WO2013127288A1 - 抗hiv-1多肽及其用途 - Google Patents
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- WO2013127288A1 WO2013127288A1 PCT/CN2013/071245 CN2013071245W WO2013127288A1 WO 2013127288 A1 WO2013127288 A1 WO 2013127288A1 CN 2013071245 W CN2013071245 W CN 2013071245W WO 2013127288 A1 WO2013127288 A1 WO 2013127288A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the invention belongs to the field of biomedicine and relates to a class of polypeptides, in particular a polypeptide of anti-HIV-1 or other related envelope virus, and the use of the polypeptide in the preparation of an HIV fusion inhibitor, in preparation for treatment or Use in the prevention of HIV-associated diseases, especially AIDS drugs, and in the preparation of drugs for the treatment or prevention of other related enveloped virus infections.
- Background technique a polypeptide of anti-HIV-1 or other related envelope virus
- Type I human immunodeficiency virus (HIV-1) is the causative agent of AIDS. There are more than 30 million infected people worldwide, causing about 2 million deaths each year, and there are about 2 million new infected people every year, which is a serious threat. A global infectious disease of human health. HIV-1 infects host cells via its envelope glycoprotein (Env)-mediated viral-cell membrane fusion. Env contains a surface subunit gpl20 and a transmembrane subunit gp41, and three Env forms a non-covalent complex embedded in the surface of the virus.
- Env envelope glycoprotein
- the surface subunit gpl20 is responsible for molecular recognition during viral infection of cells to find and access sputum cells, while functioning as a stable transmembrane subunit of gp41, and releasing gp41 at appropriate timing to initiate fusion; transmembrane subunit gp41 is a virus - a direct functional molecule of cell membrane fusion.
- transmembrane subunit gp41 is a virus - a direct functional molecule of cell membrane fusion.
- NHR and CHR C-terminal helix region
- Drugs that prevent the formation of six helices are effective in inhibiting HIV-cell membrane fusion, thereby preventing viral infection and in vivo transmission, and are used for AIDS treatment, hence the name fusion inhibitor.
- the crystal structure is shown in the six helices, and the three spiral structures formed by NHR form the inner core, forming three grooves, and the three CHRs are antiparallelly combined in the grooves.
- the exogenous CHR polypeptide binds to the HR target to form an inactive six-helix structure, prevents endogenous active helicoid formation, inhibits viral-cell fusion and viral infection, and thus acts as a fusion inhibitor.
- Typical C-peptide fusion inhibitors include C34 (US 6,150,088) and its improved polypeptides, the first marketed fusion inhibitor T20 (US 5,464,933), and the later discovered CP32 (CN1793170, CN1955190).
- C-peptide fusion inhibitors block viral infection by binding to their corresponding NHR targets; typical targets include N36 (US 6,150,088) and DP107 (US 5,656,480), which combine with C34 and CP32 to form a six-helix structure, N36 and DP107, respectively.
- NHR targets include N36 (US 6,150,088) and DP107 (US 5,656,480), which combine with C34 and CP32 to form a six-helix structure, N36 and DP107, respectively.
- the present inventors have proposed a design idea of designing an anti-HIV-1 active peptide from the corresponding envelope glycoprotein sequences of other enveloped viruses. Based on the similarity of the enveloped virus fusion mechanism, we extracted a peptide sequence of about 36 amino acids from the CHR sequence of other enveloped viral envelope glycoprotein transmembrane subunits, designed an anti-HIV-1 polypeptide, and determined its anti-HIV mediator. Guided cell-cell fusion activity, and its binding to HIV-1 gp41 NHR, to determine its target, explore new ideas for the design of HIV-1 fusion inhibitors. The present invention has thus been completed.
- a first aspect of the invention relates to a polypeptide selected from the group consisting of formula (1) to formula (42),
- Equation (4)
- Equation (6)
- X is acetyl, oligopeptide sequence, lipophilic group, polyethylene glycol or deletion; in an embodiment of the invention, X is acetyl ( CH 3 -CO- );
- Z is an amide group, an oligopeptide sequence, a lipophilic group, a polyethylene glycol or a deletion; in an embodiment of the invention, Z is an amide group (-CO-NH 2 ).
- oligopeptide sequence is 1 to 10 amino acid sequences, and may be, for example, EEE, KKK, GKK, or GQAV.
- the lipophilic group is a fatty acid and a sterol, and for example, it may be n-octanoate (C 7 H 15 -CO-0- ), laurate ( C 13 H 27 -CO-0- ), palmitate ( C 15 H 31 -CO-0- ), or cholesterol (C 27 H 46 0).
- the X and Z groups are used to improve the water solubility, secondary structure and stability of the polypeptide, enhance binding to the cell membrane to bring it closer to the target, or improve the pharmacokinetic properties of the drug.
- sequences of the above formulas (1) to (42) are derived from the CHR sequences of the fusion glycoprotein transmembrane subunit of the envelope virus, some of which introduce a tryptophan (W) residue at a key position at the amino terminus of the polypeptide.
- Base preferred is 1 bit, or 4 bits at the same time.
- formula (1) ⁇ (3) is derived from human parainfluenza virus type 3 HPIV3, and formula (4) ⁇ (6) is derived from monkey parainfluenza virus SV5, and formula (7) ⁇ (9) is derived from severe acute respiratory syndrome.
- SARS virus formula (10) ⁇ (12) from mouse hepatitis virus MHV, formula (13) ⁇ (15) from Newcastle disease virus DV, formula (16) ⁇ (18) from human metapneumovirus hMPV, Formula (19) ⁇ (21) from respiratory syncytial virus RSV, formula (22) ⁇ (24) from hepatitis E virus HeV, formula (25) ⁇ (27) from Nipah virus iV, formula (28) ⁇ (30) from measles virus MeV, formula (31) ⁇ (33) from Sendai virus SeV, formula (34) ⁇ (36) from avian pneumovirus APV, formula (37) ⁇ (39) from feline infectious peritonitis virus FIPV, formula (40) ⁇ (42) from human coron
- a polypeptide according to the first aspect of the invention which is selected from the group consisting of:
- SEQ ID NO: 1 - 42 has a truncation of 1-10, 1-5, 1-2 amino acids at the N-terminus and/or C-terminus;
- SEQ ID NO: 1 - 42 has an extension of 1-10, 1-5, 1-2 amino acids at the N-terminus and/or C-terminus; preferably, the extension is in the sequence Extension of the CHR sequence of the fusion glycoprotein transmembrane subunit of the source virus;
- SEQ ID NOs: 1 to 42 have 1-10, 1-5, 1-2 amino acid substitutions, or 1-10, 1 in the sequences of (1) and (2) above. -5, a substitution of 1-2 amino acids; preferably, the substitution is based on a different CHR sequence between different strains of the virus derived from the sequence due to the transmembrane subunit of the fusion glycoprotein;
- polypeptide obtained by chemically modifying the terminal and/or side chain of any of the polypeptides of SEQ ID NOS: 1 to 42 or the polypeptide of the above (1) to (3) is chemically modified by terminal and/or side chain Peptide.
- polypeptide of the above (1) to (4), the first amino acid and/or the fourth amino acid are replaced with a tryptophan residue, and/or the eighth amino acid is replaced with a hydrophobic amino acid residue.
- terminal and/or side chain chemical modification comprises modification at the amino terminus, the carboxy terminus or the side chain
- the modifying group includes an oligopeptide, an acetyl group, an amide group, a lipophilic group, a hydrophilic group, etc. It is to improve the stability of the polypeptide, improve the pharmacokinetics or increase the binding to the cell membrane.
- polypeptide according to the first aspect of the invention which is one or more selected from the polypeptides of any one of the first aspects of the invention.
- the polypeptide includes both a compound obtained by dehydration condensation of a 10-100 amino acid molecule directly, and a compound containing a modified group obtained by the above modification on the compound.
- the polypeptide preferably has a length of 30 to 40 amino acids, for example, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40.
- the polypeptides described above still have activity against the fusion of HIV-1 or other enveloped viruses with target cells, such as human cells, after truncation, extension, substitution and/or modification.
- the hydrophobic amino acid residue includes tryptophan, phenylalanine, isoleucine, leucine, valine, tyrosine, methionine.
- polypeptide of the present invention can be directly synthesized by a method known in the art, or can be obtained by molecular biological means by cloning a nucleotide sequence corresponding to the polypeptide into a recombinant vector and expressing it in a recombinant cell.
- the invention therefore also relates to a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of the first aspect of the invention.
- the present invention also relates to a recombinant vector comprising the nucleic acid molecule of the present invention; and a recombinant cell comprising the recombinant vector of the present invention.
- a fourth aspect of the invention relates to a fusion protein comprising the polypeptide of the first aspect of the invention or a derivative thereof, a stereoisomer, a physiologically toxic salt, or the polypeptide or derivative thereof, stereoisomer, a complex obtained by coupling or fused a physiologically toxic salt with a carrier such as another protein or a toxin or a lipid (such as a macromolecular carrier or a recombinant vector), or displaying the above polypeptide or a derivative thereof, a stereoisomer, and no physiological toxicity.
- Salt-like virus particles such as another protein or a toxin or a lipid (such as a macromolecular carrier or a recombinant vector), or displaying the above polypeptide or a derivative thereof, a stereoisomer, and no physiological toxicity.
- the polypeptide of the present invention is mainly administered by injection, and can be directly injected or made into a sustained release injection; it can also be made into an orally usable drug by improving metabolic stability, including physical and chemical modification; Topical or mucosal administration.
- compositions e.g., a pharmaceutical composition
- a composition comprising at least one polypeptide of the first aspect of the invention or a derivative thereof, a stereoisomer, a salt of no physiological toxicity, or the invention
- the composition may comprise at least one polypeptide or a derivative thereof, a stereoisomer, a physiologically toxic salt, or the fusion protein, complex or virus-like particle of the fourth aspect of the invention.
- two or more may be used to further enhance the fusion inhibitory effect; or the composition may comprise the polypeptide of the present invention or a derivative thereof, a stereoisomer, a physiologically toxic salt, and the present invention.
- Fusion proteins, complexes or viroid-like particles, as well as other fusion inhibitors such as C34, T20 or CP32.
- Another aspect of the invention relates to the polypeptide of the first aspect of the invention, or a derivative thereof, a stereoisomer, a physiologically toxic salt, the fusion protein, complex or virus-like particle of the fourth aspect, for the preparation of HIV Use in fusion inhibitors.
- the fusion inhibitor refers to a compound or composition which can prevent the formation of an active six-helix structure of HIV-1 itself and thereby inhibit fusion of a virus-cell membrane, that is, by formation An inactive six-helix structure blocks a virus-cell membrane fusion compound or composition.
- the fusion inhibitor refers to a polypeptide or a modified polypeptide, or a composition comprising a polypeptide or a modified polypeptide.
- the present invention also relates to the polypeptide of the first aspect of the present invention or a derivative thereof, a stereoisomer, a physiologically toxic salt, the fusion protein, complex or virus-like particle of the fourth aspect, which is prepared for treatment or Use in the prevention of HIV-related diseases such as AIDS drugs or other diseases caused by envelope-type viral infections.
- the HIV includes HIV-1 and HIV-2 type viruses, and in an embodiment of the present invention, the HIV is an HIV-1 type virus.
- the other envelope virus refers to a virus that utilizes the first type of fusion protein as a fusion tool, including but not limited to human parainfluenza virus type 3 HPIV3, monkey parainfluenza virus SV5, severe acute respiration Syndrome SARS virus, mouse hepatitis virus MHV, Newcastle disease virus NDV, human metapneumovirus hMP V, respiratory syncytial virus RSV, hepatitis E virus HeV, Nipah virus NiV, measles virus MeV, Sendai virus SeV, avian lung Viral APV, feline infectious peritonitis virus FIPV or human coronavirus 229E HCoV-229E.
- human parainfluenza virus type 3 HPIV3, monkey parainfluenza virus SV5 severe acute respiration Syndrome SARS virus
- mouse hepatitis virus MHV Newcastle disease virus NDV
- human metapneumovirus hMP V human metapneumovirus hMP V
- the present invention also relates to a method for preventing or treating a disease caused by an HIV infection-related disease such as AIDS or other envelope virus infection, the method comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the first aspect of the present invention A step of the polypeptide or derivative thereof, stereoisomer, non-physiological salt, fusion protein, complex or virus-like particle of the fourth aspect.
- HIV uses Class I fusion protein as a tool molecule for virus-cell membrane fusion.
- viruses that use this fusion protein, including some viruses that pose a serious threat to human health.
- the first class of fusion proteins have the following commonalities: both transmembrane subunits and surface subunits, in which transmembrane subunits are directly involved in fusion; the extramembranous portion of transmembrane subunits generally contains functional regions such as fusion peptides, NHR and CHR;
- the NHR is trimeric to form the inner core, and the CHR is anti-parallel folded into the groove formed by the NHR to form a six-helix structure.
- the energy released during the formation process promotes the fusion of the virus and the cell membrane to complete the infection [1] .
- the inventors designed a HIV fusion inhibitor based on the CHR of the fusion glycoprotein transmembrane subunit based on the virus using the first type of fusion protein as a fusion tool.
- the amino acid sequence of the envelope glycoprotein of this type of virus is known, and the functional region is also determined.
- the inventors extracted the appropriate polypeptide fragment from the CHR sequence, and based on the similarity of the fusion mechanism, it is predicted that these polypeptide fragments can be folded anti-parallel to In the groove formed by NHR, an inactive six-helix structure was formed and verified by a fusion inhibition activity experiment.
- the present invention therefore also relates to a method for obtaining a fusion inhibitor that inhibits fusion of HIV with a target cell.
- Method comprising truncating a polypeptide fragment of a suitable length from a sequence of a CHR domain of a virus other than HIV, the polypeptide fragment being capable of binding to the NHR functional region to form an inactive six-helix structure.
- the length of the CHR is about 40 amino acid residues, it is quite intuitive to take 30-40 residues of the fragment for use as an HIV fusion inhibitor, without special technique, but does not exclude computer simulation. Find the best interception effect.
- the virus other than HIV refers to a virus using the first type of fusion protein as a fusion tool, for example, an envelope virus; in one embodiment of the present invention, the virus is human parainfluenza virus type 3 HPIV3 [2] ] , monkey parainfluenza virus SV5 [3] , severe acute respiratory syndrome SARS virus [4] , mouse hepatitis virus MHV [4] , Newcastle disease virus NDV [5] , human metapneumovirus hMPV [6] , respiratory tract Cytovirus RSV [3] , Hepatitis E virus HeV [2] , Nipa virus NiV [7] , Measles virus MeV [7] , Sendai virus SeV [7] , Avian lung virus APV [8] , Cat infectious peritonitis Virus FIPV [9] or human coronavirus 229E HCoV-229E [9] .
- the virus is human parainfluenza virus type 3 HPIV
- the appropriate length is meant to comprise 30-40 amino acids, such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40.
- the present invention also relates to a fusion inhibitor obtained by the above method according to the present invention for inhibiting the fusion of HIV and target cells.
- the fusion inhibitor is selected from the group consisting of the polypeptides of the above formulas (1) to (42).
- the fusion inhibitor refers to a polypeptide or a modified polypeptide, or a composition comprising a polypeptide or a modified polypeptide.
- polypeptides represented by the formulae (1) to (42) wherein X is an acetyl group and Z is an amide group are also referred to as polypeptides 1 to 42, respectively.
- the polypeptide of the present invention shows significant anti-HIV activity
- the target action experiment shows that the polypeptide acts on the gp41 NHR region, and forms an inactive six-helix structure with NHR, destroys the structure of HIV NHR, and inhibits the fusion of HIV and target cells.
- the results of the activity experiments indicate that the mechanism of action of the polypeptide of the present invention is different from the known polypeptide fusion inhibitors containing the WWI structural template pocket binding region.
- Figure 1 shows the interaction of the polypeptide of the present invention with N46.
- the abscissa wave is the scanning wavelength, and the unit is nm; the ordinate is the CD signal, and the unit is mdeg.
- A is a polypeptide 1
- B is a polypeptide 4
- C is a polypeptide 7
- D is a polypeptide 10
- E is a polypeptide 13
- F is a polypeptide 16
- G is a polypeptide 19
- H is a polypeptide 22.
- AIDS (Acquired Immure Deficiency Syndrome) AIDS, Acquired Immune Deficiency Syndrome.
- Env envelope glycoprotein
- HIV Human immunodeficiency virus
- W is tryptophan
- N is asparagine
- A is alanine
- S is serine
- K is lysine
- L leucine
- E glutamic acid
- Q glutamine
- I is different.
- Acid H is histidine
- M is methionine
- T is threonine
- D is aspartic acid
- R is arginine
- Y is tyrosine
- F is phenylalanine
- V Proline
- P valine
- G is glycine.
- the solid phase synthesis carrier Rink amide resin used in the examples is Tianjin Nankai Synthetic Co., Ltd.; HBTU, HOBt, DIEA and Fmoc protected natural amino acids and D-type unnatural amino acids are products of Shanghai Jill Biochemical Co., Ltd. and Chengdu Nuoxin Technology Co., Ltd. . N-decylpyrrolidone
- the inventors systematically examined the fusion principle and inhibitory properties of enveloped viruses, from multiple enveloped viruses (human parainfluenza virus type 3 HPIV3 [2] , monkey parainfluenza virus SV5 [3] , severe acute respiratory syndrome).
- SARS virus [4] mouse hepatitis virus MHV [4] , Newcastle disease virus NDV [5] , human metapneumovirus hMPV [6] , respiratory syncytial virus RSV [3] , hepatitis E virus HeV [2] , Nipah virus NiV [7] , measles virus MeV [7] , Sendai virus SeV [7] , pneumovirus APV [8] , feline infectious peritonitis virus FIPV [9] and human coronavirus 229E HCoV-229E [9]
- CHR sequence of the fusion glycoprotein transmembrane subunit a corresponding polypeptide of about 36 amino acids in length is
- the designed polypeptide sequence SEQ ID NO: 1-42 is shown in Table 1, together with the virus from which it is derived.
- W tryptophan
- a standard Fmoc solid phase peptide synthesis method was employed.
- the C-terminus of the anti-HIV-1 polypeptide sequence of Example 1 designed as SEQ ID NO: 1-42 was amidated and N-terminally acetylated to give polypeptide 1-42.
- Rink Amide resin was selected and the peptide was extended from the C-terminus to the N-terminus.
- the condensing agent is HBTU/HOBt/DIEA.
- the deprotecting agent is a piperidine/DMF solution.
- the lysing agent is TFA, and the crude peptide is dissolved in water and stored in lyophilization. It was isolated and purified by medium pressure liquid chromatography or HPLC with a pure peptide content of >95%.
- Matrix-assisted laser desorption time-of-flight mass spectrometry MALDI-TOF-MS was used to determine the molecular weight of the peptide.
- the prepared lysate was added to the peptide resin under ice bath conditions, electromagnetically stirred, the resin turned orange-red, and reacted for 30 minutes under ice bath conditions, then the ice bath was removed, and the reaction was further continued at room temperature for 90 minutes to complete the reaction.
- 200 ml of cold diethyl ether was added to the reactor under vigorous stirring, and a white precipitate was precipitated, and stirring was continued for 30 min.
- the precipitate was filtered through a G4 sand filter suction funnel, washed repeatedly with cold diethyl ether for 3 times, and dried. 50 ml of double distilled water and 5 ml of acetonitrile were added to dissolve the solid sufficiently, and the mixture was suction filtered, and the filtrate was freeze-dried to obtain 1.03 g of a crude peptide.
- the crude peptide obtained was purified by medium pressure or high pressure chromatography.
- the color i column is a C18 column, and the eluent is acetonitrile, water and a small amount of acetic acid.
- the color i-column was pre-equilibrated with 200 ml of 15% acetonitrile/water/0.1% glacial acetic acid solution.
- the mixture was further equilibrated with 200 ml of the same eluent, and the eluent component was detected by high performance liquid phase.
- the acetonitrile content was gradually increased according to the test results until the purified polypeptide peak was eluted.
- the same fractions of the eluate were combined, and most of the solvent was removed by rotary evaporation, and the peptide was freeze-dried to obtain a purity of >90% by HPLC.
- Polypeptide 1-42 was synthesized by the above method (see Table 1). Polypeptide sequence, source and molecular weight
- IKEAQRLLDTV SEQ ID NO: 27
- SNKILDSIEKGN SEQ ID NO: 34
- SNKILDSIEKGN SEQ ID NO: 36
- Target cells are TZM-bl cells (US NIH AIDS reagents and reference items) Provided, catalogue number 8129), which expresses CD4 T-cell receptor and chemokine co-receptors CCR5 and CXCR4, which are recognized by HIV-1 Env, and also fluoresceinase reporter gene in cells, but not The promoter containing this gene, therefore, the luciferase background expression of the cells alone is very low.
- the effector cells are HL2/3 cells (US NIH AIDS Reagents and References Project, catalog number 1294), which express HIV-1 Env on the surface, attack target cells by Env, complete cell fusion, and also fluoresceinase in cells.
- the promoter of the reporter gene Both cells were cultured separately in DMEM containing 10% fetal bovine serum containing ampic/streptomycin double antibody at 37 ° C in an incubator containing 5% CO 2 . Both cells were adherent cells, which were harvested by trypsin/EDTA digestion. Cells were counted using a cell counting plate.
- the TZM-bl target cells were adjusted to a concentration of 750,000/ml in a medium, and added to a 96-well cell culture plate (37,500/well) at 50 ⁇ l per well, and cultured for 24 hours at 5% C0 2 at 37 °C.
- the polypeptide 1-42 or the positive control sample T20 was dissolved in phosphate buffered saline (PBS) or dissolved in an appropriate amount of DMSO, and the polypeptide concentration was measured at 280 nm by an ultraviolet light microscope. The peptide solution was then diluted to the appropriate concentration and diluted moderately in a 96-well plate (Coring) at concentrations of 1200, 300, 75, 19, 5, 1.25 ⁇ .
- PBS phosphate buffered saline
- DMSO DMSO
- the luciferase reporter gene kit (Promega) was taken out from the refrigerator, and the 5x cell lysate was diluted with double distilled water to lx lysate according to the amount, and placed at room temperature; the substrate was dissolved in the substrate buffer at room temperature. Place; At the same time, set the detection conditions of the microplate reader (Molcular Devices M5) to be set aside.
- the fused cells were taken out, the medium was discarded, and washed twice with 200 ⁇ l/well PBS, and the washing solution was removed as much as possible; then, the lysate equilibrated to room temperature was added at 50 ⁇ l/well, and the cells were fully lysed by gently shaking for 5 minutes; The lysate was added to the 96-well chemiluminescence detection plate ( Corning) at 40 ⁇ l/well, and the introduction of bubbles was avoided as much as possible; the substrate was rapidly added to the chemiluminescent ELISA plate at 40 ⁇ l/well in the dark. Immediately measure chemiluminescence on a microplate reader.
- Effective fusion of target cells and effector cells was determined based on the ratio of saturated fusion signal to background signal, with a ratio > 5 indicating efficient fusion.
- the concentration of the semi-inhibitor (IC 5 ) was determined from the concentration-chemiluminescence signal curve of the sample, and the IC 5 of the positive control sample. The value should be stable within a certain range; the ideal inhibition curve
- the signal at the medium to high concentration inhibitor should be close to the background signal, and the signal at the lowest concentration inhibitor should be close to the saturated fusion signal.
- the cell fusion inhibitory activity of polypeptide 1-42 is listed in Table 2, IC 5 of the positive control T20. It is 2 ⁇ 0.5 nM, which is consistent with the literature report [1()] .
- Table 2 Cell fusion inhibitory activity of polypeptide 1-42
- polypeptides 1-42 showed significant anti-HIV activity except for 3, 15, 21, 42 and a semi-inhibitory concentration of IC 5 . From 1 to several tens of ⁇ , all the viruses studied contain active polypeptide sequences, and the activity has room for improvement, in line with the activity standards of drug lead compounds. It can also be seen that the activity is somewhat related to the tryptophan residue we introduced, but there is no obvious rule, so the mechanism of action is different from the known polypeptide fusion inhibitors containing the WWI structural template pocket binding region. However, the above results indicate that we can improve the activity by changing the amino acid residues. For example, Compound 21 is approximately 7-fold more potent by introducing a tryptophan residue.
- Example 4 Laboratory Adaptation of Polypeptides to HIV-1 IIIB Infection Inhibition Experiment
- the laboratory adapted to the strain HIV-1IIIB, MT-2 cells (obtained through the National Institutes of Health (NIH) AIDS Research Reference Reagent Program).
- Table 3 HIV viral infection inhibitory activity of peptides
- N46 (sequence: Ac-TLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARI L- CONH 2 ) is a gp41 NHR target, wherein the sequence TLTVQARQLLSGIVQQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL is SEQ ID NO: 43.
- the circular dichroic instrument is a Biologic MOS450 spectrometer.
- the CHR polypeptides 1, 4, 7, 10, 13, 16, 19, 22 to be determined were respectively dissolved in PBS, N46 was dissolved in double distilled water, and the concentration was determined according to ultraviolet absorption at 280 nm; then 20 ⁇ M of the polypeptide PBS solution was prepared.
- the prepared samples were measured on a circular dichroic instrument.
- the scanning wavelength range of the instrument was 190-260 nm, the wavelength interval was 1 nm, the scanning speed was 100 nm/min, and the scanning was performed 4 times for averaging. First scan the buffer solution to get a blank, then scan the sample signal, and subtract the blank signal from the sample signal to get the CD. signal
- Wild, CT, et al., Peptides corresponding to a predictive alpha-helical domain of human-immunodeficiency -virus type-1 gp41 are potent inhibitors of virus-infection. Proceedings of the National Academy of Sciences of the United States of America , 1994. 91(21): p. 9770-9774.
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Abstract
本发明提供了一类多肽,其可以抑制HIV或其他相关包膜类病毒与靶细胞的融合,所述多肽具有SEQ ID NO:1-42所示的序列。本发明还提供了所述多肽在制备HIV融合抑制剂中的用途,在制备用于治疗或预防HIV感染相关疾病尤其是艾滋病的药物中的用途,以及在制备用于治疗或预防其他相关包膜类病毒感染的药物中的用途。
Description
抗 H I V- 1多肽及其用途 技术领域
本发明属于生物医学领域, 涉及一类多肽, 特别是一类抗 HIV-1或其 它相关包膜类病毒的多肽, 以及所述多肽在制备 HIV融合抑制剂中的用途, 在制备用于治疗或预防 HIV感染相关疾病尤其是艾滋病的药物中的用途,以 及在制备用于治疗或预防其它相关包膜类病毒感染药物中的用途。 背景技术
I型人免疫缺陷病毒 ( HIV-1 )是艾滋病的病原体, 现全球有超过 3000 万感染者, 每年导致约 200万人死亡, 并且每年还有新增约 200万感染者, 是一种严重威胁人类健康的全球性感染疾病。 HIV-1通过其包膜糖蛋白( Env ) 介导的病毒-细胞膜融合感染宿主细胞。 Env包含表面亚基 gpl20和跨膜亚基 gp41, 三个 Env形成非共价复合体镶嵌在病毒表面。表面亚基 gpl20负责病 毒感染细胞过程中的分子识别以找到和接近耙细胞, 同时起着稳定跨膜亚基 gp41的功能, 并在适当时机释放出 gp41以启动融合; 跨膜亚基 gp41是病毒 -细胞膜融合的直接功能分子。 病毒细胞融合过程中有一由 gp41 N-端螺旋区 和 C-端螺旋区 (NHR和 CHR )形成的六螺旋结构; 该结构的形成为病毒- 细胞膜融合提供能量, 对病毒-细胞融合至关重要。 阻止六螺旋形成的药物可 有效抑制艾滋病毒-细胞膜融合,从而阻止病毒感染和体内传播, 用于艾滋病 治疗, 因此称为融合抑制剂。
晶体结构显示在六螺旋中, 三个由 NHR形成的螺旋结构构成内核, 形 成三个沟槽,三个 CHR反平行结合在沟槽中。外源 CHR多肽可结合在 HR 靶点中形成无活性的六螺旋结构, 阻止内源的活性六螺旋体生成,抑制病毒- 细胞融合和病毒感染, 从而用作融合抑制剂。 典型的 C-肽融合抑制剂包括 C34( US 6,150,088 )及其改进多肽、首个上市融合抑制剂 T20( US 5,464,933 )、 以及后来发现的 CP32 ( CN1793170, CN1955190 ) 。 这些 C-肽融合抑制剂 通过与其对应 NHR 靶点结合阻止病毒感染; 典型的靶点包括 N36 ( US 6,150,088 )和 DP107 ( US 5,656,480 ) , 分别与 C34和 CP32结合形成六螺 旋结构, 其中 N36 和 DP107 中含有一个共同的结合口袋, 与 CHR 的 WaaWaaal (其中 a为氨基酸)模板 (又称 WWI模板 )有关键相互作用,
也是小分子融合抑制剂的热门靶点。
尽管有上市的融合抑制剂 T20 和其它临床研究中的融合抑制剂如 Sifuvirtide ( CN1334122 ) , 但由于抗药病毒株的快速出现, 使得针对抗性病 毒的融合抑制剂研发成为当务之急, 尤其是序列来源与已有融合抑制剂明显 不同的融合抑制剂。 发明内容
基于以上原因, 本发明人提出了从其它类似包膜病毒对应包膜糖蛋白序 列设计抗 HIV-1活性肽的设计思想。 我们根据包膜病毒融合机制的相似性, 从其它包膜病毒包膜糖蛋白跨膜亚基的 CHR序列连续截取约 36个氨基酸的 肽序列 , 设计出抗 HIV-1多肽, 测定其抗 HIV介导的细胞-细胞融合活性, 同时研究其与 HIV-1 gp41 NHR的结合, 确定其作用靶点, 探索研究 HIV-1 融合抑制剂设计的新思路。 由此完成了本发明。
本发明的第一方面涉及选自式 (1 ) ~式 (42 ) 所示的多肽,
式 (1 ) :
1 ) -Z; ―
式 (2 ) :
X-WIDWSIELNKAKSDLEESKEWIERSNGKLDSIGNWH ( SEQ ID NO: 2 ) -Z;
式 (3 ) :
3 ) -Z;
式 (4 ) :
X-WLDISQNLAAVNKSLSDALQHLAQSDTYLSAI ( SEQ ID NO: 4 )
-Z;
式 ( 5 ) :
X-WLDWSQNLAAVNKSLSDALQHLAQSDTYLSAK SEQ ID NO: 5 )
-Z;
式 (6 ) :
X-PLDISQNLAAVNKSLSDALQHLAQSDTYLSAI ( SEQ ID NO: 6 )
-Z;
•
5
CS vs2S0vosS0V aiSIHZaAlz3I3¾A3AZa¾l:HAx - 5
ai
αΐ αι
αι
CS αιS
卜
ί
) 9ε ί
) ε卜
) 8ε
式 ( 39 ) :
X- YLNLTGEIDDLEFRSEKLHNTTVELAILIDNINNTL( SEQ ID NO:
39 ) -Z;
式 ( 40 ) :
40 ) -Z;
式 (41 ) :
X- WLNWTSEISTLENKSAELNYTVQKLQTLIDNINSTL ( SEQ ID NO: 41 ) -Z;
式 ( 42 ) :
X- ILNLTSEISTLENKSAELNYTVQKLQTLIDNINSTL ( SEQ ID NO: 42 ) -Z;
或其衍生物、 立体异构体、 无生理毒性的盐; 其中,
X为乙酰基、 寡肽序列、 亲脂性基团、 聚乙二醇或缺失; 在本发明的实 施方案中, X为乙酰基 ( CH3-CO- ) ;
Z为酰胺基、 寡肽序列、 亲脂性基团、 聚乙二醇或缺失; 在本发明的实 施方案中, Z为酰胺基( -CO-NH2 ) 。
其中,所述寡肽序列为 1 ~ 10个氨基酸序列,例如可以为 EEE、 KKK、 GKK、 或 GQAV。
其中所述亲脂性基团为脂肪酸和甾醇, 例如可以为正辛酸酯 ( C7H15-CO-0- )、月桂酸酯( C13H27-CO-0- )、 棕榈酸酯( C15H31-CO-0- )、 或胆固醇 (C27H460)。
其中 X与 Z基团用于改善多肽的水溶性、二级结构及稳定性、增强与细 胞膜的结合使之更靠近靶点、 或改善药物的药代性质。
以上式(1 ) ~ ( 42 )的序列均来自于包膜类病毒的融合糖蛋白跨膜亚基 的 CHR序列,其中一些序列在多肽氨基末端的关键位点引入一个色氨酸 ( W ) 残基, 首选为 1位, 或者同时在 4位。
其中式(1 ) ~ ( 3 ) 来自于人副流感病毒 3型 HPIV3, 式(4 ) ~ ( 6 ) 来自于猴副流感病毒 SV5, 式(7 ) ~ ( 9 )来自于严重急性呼吸综合征 SARS 病毒, 式( 10 ) ~ ( 12 ) 来自于小鼠肝炎病毒 MHV, 式( 13 ) ~ ( 15 ) 来 自于新城疫病毒 DV,式( 16 ) ~ ( 18 )来自于人偏肺病毒 hMPV,式( 19 ) ~ ( 21 )来自于呼吸道合胞病毒 RSV, 式(22 ) ~ ( 24 )来自于戊型肝炎病毒
HeV, 式(25) ~ (27)来自于尼帕病毒 iV, 式( 28 ) ~ (30)来自于麻 疹病毒 MeV, 式(31) ~ (33)来自于仙台病毒 SeV, 式( 34 ) ~ ( 36 )来 自于禽肺病毒 APV, 式(37) ~ (39)来自于猫传染性腹膜炎病毒 FIPV, 式(40) ~ (42)来自于人冠状病毒 229E HCoV-229E。 以上病毒株均为具 有晶体结构的标准株。
根据本发明第一方面的多肽, 其选自以下多肽:
(l)SEQ ID NO: 1 - 42中的任一序列在 N端和 /或 C端有 1-10、 1-5、 1-2个氨基酸的截短;
( 2 )SEQ ID NO: 1 - 42中的任一序列在 N端和 /或 C端有 1-10、 1-5、 1-2个氨基酸的延伸; 优选地, 所述延伸是在该序列来源病毒的融合糖蛋白 跨膜亚基的 CHR序列基础上的延伸;
( 3 ) SEQ ID NO: 1~ 42中的任一序列有 1-10、 1-5、 1-2个氨基酸的 替换, 或上述(1) 和 (2) 的序列中有 1-10、 1-5、 1-2个氨基酸的替换; 优选地, 所述替换是根据该序列来源病毒的不同病毒株之间由于融合糖蛋 白跨膜亚基的 CHR序列不同而进行的替换;
( 4 ) SEQ ID NO: 1 ~ 42中的任一多肽经过末端和 /或侧链化学修饰得 到的多肽, 或上述(1) ~ (3) 的多肽经过末端和 /或侧链化学修饰得到的 多肽。
(5) 上述(1) ~ (4) 的多肽, 第一位氨基酸和 /或第四位氨基酸用 色氨酸残基替换, 和 /或第八位氨基酸用疏水氨基酸残基替换后得到的多肽。
其中所述末端和 /或侧链化学修饰包括在氨基末端、羧基末端或侧链修 饰, 修饰基团包括寡肽、 乙酰基、 酰胺基, 亲脂性基团、 亲水性基团等, 其作用是提高多肽的稳定性、 改善药代动力学或提高与细胞膜的结合。
根据本发明第一方面的多肽, 其为选自本发明第一方面任一项所述多肽 中的一种或数种。
在本发明中, 所述多肽既包括直接由 10-100个氨基酸分子脱水缩合而成 的化合物, 也包括在该化合物上经过上述修饰后得到的含有修饰基团的化合 物。
在本发明中,所述多肽的长度优选为含有 30-40个氨基酸,例如为 30、 31、 32、 33、 34、 35、 36、 37、 38、 39、 40。
以上所述多肽在经过截短、 延伸、 替换和 /或修饰后仍具有抗 HIV-1 或其它包膜类病毒与靶细胞例如人体细胞融合的活性。
在本发明中,所述疏水氨基酸残基包括色氨酸、苯丙氨酸、异亮氨酸、 亮氨酸、 缬氨酸、 酪氨酸、 曱硫氨酸。
本发明的多肽可以采用本领域公知的方法直接合成, 也可以利用分子 生物学手段通过将多肽所对应的核苷酸序列克隆入重组载体中, 在重组细 胞中表达后得到。
因此本发明还涉及核酸分子, 其含有编码本发明第一方面所述多肽的 核苷酸序列。
本发明还涉及重组载体, 其含有本发明所述的核酸分子; 以及重组细 胞, 其含有本发明所述的重组载体。
本发明的第四方面涉及包含本发明第一方面所述多肽或其衍生物、 立 体异构体、 无生理毒性的盐的融合蛋白, 或所述多肽或其衍生物、 立体异 构体、 无生理毒性的盐与其他蛋白或毒素或脂类等载体(如大分子载体或重 组载体)偶联或融合而得到的复合物, 或者展示上述多肽或其衍生物、 立体 异构体、 无生理毒性的盐的类病毒颗粒。
本发明多肽的给药方式以针剂为主, 可直接注射, 或制成緩释针剂; 也 可以通过提高代谢稳定性, 包括物理的、 化学的修饰, 使之成为可口服使用 的药物; 也包括外用或粘膜给药等。
本发明的另一方面涉及组合物 (例如药物组合物) , 其含有至少一种 本发明第一方面所述的多肽或其衍生物、 立体异构体、 无生理毒性的盐, 或者本发明第四方面所述的融合蛋白、 复合物或类病毒颗粒, 以及任选的 药学上可接受的载体或辅料。
在本发明中, 所述组合物中可以包含至少一种多肽或其衍生物、 立体 异构体、 无生理毒性的盐, 或者本发明第四方面所述的融合蛋白、 复合物 或类病毒颗粒,例如可以为两种或两种以上,以进一步提高融合抑制效果; 或者所述组合物中可以包含本发明的多肽或其衍生物、 立体异构体、 无生 理毒性的盐、 本发明所述的融合蛋白、 复合物或类病毒颗粒, 以及其它融 合抑制剂, 例如 C34、 T20或 CP32。
本发明的另一方面涉及本发明第一方面所述的多肽或其衍生物、 立体 异构体、 无生理毒性的盐、 第四方面所述的融合蛋白、 复合物或类病毒颗 粒在制备 HIV融合抑制剂中的用途。
在本发明中, 所述融合抑制剂是指可以阻止 HIV-1 自身的活性六螺旋 结构的形成进而抑制病毒 -细胞膜融合的化合物或组合物, 即是指通过形成
无活性的六螺旋结构阻止病毒 -细胞膜融合的化合物或组合物。在本发明的 实施方案中, 所述融合抑制剂是指多肽或经过修饰的多肽, 或者包含多肽 或经过修饰的多肽的组合物。
本发明还涉及本发明第一方面所述的多肽或其衍生物、 立体异构体、 无生理毒性的盐、 第四方面所述的融合蛋白、 复合物或类病毒颗粒在制备 用于治疗或预防 HIV感染相关疾病例如艾滋病的药物或者其它包膜类病毒 感染引起的疾病中的用途。
在本发明中, 所述 HIV包括 HIV-1和 HIV-2型病毒, 在本发明的实施 方案中, 所述 HIV为 HIV-1型病毒。
在本发明的实施方案中, 所述其它包膜类病毒是指利用第一类融合蛋白 作为融合工具的病毒, 包括但不限于人副流感病毒 3型 HPIV3,猴副流感病毒 SV5,严重急性呼吸综合征 SARS病毒,小鼠肝炎病毒 MHV,新城疫病毒 NDV, 人偏肺病毒 hMP V , 呼吸道合胞病毒 RSV,戊型肝炎病毒 HeV,尼帕病毒 NiV , 麻疹病毒 MeV, 仙台病毒 SeV, 禽肺病毒 APV, 猫传染性腹膜炎病毒 FIPV或 人冠状病毒 229E HCoV-229E。
本发明还涉及一种预防或治疗 HIV感染相关疾病例如艾滋病或者其它 包膜类病毒感染引起的疾病的方法, 所述方法包括给有需要的受试者以预防 或治疗有效量的本发明第一方面所述的多肽或其衍生物、 立体异构体、 无 生理毒性的盐、 第四方面所述的融合蛋白、 复合物或类病毒颗粒的步骤。
根据融合机理, HIV用第一类融合蛋白 ( Class I fusion Protein )作为病 毒 -细胞膜融合的工具分子, 利用这类融合蛋白的还有其它多种病毒, 包括一 些严重威胁人类健康的病毒。 第一类融合蛋白有下列共性: 都含有跨膜亚基 和表面亚基, 其中跨膜亚基直接参与融合; 跨膜亚基细胞膜外部分一般含有 融合肽、 NHR和 CHR等功能区; 融合过程中 NHR三聚形成内核, CHR反 平行折叠到 NHR形成的沟槽中, 形成六螺旋结构, 形成过程中放出的能量 促使病毒和细胞膜融合使病毒完成感染 [1]。 根据这些原理, 发明人针对利用 第一类融合蛋白作为融合工具的病毒, 根据其融合糖蛋白跨膜亚基的 CHR, 设计 HIV融合抑制剂。这类病毒的包膜糖蛋白的氨基酸序列已知, 功能区也 得到了确定, 发明人从其中 CHR序列中截取适当的多肽片段, 根据融合机 理的相似性, 预测这些多肽片段可以反平行折叠到 NHR形成的沟槽中, 形 成无活性的六螺旋结构 , 并用融合抑制活性实验进行验证。
因此本发明还涉及一种获得抑制 HIV 与靶细胞融合的融合抑制剂的方
法, 该方法包括从除 HIV以外的病毒的 CHR功能区序列上截取适当长度的 多肽片段,所述多肽片段能够和 NHR功能区结合形成无活性的六螺旋结构。 需要加以说明的是, 由于 CHR—般的长度约为 40个氨基酸残基, 因此截取 30-40个残基片段用作 HIV融合抑制剂相当直观, 无需特别地技术, 但不排 除进行计算机模拟以求得最好截取效果。 另外, 根据直观的逻辑, 将这些截 取片段用于 HIV 以外的与所截取病毒相同或不同病毒的融合抑制剂也应在 本发明的保护范围内。
其中所述除 HIV 以外的病毒是指利用第一类融合蛋白作为融合工具的 病毒, 例如为包膜类病毒; 在本发明的一个实施方案中, 所述病毒为人副流 感病毒 3型 HPIV3[2], 猴副流感病毒 SV5[3], 严重急性呼吸综合征 SARS病 毒[4], 小鼠肝炎病毒 MHV[4], 新城疫病毒 NDV[5], 人偏肺病毒 hMPV[6], 呼 吸道合胞病毒 RSV[3] , 戊型肝炎病毒 HeV[2], 尼帕病毒 NiV[7] , 麻疹病毒 MeV[7], 仙台病毒 SeV[7], 禽肺病毒 APV[8], 猫传染性腹膜炎病毒 FIPV[9]或 人冠状病毒 229E HCoV-229E[9]。
其中所述适当长度是指包含 30-40个氨基酸, 例如为 30、 31、 32、 33、 34、 35、 36、 37、 38、 39、 40。
本发明还涉及根据本发明所述的上述方法获得的抑制 HIV 与靶细胞融 合的融合抑制剂。
在本发明的实施方案中, 所述融合抑制剂选自上述式(1 ) ~ ( 42 )所示 的多肽。
在本发明的实施方案中, 所述融合抑制剂是指多肽或经过修饰的多肽, 或者包含多肽或经过修饰的多肽的组合物。
在本发明中, X为乙酰基、 Z为酰胺基的式(1 ) ~式(42 )所示的多 肽也分别称为多肽 1 ~ 42。 发明的有益效果
本发明的多肽显示出明显的抗 HIV活性, 靶点作用实验显示, 所述多 肽作用于 gp41 NHR区,通过与 NHR形成无活性的六螺旋结构,破坏 HIV NHR结构, 抑制 HIV与靶细胞的融合。 同时, 活性实验结果表明, 本发 明多肽的作用机制与含有 WWI 结构模板口袋结合区的已知多肽融合抑制 剂不同。
说明书附图
图 1显示了本发明多肽与 N46的相互作用
其中横坐标 wave为扫描波长, 单位为 nm; 纵坐标为 CD信号, 单位 为 mdeg。 每个小图中有四条曲线, 例如 seql、 N46分别为多肽 1, N46单 独的 CD谱, Seql/N46为多肽 1和 N46混合后测得的 CD谱, Seql+N46 为二者单独 CD谱的叠加,用于和 Seql/N46比较以确定二者是否有相互作 用。 其它小图标注的含义以此类推;
其中 A为多肽 1, B为多肽 4, C为多肽 7, D为多肽 10, E为多肽 13 , F为多肽 16, G为多肽 19, H为多肽 22。 具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述, 但是本领域技 术人员将会理解, 下列实施例仅用于说明本发明, 而不应视为限定本发明 的范围。 实施例中未注明具体条件者, 按照常规条件或制造商建议的条件 进行。 所用试剂或仪器未注明生产厂商者, 均为可以通过市购获得的常规 产品。 在本发明中使用的缩写具有下面的意义:
AIDS ( Acquired Immure Deficiency Syndrome )艾滋病, 获得性免疫 缺陷综合症。
Ala ( Alanine , Α ) 丙氛酸
Arg ( Arginine , R ) 精氛酸
Asn ( Asparagine, N ) 天冬醜胺
Asp ( Asparticacid , D ) 天冬氣酸
DCM ( Dichloromethane ) 二氯曱烷
DMF ( Ν,Ν-Dimethyl malonate ) 二曱基曱酰胺
Env ( Envelope glycoprotein ) 包膜糖蛋白
ESI-MS ( Electronic spray ion mass spectroscopy ) 电喷质 i普
Fmoc ( Fluorenylmethoxycarbonyl ) 药曱氧羧基
Gly ( Glycine, G ) 甘氨酸
Gin ( Glutamine, Q ) 谷酰胺
Glu ( Glutamic acid, E ) 谷氨酸
6-HB ( six-helix bundle ) 六螺旋体
HBTU 2- ( 1H-1-羟基苯并三唑) -1,1,3,3-四曱基六氟磷酸
His ( Histidine, H )组氨酸
HoBt ( 1-Hydroxyl benzotiazole anhydrous ) 1-幾基苯并三氮峻
NHR ( N-terminal heptad repeat ) N-端七联重复序列
CHR ( C-terminal heptad repeat ) C-端七联重复序列
HIV ( Human immunodeficiency virus ) 人免疫缺陷病毒
HIV-1 1型人免疫缺陷病毒
HPLC ( high performance liquid chromatography ) 高效液相色 i普
He ( Isoleucine, I ) 异亮氛酸
Leu ( Leucine, L ) 亮氛酸
Met ( Methionine, M ) 曱疏氨酸
Nal 正亮氨酸
Lys ( Lysine , K ) 赖氛酸
Phe ( Phenylalanine , F )笨丙氛酸
Ser ( Serine, S ) 丝氨酸
TFA ( Trifluoroacetic acid ) 三氟乙酸
Thr ( Threonie, T ) 苏氛酸
Tyr ( Tyrosine, Y ) 酷氛酸
Val ( Valine, V ) 缬氨酸
W为色氨酸、 N为天冬酰胺、 A为丙氨酸、 S为丝氨酸、 K为赖氨酸、 L为亮氨酸、 E为谷氨酸、 Q为谷氨酰胺、 I为异亮氨酸、 H为组氨酸、 M 为曱硫氨酸、 T为苏氨酸、 D为天冬氨酸、 R为精氨酸、 Y为酪氨酸、 F为 苯丙氨酸, V为缬氨酸, P为脯氨酸, G为甘氨酸。 实施例所用固相合成载体 Rink酰胺树脂为天津南开合成责任有限公司 产品; HBTU、 HOBt、 DIEA及 Fmoc保护的天然氨基酸和 D型的非天然氨 基酸为上海吉尔生化公司及成都诺新技术责任公司产品。 N-曱基吡咯烷酮
( MP )为 ACROS公司产品; TFA为北京博迈科技有限公司产品; DMF、 DCM为韩国三星公司产品; 色谱纯乙腈为 Fisher公司产品。 其它试剂如无 说明均为国产分析纯产品。
实施例 1 抗 HIV-1多肽 1-42的设计
发明人系统考察了包膜类病毒的融合原理及其抑制特性, 从多个包膜类 病毒(人副流感病毒 3型 HPIV3[2], 猴副流感病毒 SV5[3] , 严重急性呼吸综 合征 SARS病毒 [4], 小鼠肝炎病毒 MHV[4], 新城疫病毒 NDV[5], 人偏肺病毒 hMPV[6], 呼吸道合胞病毒 RSV[3], 戊型肝炎病毒 HeV[2],尼帕病毒 NiV[7],麻 疹病毒 MeV[7] ,仙台病毒 SeV[7] ,貪肺病毒 APV[8] ,猫传染性腹膜炎病毒 FIPV[9] 和人冠状病毒 229E HCoV-229E[9] ) 的融合糖蛋白跨膜亚基的 CHR序列中, 截取对应的长度约为 36个氨基酸的多肽。设计的多肽序列 SEQ ID NO: 1-42 见表 1 , 同时标明了其来源病毒。 为方便浓度测定, 我们首先在多肽氨基末 端关键位点引入一个色氨酸(W ) 残基, 首选为 1位, 或者 4位。 然后我们 根据 HIV-1 gp41 CHR 多肽序列, 在 1、 4位点再引入一个色氨酸, 形成 WaaWaaaX (其中 a为氨基酸)模板, 其中 X为疏水氨基酸残基, 使其具有 和 gp41 CHR相同的结合区模板, 考察活性。 在 Seql6, Seql7中 4位引入 W时,我们同时将 3位 F突变为 E,以保证该段序列的整体疏水性保持不变。 活性验证结果参见实施例 3。 实施例 2 抗 HIV-1多肽 1-42的制备
采用标准的 Fmoc固相多肽合成方法。 将实施例 1设计的抗 HIV-1多肽 序列 SEQ ID NO: 1-42的 C-端酰胺化, N-端乙酰化, 得到多肽 1-42。 选用 Rink Amide树脂,肽类由 C-端向 N-端延长。缩合剂为 HBTU/HOBt/DIEA。 脱保护剂为哌啶 /DMF溶液。 裂解剂为 TFA, 粗肽水溶解后冻干保存。 用中 压液相色谱法或 HPLC进行分离纯化, 纯肽含量>95%。 基质辅助激光解析 飞行时间质谱 ( MALDI-TOF-MS )确定多肽分子量。
具体微波多肽合成条件如下:
氨基酸: 0.2 M的 DMF溶液; 活化剂: 0.45M HBTU/HOBt的 DMF溶 液; 活化碱: 2M DIEA的 NMP溶液; 脱保护剂: 20% v/v哌啶的 DMF溶 液; 封闭试剂: 20% v/v乙酸酐的 DMF溶液。
称取 Rink Amide树脂 0.5g ( 0.25 mmol )置入 CEM微波多肽合成仪的 反应器中, 然后将氨基酸、 活化剂、 活化碱、 脱保护试剂、 封闭试剂按上述 条件配好后,用 CEM微波全自动多肽合成仪进行合成。完成后肽树脂用 DMF 洗涤 3遍后用无水曱醇收缩, 室温真空干燥, 得肽树脂 2.05g。
肽树脂的裂解: 将上述合成好的肽树脂称量后放入 250ml茄形瓶中, 冰
浴, 电磁搅拌。按 lg肽树脂加入 10ml的量配置裂解液(体积比:三氟乙酸: 乙二硫醇: 间苯酚: 水 =82.8:10:5:2.5 ) 。 TFA需预先冰浴降温 30min或者预 先放于冰箱中使用。 将配制好的裂解液加入到冰浴条件下的肽树脂中, 电磁 搅拌, 树脂变橙红色, 冰浴条件下反应 30min, 然后撤冰浴, 室温下再继续 反应 90min使反应完成。 剧烈搅拌下向反应器中加入冷乙醚 200ml, 析出白 色沉淀, 继续搅拌 30min; 用 G4的砂芯抽滤漏斗滤出析出物, 用冷乙醚反 复洗涤 3遍, 晾干。 加入双蒸水 50ml, 乙腈 5ml使固体充分溶解, 抽滤, 滤 液冻干得粗肽 1.03g。
所得粗肽用中压或高压色谱进行纯化。色 i普柱为 C18柱,洗脱液为乙腈, 水及少量乙酸。 具体操作步骤: 称取粗肽 lg, 加水 20ml, 乙腈 5ml溶解, 3000转 /分钟下离心 lOmin,取上清液上样。 色 i普柱预先用 15%乙腈 /水 /0.1% 冰乙酸溶液 200ml平衡, 上样后继续用 200ml同样洗脱液平衡, 高效液相检 测洗脱液成分。 根据检测结果逐步升高乙腈含量, 直至所纯化的多肽峰被洗 脱出来。合并同组分洗脱液,旋转蒸发除去大部分溶剂, 冻干得纯肽, HPLC 检测含量>90%。
纯肽经 MALDI-TOF-MS质 i瞽确定其分子量。 采用以上方法分别合成多 肽 1-42 (见表 1 ) 。 多肽序列、 来源及分子量
¾VA3NlHaia¾0INAASVN DS
88ZLZI/£10Z OAV
VFTDKVDISSQISSMNQSLQQSKDY
27 LKC132 NiV 4158.7 95.0
IKEAQRLLDTV ( SEQ ID NO: 27 )
WSLERLDVGTNLGNAIAKLEDAK
28 LKC163 MeV 4057.5 90.0
ELLESSDQILRSM ( SEQ ID NO: 28 )
WSLEWLDVGTNLGNAIAKLEDAK
29 LKC153 MeV 4087.5 95.0
ELLESSDQILRSM ( SEQ ID NO: 29 )
ISLERLDVGTNLGNAIAKLEDAKE
30 LKC133 MeV 3985.4 99.1
LLESSDQILRSM ( SEQ ID NO: 30 )
WVDISLNLADATNFLQDSKAELEK
31 LKC164 SeV 4222.3 91.0
ARKILSEVGRWY ( SEQ ID NO: 31 )
WVDWSLNLADATNFLQDSKAELE
32 LKC154 SeV KARKILSEVGRWY ( SEQ ID NO: 4295.1 97.8
32 )
PVDISLNLADATNFLQDSKAELEK
33 LKC134 SeV 4133.8 99.0
ARKILSEVGRWY ( SEQ ID NO: 33 )
WDQFNVALDQVFESVEKSQNLIDQ
34 LKC165 APV 4194.0 97.0
SNKILDSIEKGN ( SEQ ID NO: 34 )
WDQWNVALDQVFESVEKSQNLID
35 LKC155 APV 4233.5 95.0
QSNKILDSIEKGN ( SEQ ID NO: 35 )
EDQFNVALDQVFESVEKSQNLIDQ
36 LKC135 APV 4137.6 97.7
SNKILDSIEKGN ( SEQ ID NO: 36 )
WLNLTGEIDDLEFRSEKLHNTTVE
37 LKC167 FIPV 4210.4 97.6
LAILIDNINNTL ( SEQ ID NO: 37 )
WLNWTGEIDDLEFRSEKLHNTTV
38 LKC157 FIPV 4284.1 94.0
ELAILIDNINNTL ( SEQ ID NO: 38 )
YLNLTGEIDDLEFRSEKLHNTTVE
39 LKC137 FIPV 4187.5 98.9
LAILIDNINNTL ( SEQ ID NO: 39 )
HCoV WLNLTSEISTLENKSAELNYTVQK
40 LKC168 4149.5 90.8
-229E LQTLIDNINSTL ( SEQ ID NO: 40 )
HCoV WLNWTSEISTLENKSAELNYTVQK
41 LKC158 4223.8 95.4
-229E LQTLIDNINSTL ( SEQ ID NO: 41 )
HCoV ILNLTSEISTLENKSAELNYTVQKL
42 LKC138 4077.2 90.0
-229E QTLIDNINSTL ( SEQ ID NO: 42 ) 实施例 3: 多肽的抗 HIV-1融合活性检测。
我们用 HIV-1 Env介导的细胞-细胞融合模型对实施例 2制备的多肽 1-42 进行活性测定。 靶细胞为 TZM-bl细胞(美国 NIH艾滋病试剂和参照物项
目提供, 目录号为 8129 ) , 其表面表达 CD4 T-细胞受体和趋化因子辅助受 体 CCR5和 CXCR4, 可被 HIV-1 Env识别 , 同时细胞内还转录荧光素酶报 告基因,但不含该基因的启动子,因此单独细胞的荧光素酶背景表达量很低。 效应细胞为 HL2/3细胞(美国 NIH艾滋病试剂和参照物项目提供, 目录号 为 1294 ) , 其表面表达 HIV-1 Env, 由 Env进攻靶细胞, 完成细胞融合, 同 时细胞内还转录荧光素酶报告基因的启动子。两种细胞先在含有氨苄 /链霉素 双抗的含 10%胎牛血清的 DMEM中 , 37°C下在含有 5% C02的培养箱中单 独培养。 两种细胞均为贴壁细胞, 细胞胰酶 /EDTA消化后收获传代。 细胞用 细胞计数板计数。
将 TZM-bl靶细胞用培养基调整到浓度为 75万 /ml,以每孔 50μ1加入 96 孔细胞培养板中 (3.75万 /孔) , 5% C02, 37度下培养 24小时。
将多肽 1-42或阳性对照样品 T20溶于磷酸緩冲溶液生理盐水 ( PBS )中, 或加入适量 DMSO使充分溶解, 用紫外光 i普仪在 280nm处测定多肽浓度。 然后将多肽溶液稀释到适当的浓度, 在 96孔酶标板( Corning ) 中等比稀释 (浓度为 1200、 300、 75、 19、 5、 1.25 μΜ ) 。
配制 150万 /ml的 HL2/3效应细胞。
将 20μ1/孔的等比稀释的多肽 1-42加入前一天培养的 TZM-bl细胞中, 然后加入 50μ1/孔的配制好的 HL2/3效应细胞; 96孔细胞培养板的其中一排 用 PBS替代抑制剂用于测定饱和融合信号, 另一排用 DMEM培养基替代 HL2/3细胞用于测定背景信号。 5% C02, 37°C下培养 6-8小时使之充分融合。
将荧光素酶报告基因的试剂盒(Promega )从冰箱中取出, 将 5x细胞裂 解液根据用量用双蒸水稀释至 lx裂解液, 室温下放置;用底物緩冲液溶解底 物, 室温下放置; 同时将酶标仪 ( Molcular Devices M5多功能酶标仪)检测 条件设置好备用。
将融合好的细胞取出, 弃去培养基, 用 200μ1/孔 PBS洗两次, 尽量去除 清洗液; 然后以 50μ1/孔加入平衡到室温的裂解液, 轻轻震动下反应 5min使 细胞充分裂解; 将裂解液以 40μ1/孔加入 96 孔化学发光检测用酶标板板 ( Corning ) 中, 加样时尽量避免引入气泡; 避光下将底物以 40μ1/孔迅速加 入化学发光用酶标板中, 立即在酶标仪上测定化学发光。
根据饱和融合信号和背景信号的比值确定靶细胞和效应细胞的有效融合, 比值 >5 表明有效融合。 根据样品的浓度-化学发光信号曲线确定其半抑制剂 浓度(IC5。) , 阳性对照样品的 IC5。值应稳定在一定范围; 理想的抑制曲线
中高浓度抑制剂下信号应接近背景信号, 最低浓度抑制剂下信号应接近饱和 融合信号。
多肽 1-42的细胞融合抑制活性列于表 2, 阳性对照 T20的 IC5。为 2±0.5 nM, 与文献报道相符[1()]。 表 2: 多肽 1-42的细胞融合抑制活性
多肽
名称 来源病毒 序列 IC50( M)
编号
1 LKC066 HPIV3 SEQ ID NO: 1 21.6±3.9
2 LKC076 HPIV3 SEQ ID NO: 2 4.5±0.6
3 LKC107 HPIV3 SEQ ID NO: 3 >166.7
4 LKC067 SV5 SEQ ID NO: 4 34±15.5
5 LKC077 SV5 SEQ ID NO: 5 21.9±5.7
6 LKC108 SV5 SEQ ID NO: 6 63±25
7 LKC068 SARS SEQ ID NO: 7 22±1.5
8 LKC078 SARS SEQ ID NO: 8 38±10.4
9 LKC109 SARS SEQ ID NO: 9 9.8±3.5
10 LKC069 MHV SEQ ID NO: 10 14.8±9.1
11 LKC079 MHV SEQ ID NO: 11 4.4±0.5
12 LKC110 MHV SEQ ID NO: 12 10.5±1.1
13 LKC070 NDV SEQ ID NO: 13 1.38±0.16
14 LKC080 NDV SEQ ID NO: 14 46±3.7
15 LKC095 NDV SEQ ID NO: 15 »183
16 LKC072 hMPV SEQ ID NO: 16 2.2±0.9,
17 LKC081 hMPV SEQ ID NO: 17 17.0±3.1
18 LKC096 hMPV SEQ ID NO: 18 1.8±0.01
19 LKC074 RSV SEQ ID NO: 19 39±11
20 LKC082 RSV SEQ ID NO: 20 44±5
21 LKC111 RSV SEQ ID NO: 21 »200
22 LKC075 HeV SEQ ID NO: 22 94±24
23 LKC084 HeV SEQ ID NO: 23 1.07±0.3
24 LKC112 HeV SEQ ID NO: 24 92±18.3
25 LKC162 NiV SEQ ID NO: 25 26±11
26 LKC152 NiV SEQ ID NO: 26 3.9±1.2
27 LKC132 NiV SEQ ID NO: 27 43±6.1
28 LKC163 MeV SEQ ID NO: 28 136±37
29 LKC153 MeV SEQ ID NO: 29 19.9±3.1
30 LKC133 MeV SEQ ID NO: 30 40±20
31 LKC164 SeV SEQ ID NO: 31 16.7±2.7
32 LKC154 SeV SEQ ID NO: 32 5.5±1.1
33 LKC134 SeV SEQ ID NO: 33 18.9±8.9
34 LKC165 APV SEQ ID NO: 34 340±67
35 LKC155 APV SEQ ID NO: 35 66±8.4
36 LKC135 APV SEQ ID NO: 36 43±14.1
37 LKC167 FIPV SEQ ID NO: 37 41±10.5
38 LKC157 FIPV SEQ ID NO: 38 34±9.6
39 LKC137 FIPV SEQ ID NO: 39 66±42
40 LKC168 HCoV-229E SEQ ID NO: 40 13.2±3.7
41 LKC158 HCoV-229E SEQ ID NO: 41 14.5±1,7
42 LKC138 HCoV-229E SEQ ID NO: 42 »200 根据表 2的活性检测结果, 多肽 1-42除 3、 15、 21、 42外均显示出明显 抗 HIV活性, 半抑制浓度 IC5。从 1到数十 μΜ不等, 而所有研究的病毒中 都含有活性多肽序列, 且活性都有改进空间, 符合药物先导化合物的活性标 准。 同时还可看出, 活性与我们引入的色氨酸残基有些关系, 但没有明显的 规律, 因此其作用机制与含有 WWI结构模板口袋结合区的已知多肽融合抑 制剂不同。 但以上结果表明我们可以通过改变氨基酸残基进行活性改进。 例 如化合物 21仅通过引入一个色氨酸残基, 活性得到约 7倍提高。 实施例 4: 多肽的实验室适应病毒株 HIV-1IIIB感染抑制实验
1. 实验用品
实验室适应病毒株 HIV-1IIIB、 MT-2细胞(通过美国国立健康研究院 ( NIH ) 艾滋病研究参考试剂计划获得) 。
2. 实验方法
用实验室适应病毒株 HIV-1IIIB感染抑制实验考察了部分活性较好多 肽的活性。 在 200ul的 RPMI1640培养基中, 用 100 TCID50 ( 50%组织培 养感染量) 的 HIVIIIB毒株去感染 104/ml MT-2细胞, 同时加入呈浓度梯 度的不同抑制剂, 过夜培养之后弃去培养上清液, 换上新鲜的培养基。 感 染后第四天, 从每个孔中取出 lOOul培养上清液,加入等体积的 5% Triton X-100,用 ELISA方法定量检测 p24抗原。具体实验方法亦可参考 Jiang, S., et al., Antimicrobial Agent and Chemotherapy, 2004. 48(11): p. 4349-4359.
3. 实验结果
如下面的表 3所示。
表 3: 多肽的 HIV病毒感染抑制活性
我们选择细胞融合活性中活性较好的多肽, 测定其抗病毒感染活性。 结果显示,这些测试多肽显示与细胞融合活性相似的 IC50值,可以用作先 导化合物开发新型融合抑制剂。 实施例 5: 抗 HIV-1多肽 1-42与 NHR靶点的作用
我们用圆二色谱(CD )研究实施例 2制备的多肽 1、 4、 7、 10、 13、 16、 19、 22 与 gp41 NHR 靶点的相互作用, 我们选用 N46 (序列: Ac-TLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARI L-CONH2 ) 作 为 gp41 NHR 靶 点 , 其 中 序 列 TLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL为 SEQ ID NO: 43ο 圓二色 i普仪为 Biologic MOS450谱仪。 将待测定 CHR多 肽 1、 4、 7、 10、 13、 16、 19、 22分别溶于 PBS中, N46溶于双蒸水中, 根 据 280nm下紫外吸收确定浓度; 然后配制 20 μΜ的多肽 PBS溶液。
配制要检测的多肽样品: 将 N46和多肽 1、 4、 7、 10、 13、 16、 19、 22 分别以 1: 1体积比混合得二者混合样品; 如果是单独多肽样品, 将 20 μΜ 的样品与緩冲溶液以 1:1混合。 样品在 37°C下放置 30min使充分反应。 上述 实验步骤可确保样品中的多肽浓度保持一致。
将配制好的样品在圓二色 i普仪上测定,仪器扫描波长范围为 190-260nm , 波长间隔为 lnm, 扫描速度为 100nm/min, 扫描 4次进行平均。 先用緩冲溶 液扫描得到空白,然后扫描样品信号,将空白信号从样品信号中扣除得到 CD
信号
者的相互作用。 CD信号变化表明两者具有相互作用。多肽 1、 4、 7、 10、 13、 16、 19、 22分别与 N46混合后都出现 CD信号变化,表明它们均作用于 gp41 NHR,通过与 NHR形成无活性的六螺旋结构抑制 HIV与人体细胞的融合(图 1 ) 。 尽管本发明的具体实施方式已经得到详细的描述, 本领域技术人员将 会理解。根据已经公开的所有教导,可以对那些细节进行各种修改和替换, 这些改变均在本发明的保护范围之内。 本发明的全部范围由所附权利要求 及其任何等同物给出。 参考文献
1. Schibli, D.J. and W. Weissenhorn, Class I and class II viral fusion protein structures reveal similar principles in membrane fusion (Review). Molecular Membrane Biology, 2004. 21(6): p. 361-371.
2. Porotto, M., et al., Inhibition of Hendra Virus Fusion. J Virol, 2006. 80(19): p. 9837-9849.
3. Mathews, J.M., et al., The core of the respiratory syncytial virus fusion protein is a trimeric coiled coil. Journal of Virology, 2000. 74(13): p. 5911-5920.
4. Bosch, B.J., et al., Severe acute respiratory syndrome coroavirus (SARS-Co V) infection inhibition using spike protein heptad repeat-derived peptides. Proceedings of the National Academy of Sciences of the United States of America, 2004. 101(22): p. 8455-8460.
5. Young, J.K., et al., Interaction of peptides with sequences from the Newcastle disease virus fusion protein heptad repeat regions. Journal of Virology, 1999. 73(7): p. 5945-5956.
6. Miller, S.A., et al., Examination of a fusogenic hexameric core from human metapneumovirus and identification of a potent synthetic peptide inhibitor from the heptad repeat 1 region. Journal of Virology, 2007. 81(1): p. 141-149.
7. Lamb, R.A., R.G. Paterson, and T.S. Jardetzky, Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures. Virology, 2006. 344(1): p. 30-37.
8. Zhu, J.Q., et al., Biochemical and biophysical analysis of heptad repeat regions from the fusion protein of Menangle virus, a newly emergent paramyxovirus. Archives of Virology, 2003. 148(7): p. 1301-1316.
9. Xu, Y.H., et al., Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core. Journal of Biological Chemistry, 2004. 279(47): p. 49414-49419.
10. Wild, C.T., et al., Peptides corresponding to a predictive alpha-helical domain of human-immunodeficiency -virus type-1 gp41 are potent inhibitors of virus-infection. Proceedings of the National Academy of Sciences of the United States of America, 1994. 91(21): p. 9770-9774.
Claims
5
) CSOVsvsoOSSV aiS XZAZHllaaxHLilzMSZAIasax - n o ) CS SVsvsCSSV αΐSZAZ313a¾H¾lzI:H3ZA>I3x , S
CSSVSOCsoSSOSS αιS AlsMWMIAOnzIMIICLSalAx- I η ε
CSovSOCsoSS&SS αιS AlclxaxIAOMnsMICLSlχ -
S
ι
CSδόν888 αιS ΊζΙζαΙΊΊ:ΗΛ1ΛΖΊ3:ΗΖ3Ί1Ι3ζ¾χ -. 2 42 ) -Z;
或其衍生物、 立体异构体、 无生理毒性的盐; 其中,
X为乙酰基、 寡肽序列、 亲脂性基团、 聚乙二醇或缺失;
Z为酰胺基、 寡肽序列、 亲脂性基团、 聚乙二醇或缺失。
2. 权利要求 1的多肽, 其选自以下多肽:
( l )SEQ ID NO: 1 - 42中的任一序列在 N端和 /或 C端有 1-10、 1-5、 1-2个氨基酸的截短;
( 2 )SEQ ID NO: 1 - 42中的任一序列在 N端和 /或 C端有 1-10、 1-5、 1-2个氨基酸的延伸;
( 3 ) SEQ ID NO: 1 ~ 42中的任一序列有 1-10、 1-5、 1-2个氨基酸的 替换, 或上述(1 ) 和 (2 ) 的序列中有 1-10、 1-5、 1-2个氨基酸的替换;
( 4 )权利要求 1 的多肽经过末端和 /或侧链化学修饰得到的多肽, 或 上述(1 ) ~ ( 3 ) 的多肽经过末端和 /或侧链化学修饰得到的多肽。
( 5 ) 上述(1 ) ~ ( 4 ) 的多肽, 第一位氨基酸和 /或第四位氨基酸用 色氨酸残基替换, 和 /或第八位氨基酸用疏水氨基酸残基替换后得到的多肽。
3. 核酸分子, 其含有编码权利要求 1或 2所述多肽的核苷酸序列。
4. 重组载体, 其含有权利要求 3所述的核酸分子。
5. 重组细胞, 其含有权利要求 4所述的重组载体。
6. 包含权利要求 1或 2所述多肽或其衍生物、 立体异构体、 无生理毒 性的盐的融合蛋白, 或所述多肽或其衍生物、 立体异构体、 无生理毒性的 盐与其他蛋白或毒素或脂类等载体(如大分子载体或重组载体)偶联或融合 而得到的复合物, 或者展示上述多肽或其衍生物、 立体异构体、 无生理毒性 的盐的类病毒颗粒。
7. 组合物 (例如药物组合物) , 其含有至少一种权利要 1或 2所述的 多肽或其衍生物、 立体异构体、 无生理毒性的盐, 或者权利要求 6所述的 融合蛋白、复合物或类病毒颗粒,以及任选的药学上可接受的载体或辅料。
8. 权利要 1或 2所述的多肽或其衍生物、 立体异构体、 无生理毒性的 盐、 权利要求 6所述的融合蛋白、 复合物或类病毒颗粒在制备 HIV融合抑 制剂中的用途。
9. 权利要 1或 2所述的多肽或其衍生物、立体异构体、无生理毒性的盐、 权利要求 6所述的融合蛋白、 复合物或类病毒颗粒在制备用于治疗或预防 HIV感染相关疾病例如艾滋病或者其它包膜类病毒感染引起的疾病的药物中 的用途。
10. 一种预防或治疗 HIV感染相关疾病例如艾滋病或者其它包膜类病毒 感染引起的疾病的方法, 所述方法包括给有需要的受试者以预防或治疗有效 量的权利要 1或 2所述的多肽或其衍生物、 立体异构体、 无生理毒性的盐、 权利要求 6所述的融合蛋白、 复合物或类病毒颗粒的步骤。
11. 一种获得抑制 HIV与靶细胞融合的融合抑制剂的方法, 该方法包括 从除 HIV以外的病毒的 CHR功能区序列上截取适当长度的多肽片段, 所述 多肽片段能够和 NHR功能区结合形成无活性的六螺旋结构。
12. 权利要求 11的方法, 其中所述除 HIV以外的病毒是指利用第一类 融合蛋白作为融合工具的病毒, 例如为包膜类病毒; 所述病毒例如为人副流 感病毒 3型 HPIV3, 猴副流感病毒 SV5, 严重急性呼吸综合征 SARS病毒, 小鼠肝炎病毒 MHV, 新城疫病毒 NDV, 人偏肺病毒 hMPV, 呼吸道合胞病 毒 RSV,戊型肝炎病毒 He V,尼帕病毒 NiV,麻疹病毒 MeV,仙台病毒 SeV, 貪肺病毒 APV,猫传染性腹膜炎病毒 FIPV或人冠状病毒 229E HCoV-229E。
13. 权利要求 11的方法, 其中所述适当长度是指包含 30-40个氨基酸。
14. 根据权利要求 11-13所述方法获得的抑制 HIV与靶细胞融合的融合抑 制剂。
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CN107624116B (zh) * | 2015-03-31 | 2021-08-31 | 汉阳大学校产学协力团 | 抗癌活性的肽以及含有其作为活性成分的预防和治疗癌症的药物组合物和膳食补充组合物 |
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