CN104311644B - Polypeptide improves the purposes of method, pharmaceutical composition and the polypeptide of polypeptide stability in medicine preparation - Google Patents
Polypeptide improves the purposes of method, pharmaceutical composition and the polypeptide of polypeptide stability in medicine preparation Download PDFInfo
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- CN104311644B CN104311644B CN201410535702.XA CN201410535702A CN104311644B CN 104311644 B CN104311644 B CN 104311644B CN 201410535702 A CN201410535702 A CN 201410535702A CN 104311644 B CN104311644 B CN 104311644B
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- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of polypeptides of separation, and with the high property of enzyme stability, the polypeptide is (a) SEQ ID NO：What 1~4 any one of them amino acid sequence was obtained through following at least one modification：(1) amino acid sequence with side chain modification compared with (a)；(2) compared with (1) and (a), the insertion with one or several amino acid and/or the amino acid sequence of replacement and/or missing；(3) compared with (1) and (a), the amino acid sequence with non-natural amino acid displacement；(4) compared with (1)~(3) and (a), there is the amino acid sequence for the N-terminal acetyl group being modified；And (5) are compared with (1)~(4) and (a), are the amino acid sequence of amide with C-terminal.The polypeptide of the present invention can effectively inhibit the film fusion process of HIV.
Description
Technical Field
The invention relates to the field of medicines, in particular to an isolated polypeptide and application thereof, and more particularly to an isolated polypeptide, a method for improving the stability of the polypeptide, a pharmaceutical composition and application of the isolated polypeptide in preparation of medicines.
Background
Acquired Immunodeficiency syndrome (also known as "AIDS"), is a fatal infectious disease caused by Human Immunodeficiency Virus (HIV).
Entry of HIV into human cells can be divided into three processes. An adsorption process: the HIV surface glycoprotein gp120 binds to the human cell surface receptor CD41, thereby allowing HIV to adsorb to the human cell surface. And (3) a drawing-in process: the HIV surface glycoprotein gp120 further binds to the human cell co-receptor CCR5 or CXCR4, thereby drawing HIV closer to the human cell membrane. And (3) fusion process: the conformation of the HIV surface transmembrane subunit gp41 is changed, and then the fusion peptide at the N terminal is inserted into the host cell membrane. And finally, starting the fusion of the virus envelope and the human cell membrane to finish the virus entering the host cell.
The AIDS fusion inhibitor is a new generation medicine for preventing and treating AIDS, which can cut off infection of AIDS at the early stage of AIDS infection. It can block HIV from entering human cell by blocking the fusion process of HIV and human cell membrane. The HIV fusion inhibitor takes HIV surface transmembrane glycoprotein gp41 as a target, and inhibits the formation of gp41 hexaspira (6-HB) so as to block the membrane fusion process.
However, existing HIV membrane fusion inhibitors remain to be improved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. To this end, it is an object of the present invention to propose polypeptides capable of effectively inhibiting the membrane fusion process of HIV.
According to one aspect of the invention, the invention provides an isolated polypeptide having the property of high enzymatic stability. According to an embodiment of the invention, the polypeptide is (a) SEQ ID NO: 1 to 4, wherein the amino acid sequence is modified by at least one of the following: (1) an amino acid sequence having a side chain modification compared to (a); (2) an amino acid sequence having insertion and/or substitution and/or deletion of one or several amino acids as compared with (1) and (a); (3) an amino acid sequence having an unnatural amino acid shift as compared to (1) and (a); (4) an amino acid sequence having a modified N-terminal acetyl group as compared with (1) to (3) and (a); and (5) an amino acid sequence having an amide at the C-terminus as compared with (1) to (4) and (a). According to the embodiment of the invention, the polypeptide can effectively inhibit the membrane fusion process of HIV and has the activity of inhibiting the fusion process of HIV membrane and human cell surface. In addition, the inventors surprisingly found that the polypeptide enzyme according to the embodiment of the present invention has good stability and high pharmacokinetic stability, and can effectively inhibit the membrane fusion of HIV, thereby further improving the efficiency of inhibiting HIV-infected cells.
According to another aspect of the present invention, there is provided a method for increasing the stability of a polypeptide enzyme. According to an embodiment of the present invention, the method may comprise forming a covalent bond between two amino acid side chains of said polypeptide, wherein said covalent bond is selected from one of a carbon-carbon double bond, a carbon-carbon single bond, a carbon-sulfur bond, a sulfur-carbon bond, an amide bond or a carbon-oxygen bond. According to a specific example of the present invention, the bond is a carbon-carbon double bond. The inventor surprisingly found that the polypeptide modified by the method has good stability of the polypeptide enzyme, and the method can be applied to improving the pharmacological stability of the polypeptide drug according to the embodiment of the invention.
According to yet another aspect of the present invention, there is provided a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises: the polypeptide of the invention. The inventor surprisingly found that the pharmaceutical composition can effectively inhibit the membrane fusion process of HIV and has the activity of inhibiting the fusion process of HIV membrane and human cell surface. In addition, the inventors surprisingly found that the polypeptide enzyme according to the examples has good stability and high pharmacokinetic stability, and can effectively inhibit the membrane fusion of HIV, thereby further improving the efficiency of inhibiting HIV-infected cells.
According to a further aspect of the invention, there is also provided the use of a polypeptide according to the invention in the manufacture of a medicament for the treatment or prophylaxis of AIDS or for the inhibition of HIV membrane fusion. The inventor surprisingly found that the HIV inhibitor can effectively inhibit the membrane fusion process of HIV and has the activity of inhibiting the fusion process of HIV membrane and human cell surface. In addition, the inventors surprisingly found that the polypeptide enzyme according to the examples has good stability and high pharmacokinetic stability, and can effectively inhibit the membrane fusion of HIV, thereby further improving the efficiency of inhibiting HIV-infected cells. According to embodiments of the present invention, the polypeptide of the present invention can be used for the treatment or prevention of AIDS, or the drug is used for the inhibition of HIV membrane fusion.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a chromatogram picture of a polypeptide circular dichroism chromatogram according to an embodiment of the present invention; and
FIG. 2 shows a schematic representation of the chymotrypsin degradation kinetics of a polypeptide according to one embodiment of the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
According to one aspect of the invention, the invention provides an isolated polypeptide having the property of high enzymatic stability. In accordance with an embodiment of the present invention,
the polypeptide is (a) SEQ ID NO: 1 to 4 of the amino acid sequence
Wherein the polypeptide is obtained by at least one of the following modifications, SEQ ID NO: 1-4 are as follows:
(1) compared with (a), the polypeptide has an amino acid sequence with modified side chain, and the polypeptide has the activity of inhibiting HIV membrane fusion. According to an embodiment of the invention, the side chain modification comprises at least one of: side chain extension, side chain shortening, heteroatom replacement. Thus, stable covalent bonds are easily formed between the amino acid side chains.
It should be noted that the term "side chain modification" as used herein is not particularly limited as long as it is modified to have the activity of inhibiting HIV membrane fusion. Preferably, the side chain is a linker arm side chain that forms a covalent bond. Side chain extension means: increasing the number of carbon atoms of the side chain; side chain shortening means: reducing the number of carbon atoms of the side chain; heteroatom substitution means: at least one carbon atom is replaced by at least one selected from the group consisting of an N atom, a P atom and an O atom;
the term "polypeptide" as used herein is to be understood in a broad sense and refers to a compound comprising at least two amino acids joined by a peptide bond, which may contain optionally modified amino acids or non-amino acid structures. In addition, herein, "polypeptide", "protein", and "peptide chain" may be used interchangeably, as not explicitly stated.
The term "isolated" as used herein refers to a material, such as a polypeptide or protein, that is separated from its naturally occurring environment. The isolated material optionally comprises material not found in its natural environment.
(2) An amino acid sequence having insertion and/or substitution and/or deletion of one or several amino acids as compared with (1) and (a), and the polypeptide has an activity of inhibiting HIV membrane fusion.
It is to be noted that the term "substitution" as used herein is not particularly limited in meaning as long as the substituted amino acid still has an activity of inhibiting HIV membrane fusion. According to the embodiments of the present invention, the amino acid to be substituted may be either a common amino acid or a conformational D-type amino acid, a naturally occurring rare amino acid, or an artificially modified amino acid.
(3) Compared with (1) and (a), has an amino acid sequence with unnatural amino acid shifts, and the polypeptide has the activity of inhibiting HIV membrane fusion. I.e., a polypeptide in which the position of the unnatural amino acid insertion in the polypeptide has been altered, and which still has activity in inhibiting HIV membrane fusion.
The kind and structure of the term "unnatural amino acid" used herein are not particularly limited as long as the resulting polypeptide still has activity of inhibiting HIV membrane fusion, and unnatural amino acids which easily form covalent bonds are preferred. According to an embodiment of the invention, the unnatural amino acids are all (S) -2- (4' -pentenyl) -alanine. Thus, the amino acid side chain is easy to form a covalent bond, and the stability of the covalent bond is good.
(4) Compared with (1) - (3) and (a), the polypeptide has a modified amino acid sequence of N-terminal acetyl group, and has HIV membrane fusion inhibiting activity. Namely, the polypeptide obtained by modifying the N-terminal acetyl group of the polypeptides in the above (1) to (3) and (a) and still has the activity of inhibiting HIV membrane fusion.
(5) Compared with the (1) - (4) and (a), the polypeptide has an amino acid sequence with an amide at the C terminal, and has the activity of inhibiting HIV membrane fusion. Namely, a polypeptide obtained by changing the C-terminal of the polypeptide in (1) to (5) above to an amide, which still has an activity of inhibiting HIV membrane fusion.
According to an embodiment of the invention, the invention has at least one of the following advantages: strong activity for inhibiting HIV membrane fusion, good stability of polypeptidase and high pharmacokinetics stability. For example, the amino acid sequence of the modified polypeptide may be as follows:
the inventors surprisingly found that the polypeptide enzyme listed in the above table has good stability and high pharmacokinetics stability, and can effectively inhibit the membrane fusion of HIV, thereby efficiently inhibiting HIV infected cells.
According to embodiments of the invention, the polypeptide further comprises at least two unnatural amino acids, and a covalent bond (e.g., a carbon-carbon double bond, a carbon-carbon single bond, a carbon-sulfur bond, a sulfur-carbon bond, an amide bond, a carbon-oxygen bond) is formed between the side chains of the unnatural amino acids. Therefore, the polypeptide has good enzyme stability and high pharmacokinetics stability.
According to an embodiment of the invention, two of said covalent bonds are formed between side chains of said unnatural amino acid. Therefore, the stability of the polypeptide is better, and the HIV membrane fusion inhibition activity of the polypeptide is further improved.
According to an embodiment of the invention, the covalent bond is selected from one of a carbon-carbon double bond, a carbon-carbon single bond, a carbon-sulfur bond, a sulfur-carbon bond, an amide bond or a carbon-oxygen bond. According to a specific example of the present invention, the covalent bond is a carbon-carbon double bond. Therefore, the stability of the covalent bond in the polypeptide molecule is good.
according to the embodiment of the invention, the polypeptide has α -helical conformation, so that the polypeptide is not easily recognized by protease in vivo and is not easily degraded by the protease.
According to another aspect of the present invention, there is also provided a method for improving the stability of a polypeptide enzyme. According to an embodiment of the present invention, the method may comprise forming a covalent bond between two amino acid side chains of said polypeptide, wherein said covalent bond is selected from one of a carbon-carbon double bond, a carbon-carbon single bond, a carbon-sulfur bond, a sulfur-carbon bond, an amide bond or a carbon-oxygen bond. According to a specific example of the present invention, the bond is a carbon-carbon double bond. The inventor surprisingly found that the polypeptide modified by the method has good stability of the polypeptide enzyme and high pharmacokinetics stability.
According to yet another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises: the polypeptide of the invention. The inventor surprisingly finds that the pharmaceutical composition is not easily degraded by proteolytic enzyme in vivo, and the polypeptide enzyme has good stability and high pharmacokinetics stability.
According to an embodiment of the invention, the pharmaceutical composition further comprises at least one of an excipient, an additive, and a pharmaceutically acceptable carrier.
Herein, it is to be noted that the "pharmaceutically acceptable carrier" refers to a carrier medium which does not significantly change the biological activity of the added active ingredient (e.g., the polypeptide having an activity of inhibiting HIV membrane fusion of the present invention). Pharmaceutically acceptable carriers include, but are not limited to, one or more of the following: water, buffer solution, physiological saline, 0.3% glycine, alcoholic solution, isotonic water solution; one or more of the following, such as glycerol; an oil; salts such as sodium, potassium, magnesium, ammonium and phosphate; a carbonate ester; a fatty acid; sugars (e.g., mannitol); a polysaccharide; a polymer; an excipient; and preservatives and/or stabilizers and the like.
According to one aspect of the invention, there is provided the use of a polypeptide according to the invention in the manufacture of a medicament for the treatment or prophylaxis of AIDS or for the inhibition of HIV membrane fusion.
The pharmaceutical compositions or medicaments of the invention include, for example, compositions suitable for oral, rectal, nasal, topical, peritoneal and parenteral (including intramuscular, subcutaneous or intravenous) administration. Or compositions for administration by inhalation or insufflation. Can be administered locally or systemically. Preferably by oral or intravenous route. The pharmaceutical compositions of the present invention include any pharmaceutical dosage form identified in the art, such as capsules, pills, tablets, powders, multiparticulate formulations (e.g., beads, granules or crystals), aerosols, sprays, foams, solutions, dispersions, tinctures, syrups, elixirs, suspensions, water-in-oil emulsions such as ointments, and oil-in-water emulsions such as creams, lotions and balms.
The inventor surprisingly finds that the polypeptide is not easily degraded by proteolytic enzyme in vivo, the stability of the polypeptide is good, the pharmacokinetics stability is high, the inhibition effect on HIV membrane fusion is strong, and the polypeptide can be used for treating or preventing AIDS or used for inhibiting HIV membrane fusion.
The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.
The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.
For the purpose of more clearly illustrating the following examples, the following are provided for the symbolic abbreviations of the raw materials used in the examples, and the parts where the symbolic abbreviations are used refer to the corresponding parts of the symbolic abbreviations in the examples, which are as follows:
example 1
In the examples of the present invention, the polypeptides provided by the present invention were obtained by standard Fmoc solid phase synthesis strategy. In order to mask the negative charge of the C-terminal, the carboxyl group of the last amino acid of the C-terminal was changed to an amide using Rink Amind AM resin. The amino acids adopted in the solid phase synthesis process are standard Fmoc amino acids and Fmoc-S5-OH. After the linear peptide synthesis is completed, the first generation Grubbs is used for catalyzingThe agent performs an olefin metathesis reaction on the resin. TFA (trifluoroacetic acid)/TIPS (triisopropylsilyl)/H was then used2O cleaving the polypeptide from the resin. And finally, purifying the polypeptide by using HPLC, and detecting by ESI-MS. The specific synthesis steps are as follows:
1. amino acid condensation: 300mg of Rink Amide AM resin having a degree of substitution of 0.33mmol/g was weighed into a 80mL polypeptide synthesis tube, and mixed solvent of 2mL of DMF (dimethylformamide) and 6mL of DCM (dichloromethane) was added for swelling. After 0.5h the solvent was pumped dry with water. The resin was washed sequentially with DMF (5mL × 5), DCM (5mL × 5), DMF (5mL × 5). The Fmoc protecting group was then removed using 5mL of 20% piperidine DMF solution (5min +10 min). The above washing of the resin, amount and method was repeated as described above. Finally, an amino acid reaction solution (Fmoc-AA-OH 4equiv., HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate) which was activated in advance for one minute was added thereto, and the mixture was reacted for 1.5 hours, wherein the HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate) was 3.9equiv. (Equivalent, the same applies hereinafter), HOBt (1-hydroxybenzotriazole) 4equiv., DIEA (N, N-diisopropylethylamine) 8equiv., and DMF5 mL). The condensation of the natural amino acids on the resin is carried out as described above. Condensation of unnatural amino acids Fmoc-S5The combination of reaction liquid is Fmoc-S when the-OH is performed5-OH 2equiv., HATU (2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate) 1.9equiv., HOBt 2equiv, DIEA 4equiv, DMF 3ml, reaction time 2 h.
2. Olefin metathesis: after the polypeptide chains are assembled on the resin, an olefin metathesis reaction is carried out. Under the protection of argon, a solution of Grubs' catalyst in 1, 2-dichloroethane (40mg/5mL) was added and the reaction was carried out at room temperature. After 2h, a solution of Grubs' catalyst in 1, 2-dichloroethane was added again. After reacting for 2h, washing with DMF and DCM respectively.
3. Acetylation modification: the N-terminal of the polypeptide is acetylated and modified, and the specific operation is as follows. After olefin metathesis, Fmoc of the last amino acid in the polypeptide chain was removed, washed, and 5mL of acetylating reagent (Ac) was added2O (acetic anhydride) DIEA DMF 1:1:8, v: v: v) was reacted, 5min later acetylation reagent was added again, and finally DMF, DCM was used again for washing.
4. Protecting group cleavage: 10mL of a cleavage reagent (TFA: TIPS: water: 95:2.5:2.5, v: v: v) was added to the polypeptide synthesis tube, and the reaction was carried out at room temperature. After 2.5h the cleavage reagent was transferred to a 50mL centrifuge tube and the TFA in the centrifuge tube was blown to 2mL using Ar gas. Then, 40mL of glacial ethyl ether was added to the centrifuge tube, and the mixture was precipitated and centrifuged. Finally the ether solution was poured off leaving a precipitate.
5. Polypeptide separation and identification: the analysis of the crude peptide was performed by using SHIIMAZU high performance liquid chromatography, GRACE Vydac C184.6X 250mm high performance liquid chromatography column with mobile phases A acetonitrile containing 0.1% TFA and B water containing 0.1% TFA, and gradient elution was performed from 10% B to 80% B at 30min flow rate of 1 mL/min. After the polypeptide was identified by ESI mass spectrometry, the crude peptide was isolated and purified using a GRACE Vydac C18 high performance liquid semi-preparative column.
The solid phase synthesis route of the polypeptide is as follows:
through the method, 12 polypeptides (SFT-0, SFT-1, SFT-2; SC34EK-0, SC34EK-1, SC34 EK-2; MT-SC22EK-0, MT-SC22EK-1A, MT-SC22EK-1B, MT-SC22EK-2, CP32M-0 and CP32M-1) in total of four types are synthesized, wherein 0, 1 and 2 respectively indicate the number of formed stapling structures (namely covalent bonds). Wherein SFT-0, SC34EK-0, MT-SC22EK-0 and CP32M-0 are respectively SEQ ID NO: 1-4, and the rest polypeptides are polypeptide derivatives obtained by modifying the four polypeptides. The specific amino acid sequence of the polypeptide is as follows:
example 2
The physicochemical properties of the 12 polypeptides (SFT-0, SFT-1, SFT-2; SC34EK-0, SC34EK-1, SC34 EK-2; MT-SC22EK-0, MT-SC22EK-1A, MT-SC22EK-1B, MT-SC22EK-2, CP32M-0, CP32M-1) obtained in example 1 were analyzed by circular dichroism.
1. Experimental methods
Each of 12 polypeptides was dissolved in phosphate buffer at pH 6.8 to give a test solution at a concentration of 50 μ M. The test solution was transferred to a 0.1mm quartz cuvette at 25 ℃ and the circular dichroism spectrum of the polypeptide was tested using a Jasco-715 spectroscopic polarimeter. Spectroscopic polarimeter measurement parameters: wavelength 190-260nm, step size 0.5nm, and 3 times of accumulation.
2. Results of the experiment
the typical α -helix has a positive absorption peak at 195nm and negative absorption peaks at both 208nm and 222nm of the circular dichroism spectrum, it can be seen from FIG. 1 that 12 polypeptides have double negative peaks at 208nm and 222nm and a positive absorption peak at 195nm, indicating that the synthesized polypeptides all have α -helix conformation]222And (6) determining. By contrast to CD absorption peak at 222nm [ theta ]]222, using the calculation formula of the degree of helicity of the polypeptide [ theta ]]222/[θ]maxThe degree of helicity of the polypeptide can be calculated. The degree of helicity of the four types of 12 polypeptides is shown in FIG. 1.
Example 3 (enzyme stabilizing Properties of the polypeptide)
In this example, 7 of the 12 polypeptides obtained in example 1 (SC34EK-0, SC34EK-1, SC34 EK-2; MT-SC22EK-0, MT-SC22EK-1A, MT-SC22EK-1B, MT-SC22EK-2) were analyzed for their enzyme stability by high performance liquid chromatography.
1. Experimental methods
The 7 polypeptides were prepared as DMSO stock solutions at a concentration of 1mM, respectively. 50. mu.L of the stock solution was added to 1950. mu.L of phosphate buffer (0.05mol/L Na)2HPO4，2mM CaCl2pH 7.8, 0.5 ng/. mu.Lcymoprisin) at 30 deg.CAnd carrying out enzymolysis reaction. At different time periods, 100. mu.L of the enzymolysis reaction solution is added with 20. mu.L of 1M hydrochloric acid to quench the enzymolysis reaction. The degradation kinetic process of the enzymolysis reaction liquid of the polypeptide at different time periods is obtained by monitoring the enzymolysis reaction liquid of the polypeptide by using high performance liquid chromatography.
2. Results of the experiment
The chymotrypsin degradation kinetics curves of 7 polypeptides are shown in FIG. 2. Obtaining the half-life of the polypeptide according to the chymotrypsin degradation kinetic curve of each polypeptide, wherein the half-life of each polypeptide is as follows: the half-life of SC34EK-0 is 55min, the half-life of SC34EK-1 is 109min, the half-life of SC34EK-2 is 101min, the half-life of MT-SC22EK-0 is 125min, the half-life of MT-SC22EK-1A is 405min, the half-life of MT-SC22EK-1B is 725min, and the half-life of MT-SC22EK-2 is 3665 min.
By comparing the half-lives of the two types of polypeptides it was found that:
1. the half-life sequences of the two types of polypeptides are SC34EK-2> SC34EK-1> SC34 EK-0; MT-SC22EK-2> MT-SC22EK-1B > MT-SC22EK-1A > MT-SC22 EK-0. The stability of the polypeptide to the chymotrypsin can be effectively enhanced by carrying out skeleton toughening structure modification.
2. Polypeptide MT-SC22EK series half-life containing 24 amino acids > polypeptide SC34EK series half-life containing 34 amino acids. Thus, the stability of the short peptide to the chymotrypsin is higher than that of the long peptide.
Example 4
In this example, the activity of the polypeptide to inhibit infection by the HIV pseudovirus was tested by the HIV test system.
1. Experimental methods
The 12 polypeptides (SFT-0, SFT-1, SFT-2; SC34EK-0, SC34EK-1, SC34 EK-2; MT-SC22EK-0, MT-SC22EK-1A, MT-SC22EK-1B, MT-SC22EK-2, CP32M-0 and CP32M-1) obtained in example 1 were each prepared as DMSO stock solutions at a certain concentration. Three HIV pseudovirus Chinese epidemic strains with different subtypes and three HIV pseudovirus standard strains are used for detecting the activity of polypeptide inhibiting virus infection, wherein CNE6 and CNE11 are B' subtype, CNE15 and CNE30 are BC subtype, CNE5 and CNE55 are CRF01_ AE subtype, sf162, JRFL and HXB2 are HIV standard strain B subtype (the construction method of the strains is referred to as Hong Shang, Xiaoxu Han, Xuanling Shi, et al.J Biol chem.2011Apr 22; 286(16) 14531-41, provided by Zhanghua university Productus chun laboratories). The 9 HIV pseudoviruses all contain a luciferase reporter gene detection system, 12 polypeptides are detected by using the 9 pseudoviruses respectively, and the capacity of HIV pseudoviruses to infect cells is obtained by measuring the activity of the reporter gene luciferase.
Packaging and detection methods for HIV pseudoviruses: infectious but replication-incompetent HIV pseudoviruses were prepared by co-transfecting 293T cells with a eukaryotic expression plasmid (pcDNA3.1+, available from Invitrogen) capable of expressing full-length membrane proteins of various subtypes of HIV and a backbone plasmid pNL4-3R-E-luciferase (ref: He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR.J Virol 69: 6705-6711, 1995). The HIV pseudovirus is incubated with the polypeptide, then the supernatant is infected with HIV susceptible cells GHOST (3) X4/R5, and the HIV pseudovirus infection capacity is determined by measuring the activity of the reporter gene luciferase. In the GHOST (3) X4/R5 experiment of the susceptible cells of the supernatant infected with HIV, if the polypeptide has the capability of inhibiting GHOST (3) X4/R5 of the HIV infected susceptible cells, the number of viruses capable of entering GHOST (3) X4/R5 is obviously reduced, and the expression quantity of luciferase carried by HIV pseudoviruses in GHOST (3) X4/R5 is also reduced, so the virus quantity of the infected cells can be quantitatively detected by measuring the activity of the luciferase in GHOST (3) X4/R5, and the capability of the polypeptide in inhibiting HIV infection can also be reflected.
2. Results of the experiment
The inhibition curve of each polypeptide to 9 types of HIV pseudoviruses can be obtained by detecting the activity of luciferase, so that an activity parameter (namely, the half inhibition concentration, the lower the half inhibition concentration, the stronger the inhibition activity to HIV infection) for representing the inhibition of the polypeptide to HIV pseudovirus infection can be obtained, and specific results are shown in tables 1-4.
TABLE 1 half inhibitory concentration (IC50, nM) of SFT series of polypeptides against HIV pseudovirus
| Pseudovirus name | Subtype of cell | 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 2 median inhibitory concentration (IC50, nM) of SC34EK series polypeptides against HIV pseudovirus
| Pseudovirus name | Subtype of cell | 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 3 median inhibitory concentration (IC50, nM) of MT-SC22EK series polypeptides against AIDS pseudovirus
TABLE 4 half inhibitory concentration (IC50, nM) of CP32M series of polypeptides against HIV pseudovirus
| Pseudovirus name | Subtype of cell | 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 |
By comparing the median inhibitory concentrations of the four types of polypeptides it was found that:
(1) the order of median inhibitory concentration for the four types of polypeptides was: SC34EK > MT-SC22EK > SFT > CP 32M. The half inhibitory concentration of the polypeptide without modification is 2-10 times of that of the polypeptide modified by a skeleton toughened structure (namely, the polypeptide forming covalent bonds in polypeptide molecules).
(2) For the polypeptide with the number of amino acids larger than 30, the activity of the peptide modified by carrying out the skeleton toughening structure twice is lower than that of the peptide modified by carrying out the skeleton structure once. For the polypeptide with the number of amino acids less than 30, the peptide activity of the peptide subjected to the framework toughening structure modification twice is higher than that of the peptide subjected to the framework toughening structure modification once.
(3) The 12 polypeptides have the activity of inhibiting HIV membrane fusion process, wherein the activity of SC34EK-1 and MT-SC22EK-2 is the best.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (5)
1. An isolated polypeptide having the property of high enzymatic stability, wherein the sequence of said polypeptide is as shown in the following table,
wherein,
s of the polypeptide sequence5Is (S) -2- (4 '-pentenyl) -alanine, and the side chains of the (S) -2- (4' -pentenyl) -alanine form covalent bonds which are carbonA carbon double bond.
2. The polypeptide of claim 1, wherein said polypeptide has activity in inhibiting HIV membrane fusion.
3. the polypeptide of claim 1, wherein the polypeptide has an α -helical conformation.
4. A pharmaceutical composition, comprising: the polypeptide of any one of claims 1 to 3.
5. Use of a polypeptide according to any one of claims 1 to 3 for the preparation of a medicament for the treatment or prophylaxis of aids or for the inhibition of HIV membrane fusion.
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| 订书肽的合成与应用;高帅;《化学进展》;20131225;第26卷(第1期);摘要,第100页左栏倒数第2段,右栏第2段,第100-101页跨页段,第102页左栏首段，第103页左栏首段，第104页左栏倒数第2段至右栏第2段，第106页右栏至第107页左栏第一段第4.2部分，图1-2，表3 * |
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