ [θ]
max, the helix degree of polypeptide can be calculated.The helix degree of Four types 12 polypeptide is shown in Fig. 1.
Embodiment 3 (the enzyme stability matter of polypeptide)
In the present embodiment, 7 (SC34EK-0, SC34EK-1, SC34EK-2 in 12 polypeptide adopting high-efficient liquid phase chromatogram technique analysis embodiment 1 to obtain; MT-SC22EK-0, MT-SC22EK-1A, MT-SC22EK-1B, MT-SC22EK-2) enzyme stability matter.
1, experimental technique
Above-mentioned 7 polypeptide are prepared as the DMSO storage liquid that concentration is 1mM respectively.Above-mentioned storage liquid 50 μ L is joined (0.05mol/L Na in 1950 μ L phosphoric acid buffers respectively
2hPO
4, 2mM CaCl
2, pH=7.8,0.5ng/ μ L chymotrypsin) and at 30 DEG C, carry out enzyme digestion reaction.Get 100 μ L enzyme digestion reaction liquid respectively in different time sections and add 20 μ L1M hydrochloric acid enzyme digestion reactions.By using the enzyme digestion reaction liquid of high performance liquid chromatography monitoring different time sections polypeptide, obtain its kinetic process of degradation.
2, experimental result
Article 7, the Chymotrypsin degradation kinetics curve of polypeptide as shown in Figure 2.The transformation period of polypeptide is obtained according to the Chymotrypsin degradation kinetics curve of each polypeptide, the transformation period of each polypeptide is as follows: the SC34EK-0 transformation period is 55min, the SC34EK-1 transformation period is 109min, the SC34EK-2 transformation period is 101min, the MT-SC22EK-0 transformation period is 125min, the MT-SC22EK-1A transformation period is 405min, the MT-SC22EK-1B transformation period be 725min, the MT-SC22EK-2 transformation period is 3665min.
Found by the transformation period of comparing two type polypeptide:
1, the polypeptide transformation period order of two types is SC34EK-2>SC34EK-1>SC34EK-0; MT-SC22EK-2>MT-SC22EK-1B>MT-SC22EK-1A>MT-SC22EK-0.Illustrate that carrying out skeleton tempering structural modification effectively can strengthen the stability of polypeptide to Chymotrypsin.
2,34 amino acid whose polypeptide SC34EK series transformation period are contained containing 24 amino acid whose polypeptide MT-SC22EK series transformation period >.Small peptide is described to the stability of Chymotrypsin higher than long peptide.
Embodiment 4
In the present embodiment, by HIV detection system, detect the activity that polypeptide suppresses HIV pseudovirus to infect.
1, experimental technique
12 polypeptide (SFT-0, SFT-1, SFT-2 respectively embodiment 1 being obtained; SC34EK-0, SC34EK-1, SC34EK-2; MT-SC22EK-0, MT-SC22EK-1A, MT-SC22EK-1B, MT-SC22EK-2, CP32M-0 and CP32M-1) be prepared as certain density DMSO storage liquid.The detection using the HIV pseudovirus Chinese epidemic strain of three kinds of different subtypes and three kinds of HIV pseudovirus type strains to carry out polypeptide to suppress viral infectivity, wherein CNE6 and CNE11 is B ' hypotype, CNE15 and CNE30 is BC hypotype, CNE5 and CNE55 is CRF01_AE hypotype, sf162, JRFL and HXB2 are HIV type strain B hypotype (the construction process reference of bacterial strain: Hong Shang, Xiaoxu Han, Xuanling Shi, et al.J Biol Chem.2011Apr 22; 286 (16): 14531-41., teaching laboratory by Tsing-Hua University Zhang Linqi provides).These 9 kinds of HIV pseudoviruss, all containing luciferase reporter gene detection system, use these 9 kinds of pseudoviruss to detect 12 polypeptide respectively, are obtained the ability of HIV pseudovirus cells infected by the activity measuring reporter gene luciferase.
The packaging of HIV pseudovirus and detection method: utilize the eukaryon expression plasmid (pcDNA3.1+ that can express various hypotype HIV total length membranin, purchased from Invitrogen) and skeleton plasmid pNL4-3R-E-luciferase (reference: He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR.J Virol 69:6705 – 6711,1995.) cotransfection 293T cell, preparation has infectivity but does not have the HIV pseudovirus of replication, and its infectivity is similar with live virus.After utilizing the HIV pseudovirus homopolypeptide of preparation jointly to hatch, permissive cell GHOST (3) X4/R5 of Supernatant infection HIV again, carries out the determination of HIV pseudovirus infection ability by the activity measuring reporter gene luciferase.Wherein, in permissive cell GHOST (3) the X4/R5 experiment of Supernatant infection HIV, if polypeptide has the ability of GHOST (3) X4/R5 suppressing HIV permissive cell, the viral load that then can enter GHOST (3) X4/R5 will significantly reduce, the expression amount of luciferase in GHOST (3) X4/R5 that then HIV pseudovirus itself carries also can reduce, so just can be detected the virus quantity of cells infected quantitatively by the activity measuring luciferase in GHOST (3) X4/R5, the ability that polypeptide suppresses HIV also can be reflected.
2, experimental result
Each polypeptide can be obtained by the activity detecting luciferase and curve is suppressed to 9 type HIV pseudoviruss, thus obtain reactivity parameter (that is: the half-inhibition concentration that can characterize the infection of polypeptide suppression HIV pseudovirus, half-inhibition concentration lower suppression HIV activity is stronger), concrete outcome is in Table 1-table 4.
Table 1SFT series polypeptide is to the half-inhibition concentration (IC50, nM) of AIDS pseudovirus
| Pseudovirus title | Hypotype | SFT-0 | SFT-1 | SFT-2 |
| CNE6 | B’ | 4.04±0.55 | 1.98±0.56 | 3.74±1.12 |
| CNE11 | B’ | 7.38±1.31 | 4.57±0.72 | 7.45±2.24 |
| CNE15 | BC | 40.04±1.77 | 4.84±1.10 | 5.12±0.16 |
| CNE30 | BC | 51.05±5.15 | 29.67±4.27 | 18.89±4.82 |
| CNE5 | CRF01_AE | 7.36±0.08 | 6.25±1.71 | 4.48±1.77 |
| CNE55 | CRF01_AE | 11.26±0.99 | 11.91±3.41 | 15.28±3.62 |
| sf162 | B | 5.03±0.09 | 7.14±0.09 | 13.84±0.50 |
| JRFL | B | 8.18±1.29 | 5.88±1.48 | 1.92±1.01 |
| HXB2 | B | 0.47±0.13 | 0.27±0.04 | 0.14±0.04 |
Table 2SC34EK series polypeptide is to the half-inhibition concentration (IC50, nM) of AIDS pseudovirus
| Pseudovirus title | Hypotype | SC34EK-0 | SC34EK-1 | SC34EK-2 |
| CNE6 | B’ | 1.46±0.13 | 0.55±0.14 | 0.51±0.14 |
| CNE11 | B’ | 9.46±2.27 | 1.02±0.01 | 7.16±0.32 |
| CNE15 | BC | 6.54±1.50 | 1.01±0.55 | 3.28±1.33 |
| CNE30 | BC | 41.49±7.69 | 6.35±0.21 | 31.13±0.30 |
| CNE5 | CRF01_AE | 19.28±8.21 | 2.48±0.96 | 8.48±1.82 |
| CNE55 | CRF01_AE | 16.35±4.37 | 2.91±0.35 | 14.46±2.72 |
| sf162 | B | 9.31±4.59 | 3.55±0.24 | 6.75±1.35 |
| JRFL | B | 8.44±6.90 | 3.31±1.67 | 6.01±1.01 |
| HXB2 | B | 2.08±1.34 | 0.04±0.02 | 0.33±0.23 |
Table 3MT-SC22EK series polypeptide is to the half-inhibition concentration (IC50, nM) of AIDS pseudovirus
Table 4CP32M series polypeptide is to the half-inhibition concentration (IC50, nM) of AIDS pseudovirus
| Pseudovirus title | Hypotype | CP32M-0 | CP32M-1 |
| CNE6 | B’ | 27.35±7.99 | 15.84±3.87 |
| CNE11 | B’ | 422.90±197.00 | 19.65±1.53 |
| CNE15 | BC | 56.50±31.20 | 42.28±1.62 |
| CNE30 | BC | 920.01±400.31 | 127.55±14.50 |
| CNE5 | CRF01_AE | 1.12±0.45 | 27.19±6.45 |
| CNE55 | CRF01_AE | 3.90±0.10 | 54.10±4.80 |
| sf162 | B | 126.13±43.49 | 34.38±1.41 |
| JRFL | B | 620.22±361.67 | 25.14±6.33 |
| HXB2 | B | 14.02±11.59 | 4.15±0.50 |
Found by the half-inhibition concentration comparing four type polypeptide:
(1) the polypeptide half-inhibition concentration order for Four types is: SC34EK>MT-SC22EK>SFTGreatT.G reaT.GTCP32M.Half-inhibition concentration not through the polypeptide modified is through the 2-10 of the polypeptide (namely forming the polypeptide of covalent linkage in peptide molecule) of skeleton tempering structural modification doubly.
(2) amino acid number is greater than to the polypeptide of 30, the activity of carrying out twice skeleton tempering structural modification peptide will lower than carrying out a skeleton structure modified peptides.Amino acid number is less than to the polypeptide of 30, the peptide activity of carrying out twice skeleton tempering structural modification is higher than carries out a skeleton tempering structural modification peptide.
(3) above-mentioned 12 polypeptide all have suppression HIV film fusion process activity, and wherein, the activity of SC34EK-1 and MT-SC22EK-2 is best.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. an isolated polypeptide, has the character that enzyme stability is high, it is characterized in that, described polypeptide is
Aminoacid sequence described in any one of (a) SEQ ID NO:1 ~ 4
Obtain through following at least one modification:
(1) there is the aminoacid sequence that side chain is modified compared with (a);
(2) compare with (a) with (1), there is the aminoacid sequence of one or several amino acid whose insertion and/or replacement and/or disappearance;
(3) compare with (a) with (1), there is the aminoacid sequence of alpha-non-natural amino acid displacement;
(4) compare with (a) with (1) ~ (3), there is the aminoacid sequence that adorned N holds ethanoyl; And
(5) compare with (a) with (1) ~ (4), there is the aminoacid sequence that C end is acid amides.
2. polypeptide according to claim 1, is characterized in that, described polypeptide has the activity suppressing HIV film to merge.
3. polypeptide according to claim 1, is characterized in that, described side chain modify comprise following one of at least: side chain increases, side chain shortens, heteroatoms is replaced,
Optionally, described heteroatoms replace be selected from atom N, P atom and O atom one of at least replace at least one carbon atom.
4. polypeptide according to claim 1, is characterized in that, described polypeptide comprises at least two alpha-non-natural amino acids further, and forms covalent bonding between the side chain of described alpha-non-natural amino acid.
5. polypeptide according to claim 4, is characterized in that, is formed with two covalent bondings between the side chain of described alpha-non-natural amino acid,
Optionally, described covalent bonding be selected from carbon-carbon double bond, carbon-carbon single bond, carbon-sulfur bond, sulphur carbon bond, amido linkage or carbon-oxygen bond one of, preferably, described covalent bonding is carbon-carbon double bond.
6. polypeptide according to claim 4, is characterized in that, described alpha-non-natural amino acid is (S)-2-(4 '-pentenyl)-L-Ala.
7. polypeptide according to claim 1, is characterized in that, described polypeptide has alpha-helix conformation.
8. one kind is improved the method for polypeptidase stability, it is characterized in that, be included between described polypeptide two amino acid side chains and form covalent bonding, wherein, described covalent bonding be selected from carbon-carbon double bond, carbon-carbon single bond, carbon-sulfur bond, sulphur carbon bond, amido linkage or carbon-oxygen bond one of, preferably, described bonding is carbon-carbon double bond.
9. a pharmaceutical composition, is characterized in that, comprising: the polypeptide described in any one of claim 1 ~ 7.
10. the polypeptide described in any one of claim 1 ~ 7 is preparing the purposes in medicine, and described medicine is used for the treatment of or prevents AIDS, or described medicine merges for suppressing HIV film.
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| WO2017211317A1 (en) * | 2016-06-08 | 2017-12-14 | 胡卓伟 | Polypeptide and application thereof in manufacturing pharmaceutical product for treating and/or preventing tumor |
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| US20130108653A1 (en) * | 2009-12-22 | 2013-05-02 | Shervin Bahrami | Bivalent molecules for hiv entry inhibition |
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| US20130108653A1 (en) * | 2009-12-22 | 2013-05-02 | Shervin Bahrami | Bivalent molecules for hiv entry inhibition |
Non-Patent Citations (2)
| Title |
|---|
| HUIHUI CHONG: "Short-peptide fusion inbibitors with high potency against wild-type and enfuvirtide-resistant HIV-1", 《THE FASEB JOURNAL》 * |
| 高帅: "订书肽的合成与应用", 《化学进展》 * |
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| WO2017211317A1 (en) * | 2016-06-08 | 2017-12-14 | 胡卓伟 | Polypeptide and application thereof in manufacturing pharmaceutical product for treating and/or preventing tumor |
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