WO2019223642A1 - 抗病毒多肽及其药物组合物和用途 - Google Patents
抗病毒多肽及其药物组合物和用途 Download PDFInfo
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- WO2019223642A1 WO2019223642A1 PCT/CN2019/087584 CN2019087584W WO2019223642A1 WO 2019223642 A1 WO2019223642 A1 WO 2019223642A1 CN 2019087584 W CN2019087584 W CN 2019087584W WO 2019223642 A1 WO2019223642 A1 WO 2019223642A1
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Images
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
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This application relates to the field of biomedicine, and in particular, to antiviral polypeptides and pharmaceutical compositions and uses thereof.
- HIV Human immunodeficiency virus
- SARS coronavirus SARS coronavirus
- H5N1 highly pathogenic avian influenza virus
- MERS-CoV Middle East respiratory syndrome Coronary disease
- EBOV Ebola virus
- Viruses can be divided into enveloped and non-enveloped viruses. Fusion of the virus to the target cell membrane is a necessary step for all enveloped virus infections. Retroviruses typified by HIV, filamentous viruses typified by EBOV, coronavirus typified by SARS-CoV and MERS-CoV, orthomyxovirus typified by influenza virus, etc., although their forms are quite different, However, they are all type I enveloped viruses with similar virus-host cell membrane fusion mechanisms. During the invasion of this type of virus, the fusion protein on its envelope will be intramolecularly folded to form a stable six-helix bundle (6HB) structure, which shortens the distance between the virus and the host cell membrane.
- 6HB six-helix bundle
- the released energy further promotes the fusion of the two membranes.
- the active polypeptide designed based on the above mechanism can efficiently and specifically bind to the fusion protein in a short time before folding, thereby preventing the formation of 6HB and achieving the purpose of inhibiting the fusion of the virus and the host cell membrane.
- the peptide T20 derived from the HIV fusion protein was approved by the US FDA as the first HIV fusion inhibitor to be used clinically.
- the discovery of T20 marks the confirmation of type I enveloped virus fusion protein as a drug target, and also opens up a new field of using peptide drugs to control the virus epidemic.
- 6HB is essentially a coiled coil structure formed by the interaction of alpha helices; each alpha helix can be divided into a hydrophobic core region located inside the coiled helix and exposed to Hydrophilic surface in solvent; this amphiphilic structure of the alpha helix is an intrinsic driving force for the formation of coiled helix.
- the C-peptide sequence that has a one-to-one residue and specifically binds to the target is easily replaced by a universal sequence that forms an alpha helix.
- the "lipid raft" microdomain on the cell membrane which is rich in cholesterol and sphingosyl esters, is the site where many pathogens enter the host cell, and it is also the site of budding of viral particles.
- the fatty acylation of the fusion protein helps it target the "lipid raft” region of the cell membrane, which in turn affects the virus's ability to invade and the assembly and release of virus particles.
- C peptides Utilizing the "anchoring" ability of lipid membranes to covalently bond lipid molecules such as fatty acids, cholesterol, or sphingosine to C peptides, C peptides can be enriched in "lipid rafts", effectively improving targets Nearby drug concentrations in turn enhance the antiviral activity of the C-peptide.
- the present application provides a compound of formula (I) or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof,
- X a represents a hydrophobic amino acid, and each X a is the same or different;
- X d represents a hydrophobic amino acid, and each X d is the same or different;
- X e is selected from the following amino acids: Ser, Asn, Gln, Glu, Asp, Lys, Arg, His, Tyr, Trp, Met, and Cys, each X e being the same or different;
- E is Glu
- R 1 represents cholesterol, steroids, sphingosine, and fatty acids (such as C 6-24 fatty acids);
- L 1 is selected from glycine, ⁇ -alanine ( ⁇ -Ala), ⁇ -aminobutyric acid (GABA), 6-aminohexanoic acid (6-Aca), and NH 2- (CH 2 CH 2 -O) n- CH 2 CH 2 -COOH, where n is an integer selected from 1-25;
- n is selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10.
- X a is an amino acid residue that can have a hydrophobic interaction with the transmembrane subunit NHR region of a type I enveloped virus fusion protein.
- X a is selected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, and Met, each X a being the same or different.
- X a is selected from Ala, Val, Leu, Ile, Phe, and Tyr, each X a being the same or different.
- X a is Ile.
- X d is an amino acid residue that can have a hydrophobic interaction with the transmembrane subunit NHR region of a type I enveloped virus fusion protein.
- X d is selected from Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp and Met, each X d being the same or different.
- X d is selected from Ala, Val, Leu, Ile, Phe, and Tyr, each X d being the same or different.
- X d is Ile.
- X e is an amino acid residue that can interact polarly with the NMR region of a transmembrane subunit of a type I enveloped virus fusion protein.
- X e is selected from Ser, Gln, Glu, Lys, His, Tyr, and Trp, each X e being the same or different.
- X e is Gln.
- R 1 is selected from C 6-24 saturated fatty acids.
- R 1 is selected from caprylic acid, capric acid, lauric acid, myristic acid, and palmitic acid.
- L 1 is selected from ⁇ -alanine ( ⁇ -Ala), 6-aminocaproic acid (6-Aca), and NH 2- (CH 2 CH 2 -O) 7 -CH 2 CH 2 -COOH.
- m is selected from 3, 4, 5, 6, 7, and 8.
- m is selected from 4, 5, and 6.
- n is 5.
- the compound of formula (I) is selected from:
- a is ⁇ -Ala
- z is 6-Aca
- p is NH 2- (CH 2 CH 2 -O) 7 -CH 2 CH 2 -COOH
- C 8 is caprylic acid
- C 10 is capric acid
- C 12 is Lauric acid
- C 14 is myristic acid
- C 16 is palmitic acid.
- the compound of the invention has at least 90% identity with the compound of formula (I), preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% Identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity.
- the present application provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound of formula (I) or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt thereof, and a One or more pharmaceutically acceptable carriers or excipients.
- the pharmaceutical composition of the present application can be made into tablets, sustained-release tablets, controlled-release tablets, dragees, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules , Syrups or elixirs, drip pills, pellets, or oral solutions.
- Pharmaceutical compositions for oral use may also contain, for example, one or more colorants, sweeteners, flavoring agents, and / or preservatives.
- Suitable excipients for tablets include, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; disintegrants such as corn starch and alginic acid; binders such as starch; lubricants such as magnesium stearate , Stearic acid or talc; preservatives such as ethyl or propyl paraben; and antioxidants such as ascorbic acid and the like.
- the tablets may be uncoated or they may be coated to alter their disintegrating effect and subsequent absorption of the active ingredient in the gastrointestinal tract or to improve their stability and / or appearance, which can be used in any case Conventional coating agents and methods well known in the art.
- Suitable excipients for hard capsules include inert solid diluents such as calcium carbonate, calcium phosphate or kaolin, and the like.
- Suitable excipients for soft capsules include water or oils such as peanut oil, liquid paraffin or olive oil, and the like.
- Aqueous suspensions generally contain the active ingredient in the form of micronized powder and one or more dispersants, wetting agents or suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl ester Cellulose, sodium alginate, polyvinyl-pyrrolidone, tragacanth gum, gum arabic, etc .; dispersants or wetting agents, such as condensates of lecithin or alkenyl oxide with fatty acids (such as polyoxyethylene stearate) ), Or the condensation product of ethylene oxide with a long-chain fatty alcohol, such as ethylene cetyl heptyl alcohol, or the condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol, such as polyoxygenation Ethylene sorbitol monooleate, or the condensation product of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan mono
- Aqueous suspensions may also contain one or more preservatives (e.g., ethyl or propyl parabens), antioxidants (e.g., ascorbic acid), colorants, flavoring agents, and / or sweeteners (e.g., Sucrose, saccharin and aspartame).
- preservatives e.g., ethyl or propyl parabens
- antioxidants e.g., ascorbic acid
- colorants e.g., ascorbic acid
- flavoring agents e.g., ascorbic acid
- sweeteners e.g., Sucrose, saccharin and aspartame
- Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or a mineral oil (such as liquid paraffin). Oily suspensions may also contain thickening agents such as beeswax, solid paraffin or cetyl alcohol. Sweeteners and flavoring agents as described above may be added to enhance the mouthfeel of the oral formulation.
- the pharmaceutical composition can be preserved by the addition of an antioxidant such as ascorbic acid.
- the pharmaceutical composition of the present application may also take the form of an oil-in-water emulsion.
- the oily phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture thereof.
- Suitable emulsifiers may be, for example, natural gums such as acacia or astragalus gum, natural phospholipids such as soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides (such as sorbitan monooleate), And a condensation product of the partial ester and ethylene oxide, such as polyethylene oxide sorbitan monooleate.
- Emulsions may also contain sweeteners, flavoring agents, preservatives, and the like.
- Syrups and elixirs may be formulated with sweeteners (such as glycerol, propylene glycol, sorbitol, aspartame, or sucrose), and may also contain a demulcent, a preservative, a flavoring agent, and / or a coloring agent.
- sweeteners such as glycerol, propylene glycol, sorbitol, aspartame, or sucrose
- the pharmaceutical composition When administered parenterally (e.g., intravenously, subcutaneously or intramuscularly), the pharmaceutical composition can be prepared as a sterile aqueous or oily solution, sterile powder, liposome, emulsion, microemulsion, Nanoemulsions or microcapsules.
- the pharmaceutical composition may also be in the form of a sterile aqueous or oily suspension for injection, which may be formulated according to known methods using one or more suitable dispersing, wetting agents and / or suspending agents, these The reagents are as described above.
- the sterile injectable preparation may also be a sterile aqueous or oleaginous suspension for injection in a diluent or solvent, which is non-toxic and gastrointestinal acceptable, such as a solution in 1,3-butanediol .
- the amount of active ingredient which is combined with one or more excipients to produce a single dosage form can be determined depending on the host treated and the particular route of administration.
- formulations for oral administration to humans generally contain, for example, 0.5 mg to 2 g of active ingredient and appropriate and conventional amounts of excipients (approximately 5-98% of the total weight of the composition).
- a unit preparation generally contains about 1 mg to 500 mg of the active ingredient.
- the dosage of the pharmaceutical composition for therapeutic or preventive purposes should be adjusted according to the nature and severity of the disorder, the age and sex of the animal or patient, and the route of administration.
- the pharmaceutical composition When used for treatment or prevention, it is generally administered in a daily dose in the range of, for example, 1 mg to 100 mg / kg of body weight, and divided doses may be administered if necessary.
- lower dosages are used for parenteral administration, for example, intravenous administration generally uses dosages in the range of, for example, 1 mg to 10 mg / kg of body weight.
- the present application provides a compound of formula (I) or a compound having at least 80% identity, a stereoisomer, or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the preparation of an inhibitory virus and target cell membrane. Use in fused medicine.
- the virus is an enveloped virus.
- the virus is a type I enveloped virus.
- the virus is MERS-CoV or HIV (eg, HIV-1).
- the target cells are cell lines or cells from a subject.
- the application provides the use of a compound of formula (I) or a compound having at least 80% identity, a stereoisomer or a pharmaceutically acceptable salt thereof, as described above, for inhibiting the fusion of a virus with a target cell membrane in vitro .
- the virus is an enveloped virus.
- the virus is a type I enveloped virus.
- the virus is MERS-CoV or HIV (eg, HIV-1).
- the target cells are cell lines or cells from a subject.
- the present application provides a compound of formula (I) or a compound having at least 80% identity, a stereoisomer, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, for the prevention or treatment of viruses.
- a compound of formula (I) or a compound having at least 80% identity, a stereoisomer, or a pharmaceutically acceptable salt or pharmaceutical composition thereof for the prevention or treatment of viruses.
- the virus is an enveloped virus.
- the virus is a type I enveloped virus.
- the virus is MERS-CoV or HIV (eg, HIV-1).
- the disease associated with a viral infection is selected from AIDS and Middle East Respiratory Syndrome.
- the application provides a compound of formula (I) or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof for use in inhibiting viruses and Target cell membrane fusion.
- the virus is an enveloped virus.
- the virus is a type I enveloped virus.
- the virus is MERS-CoV or HIV (eg, HIV-1).
- the target cells are cell lines or cells from a subject.
- the compound, or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof is used in an in vivo method.
- the present application provides a compound of formula (I) as described above or a compound having at least 80% identity therewith, a stereoisomer or a pharmaceutically acceptable salt or pharmaceutical composition thereof for use in prevention or treatment Diseases related to viral infections.
- the virus is an enveloped virus.
- the virus is a type I enveloped virus.
- the virus is MERS-CoV or HIV (eg, HIV-1).
- the disease associated with a viral infection is selected from AIDS and Middle East Respiratory Syndrome.
- the present application provides a method for inhibiting the fusion of a virus with a target cell, which comprises administering to the target cell an effective amount of a compound of formula (I) described above or a compound having at least 80% identity with the same, Stereoisomer or pharmaceutically acceptable salt or pharmaceutical composition step.
- the virus is an enveloped virus.
- the virus is a type I enveloped virus.
- the virus is MERS-CoV or HIV (eg, HIV-1).
- the target cells are cell lines or cells from a subject.
- the method is performed in vivo.
- the method is performed in vitro.
- the present application provides a method of preventing or treating a disease associated with a viral infection, comprising administering to a subject in need thereof an effective amount of a compound of formula (I) Steps of an identical compound, a stereoisomer thereof, or a pharmaceutically acceptable salt or pharmaceutical composition.
- the disease associated with a viral infection is selected from AIDS and Middle East Respiratory Syndrome.
- the application provides an antiviral method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a compound having at least 80% identity therewith, Isomer or pharmaceutically acceptable salt or step of a pharmaceutical composition.
- the virus is an enveloped virus.
- the virus is a type I enveloped virus.
- the virus is MERS-CoV or HIV (eg, HIV-1).
- fatty acid refers to an aliphatic carbon chain containing one carboxyl group at one end. According to the degree of carbon chain saturation, it can be divided into saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids. Among them, the saturated fatty acids have the structural formula of C x H 2x + 1 COOH. According to the length of the carbon chain, it can be divided into short-chain fatty acids (the number of carbon atoms on the carbon chain is less than 6), medium-chain fatty acids (the number of carbon atoms on the carbon chain is 6-12), and long-chain fatty acids (the carbon on the carbon chain) The number of atoms is greater than 12).
- hydrophobic amino acid mainly includes tyrosine, tryptophan, phenylalanine, valine, leucine, isoleucine, proline, methionine and Alanine.
- the term "identity" is used to refer to a sequence match between two polypeptides or between two nucleic acids.
- a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or two Each position of the polypeptide is occupied by lysine)
- the molecules are identical at that position.
- the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of compared positions x 100. For example, if 6 of the 10 positions of two sequences match, the two sequences are 60% identical.
- the DNA sequences CTGACT and CAGGTT share 50% identity (3 positions out of a total of 6 positions match).
- comparisons are made when two sequences are aligned to produce maximum identity.
- Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc).
- the algorithm of E.Meyers and W.Miller Comput.Appl. Biosci., 4: 11-17 (1988)
- the PAM120 weight residue table is used.
- the term "subject" refers to an animal, particularly a mammal, preferably a human.
- the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect.
- a prophylactically effective amount refers to an amount sufficient to prevent, prevent, or delay the onset of a disease
- a therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent a disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such an effective amount.
- the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the manner in which the drug is administered, and other treatments administered concurrently and many more.
- pAAV-IRES-EGFP-MERS-S plasmid refers to a pAAV-IRES-EGFP reporter gene plasmid expressing the MERS-CoVS2 protein.
- Figure 1 shows a laser confocal photo of the N-terminally conjugated green fluorescent label NBD compound (denoted as NBD-IIQ) interacting with target cells; of which, Figure A is the laser interacting NBD-IIQ with Huh-7 cells Confocal photo, Figure B is a laser confocal photo of the interaction between NBD-IIQ and TZM-b1 cells.
- NBD-IIQ N-terminally conjugated green fluorescent label NBD compound
- HIV Human Immunodeficiency Virus
- MALDI-TOF-MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
- MERS-CoV Middle East Respiratory Syndrome Coronavirus
- NHR N-terminal heptad repeat
- NMP N-Methyl pyrrrolidone
- TFA trifluoroacetic acid
- Trp Tryptophan
- the Rink amide resin used as a solid-phase synthesis carrier in the examples is a product of Tianjin Nankai Synthesis Co., Ltd.
- Natural amino acids protected by HBTU, HOBt, DIEA, and Fmoc or D-type unnatural amino acids are the products of Shanghai Jier Biochemical Company and Beijing Okinas Technology Co., Ltd.
- N-methylpyrrolidone (NMP) and trifluoroacetic acid (TFA) are products of Beijing Bailingwei Technology Co., Ltd.
- DMF and DCM are products of Sinopharm Chemical Reagent Co., Ltd.
- Chromatographically pure acetonitrile is a Fisher product.
- Other reagents are domestically produced analytical products unless otherwise specified.
- Peptide synthesis was performed using standard Fmoc solid-phase methods. With Rink Amide resin, the peptide chain is extended from the C-terminus to the N-terminus.
- the condensing agent is HBTU / HOBt / DIEA.
- the deprotecting agent was a piperidine / DMF solution.
- the lysate was trifluoroacetic acid (TFA).
- the crude peptide was dissolved in water and lyophilized. It was separated and purified by medium pressure liquid chromatography or high pressure liquid chromatography (HPLC), and the pure peptide content was greater than 90%.
- Matrix-assisted laser analytical time-of-flight mass spectrometry MALDI-TOF-MS was used to determine the molecular weight of the peptide sequence.
- the peptide sequence was synthesized using a CEM microwave peptide synthesizer.
- Blocking reagent 20% v / v acetic anhydride in DMF.
- Rink Amide resin 0.23 g (0.1 mmol) of Rink Amide resin was weighed and placed in the reactor of CEM microwave peptide synthesizer, and then protected amino acids, condensation reagents, activated bases, deprotection reagents, and blocking reagents were configured according to the above concentrations. Automatic peptide synthesizer for synthesis. After completion, the peptide resin was transferred to a peptide solid-phase synthesis reactor, washed twice with DMF, anhydrous methanol, and DCM, respectively, and dried under vacuum at room temperature to obtain 1.25 g of the peptide resin.
- Peptide resin [containing Lys (Dde) special amino acid)] swell resin with a little DCM for 20 min, and drained.
- Dde protection group removal reagent 2ml of hydrazine hydrate is dissolved in 40ml of DMF in a volume ratio of 20: 1. The deprotection reagent was added to the resin, stirred at room temperature for 3 minutes, dried, and repeated 4 times to remove the reaction.
- Peptide resin lysis Weigh 1.31g of peptide resin synthesized by a microwave synthesizer, put it into a 250ml eggplant-shaped bottle, and ice bath. A lysate was prepared by adding 10 ml of 1 g of peptide resin. The TFA needs to be cooled in the ice bath for 30 minutes or stored in the refrigerator before use; add the prepared lysate to the peptide resin under the ice bath condition, and slowly stir the electromagnetically, the resin turns orange-red, and react for 30 minutes in the ice bath condition, and then Remove the ice bath and continue to stir the reaction for 150 minutes. The reaction is complete. Add 200 ml of ice ether cooled at 4 ° C under vigorous stirring to precipitate a white precipitate.
- Crude peptides are purified by medium pressure or high pressure chromatography.
- the chromatographic column was a C8 column, and the eluents were acetonitrile, water, and a small amount of TFA.
- the column was equilibrated with 200 ml of a 20% acetonitrile / water / 0.1% TFA solution in advance.
- a ⁇ -Ala; z: 6-Aca; p: NH 2- (CH 2 CH 2 -O) 7 -CH 2 CH 2 -COOH; C 8 : caprylic acid, C 10 : capric acid, C 12 : Lauric acid, C 14 : myristic acid, C 16 : palmitic acid.
- sequence information of the polypeptide compounds 21 and 22 used as a positive control is as follows, wherein compound 21 is a specific anti-MERS-CoV polypeptide HR2P, and compound 22 is a specific anti-HIV-1 polypeptide T20:
- Example 1 The polypeptide isolated in Example 1 was dissolved in PBS (pH 7.4) and diluted to a final concentration of 10 ⁇ M for testing.
- a MOS-450 circular dichroic analyzer was used for spectral scanning of the peptide solution, and the parameters were set: the scanning wavelength was 180-280nm, the scanning optical path was 1mm, the scanning unit was 1nm, and the scanning repetition number of each sample was 3 times.
- Polypeptides with a typical ⁇ -helical structure appear on the CD spectrum as negative peaks at 208 nm and 222 nm and positive peaks at 195 nm, while ⁇ -helicality is based on a molar ellipticity of -33,000 (deg ⁇ cm 2 at 222 nm).
- Dmol -1 is 100% alpha helicity to calculate the percentage helix content of the polypeptide.
- Example 3 Evaluation of peptide-inhibited MERS-CoV S2 protein-mediated cell-cell fusion activity
- Cells 293T cells (derived from ATCC (Manassas, VA, USA))
- the fourth day 36-48 hours after transfection, the cells can express strong fluorescence, and subsequent fusion inhibition tests can be performed.
- Huh-7 cells (derived from the cell bank of the Chinese Academy of Sciences) were digested 6 to 12 hours before fusion, and cell suspensions were prepared, and 4 to 5 ⁇ 10 4 cells were added to each well. Culture at 37 ° C until use.
- the drug plate were added 10 4 per well 293T / MERS / EGFP cells.
- a drug-free culture well was set as a positive control (293T / MERS / EGFP + Huh-7), and a 293T / EGFP cell well was used as a negative control (293T / EGFP + Huh-7).
- the final volume was controlled at 110 ⁇ L.
- Example 3 Evaluation of compound-inhibited HIV-1 Env protein-mediated cell-cell fusion activity
- HIV-1 IIIB infected H9 cells H9 / HIV-1 IIIB
- the fluorescent reagent Calcein-AM Molecular Probes, Inc., Eugene, OR
- the test compound was diluted from 250 ⁇ g / ml by a two-fold gradient. Fusion and unfused Calcein-labeled HIV-1 infected cells were counted using a reverse fluorescence microscope (Zeiss, Germany). Calculate the IC 50 value.
- the compounds of the present application can effectively inhibit the cell fusion mediated by the MERS-CoV protein or HIV-1 Env protein, among which compounds 12-18 inhibit the MERS-CoV fusion activity and positively control specific anti-MERS- CoV polypeptide HR2P (compound 21) is equivalent; and its HIV-1 fusion inhibiting activity is comparable to that of positive control-specific anti-HIV-1 polypeptide T20 (compound 22).
- Huh-7 cells were digested 12 hours in advance, and a single cell suspension was prepared using DMEM and the cell concentration was adjusted.
- Resuscitation / freezing of cells Remove the cell cryopreservation tube from liquid nitrogen, quickly raise the temperature in a 37 ° C water bath, remove 1 ml of the cell cryopreservation solution, add it to a 15 ml centrifuge tube, add 1 ml of culture medium, and centrifuge (800 rpm, 10 min ), Remove the culture medium, re-add 1 ml of fresh culture medium, and gently blow the cells to uniformly suspend the cells. The cell suspension is completely transferred to a 75 cm 2 culture flask containing 15 ml of culture medium, and cultured at 37 ° C. and 5% CO 2 .
- cryopreservation solution added blow homogeneous suspension cells (10 6 / ml), dispensed into vials (1ml / tube) were placed in 4 °C (30min) , -20 ° C (2h), -80 ° C (12h), -196 ° C / liquid nitrogen.
- Subculture Remove the cell culture flask, pour the medium, add 2ml of digestion solution, and shake it evenly on the cell surface. Pour off the digestion solution, add 2ml digestion solution (trypsin / EDTA) again, and spread Dilute well at 37 ° C for 2min, add 4ml medium to stop digestion, remove all liquid, centrifuge, discard the supernatant, add 4ml medium and gently blow to uniformly suspend the cells, take 10 ⁇ l count, take 4-5 ⁇ 10 5 cells and place Subculture in flasks.
- 2ml digestion solution trypsin / EDTA
- the second group add NBD-IIQ to TZM-b1 cell culture dish, incubate at 37 ° C for 1h, pour off the supernatant, wash twice with PBS, fix the sample with 3% paraformaldehyde, and save at 4 ° C in the dark To be observed under a confocal microscope.
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Abstract
本申请提供抗病毒多肽及其药物组合物和用途,具体地,本申请提供式(I)所示化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,其药物组合物及其在抑制病毒与靶细胞融合和在预防或治疗与此相关的疾病中的用途,Ac-[XaEEXdXeKK]m-L1-K(R1)-NH2 (I)。
Description
本申请是以CN申请号为201810499377.4,申请日为2018年5月23日的申请为基础,并主张其优先权,该CN申请的公开内容在此作为整体援引加入本申请中。
本申请涉及生物医药领域,具体涉及抗病毒多肽及其药物组合物和用途。
在近几个世纪以来,新发传染病中有50%以上是病毒性传染病。它们出现突然、来势凶猛、人们对其全无认识或知之甚少,大多无针对性药物和防控手段。以首次发现于上世纪80年代并持续威胁至今的人类免疫缺陷病毒(HIV),以及近几年相继爆发的SARS冠状病毒(SARS-CoV)、H5N1型高致病性禽流感病毒、中东呼吸综合征冠状病(MERS-CoV)和埃博拉病毒(Ebola virus,EBOV)为例,无不因其传染快、死亡率高,对人类健康、社会稳定和经济发展频频造成严重的影响。针对病毒感染,尤其是针对突发或未知病原体,目前尚无类似于抗生素一样广谱、高效的药物治疗手段。这些事实均使得研发广谱抗病毒药物,突破抗病毒药物只针对单一病原体的瓶颈与限制,消除公众在面临突发病毒时的过度紧张与恐慌,成为国际医药界备受瞩目的热点之一。
病毒可分为包膜病毒和无包膜病毒。病毒与靶细胞膜间的融合是所有包膜病毒感染的必须步骤。以HIV为代表的反转录病毒、以EBOV为代表的丝状病毒、以SARS-CoV和MERS-CoV为代表的冠状病毒、以流感病毒为代表的正粘病毒等,虽然它们的形态迥异,但它们均属于I型包膜病毒,具有相似的病毒–宿主细胞膜融合机制。这类病毒在入侵过程中,其包膜上的融合蛋白会发生分子内折叠,形成稳定的六股α螺旋束(six-helix bundle,6HB)结构,拉近病毒与宿主细胞膜的距离,此过程释放出来的能量进一步促使两种膜融合。以上述机制为基础设计的活性多肽可以在融合蛋白折叠前的短时间内,高效、特异地与其结合,从而阻止6HB的形成,达到抑制病毒与宿主细胞膜融合的目的。2003年,衍生于HIV融合蛋白的多肽T20经美国FDA批准成为第一个临床使用的HIV融合抑制剂。T20的发现标志着I型包膜病毒融合蛋白 作为药物靶标的确认,同时也开辟了利用肽类药物控制病毒流行的新领域。
无论是完全衍生于CHR的功能区的C肽还是经过人工修饰的C肽,均通过单股α螺旋活性构象与靶标NHR三股α螺旋结合,C肽螺旋一侧的疏水残基与NHR三股α螺旋构成的疏水腔形成相互作用。从前期研究获得的信息表明:6HB本质上是一种由α螺旋彼此作用形成的卷曲螺旋结构(Coiled coil);其中的每股α螺旋都可以分为位于卷曲螺旋内部的疏水核心区和暴露于溶剂中的亲水表面;α螺旋的这种两亲性结构是卷曲螺旋形成的内在驱动力。因此,在融合抑制多肽的设计中,残基一一对应的、与靶标特异性结合的C肽序列易于被形成α螺旋的通用序列所替代。这些发现提示我们存在设计具有广谱抗病毒α螺旋结构多肽的可能性。
发明内容
细胞膜上富含胆固醇和鞘酯的“脂筏”微结构域,是多种病原体进入宿主细胞的位点,也是病毒粒子的出芽部位。在I型包膜病毒的感染过程中,融合蛋白的脂酰化有助于其靶向细胞膜的“脂筏”区,进而影响病毒的侵入能力,以及病毒颗粒的装配与释放。利用脂类分子的这种细胞膜“锚定”能力,在C肽上共价键连脂肪酸、胆固醇、或鞘氨醇等脂质分子,可以将C肽富集于“脂筏”,有效提高靶标附近的药物浓度进而增强C肽的抗病毒活性。
基于I型包膜病毒相似的病毒–宿主细胞膜融合机制和六股α螺旋束(6HB)的本质特征,将可以靶向病毒NHR的人工设计α螺旋肽与具有膜锚定功能的脂质分子共缀,所设计的缀合物可以有效抑制同属于I型包膜病毒的MERS-CoV和HIV 6HB结构的形成,从而发挥广谱抗病毒活性。
因此,在一个方面本申请提供式(I)所示化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,
Ac-[X
aEEX
dX
eKK]
m-L
1-K(R
1)-NH
2
(I)
其中,X
a表示疏水性氨基酸,各X
a相同或不同;
X
d表示疏水性氨基酸,各X
d相同或不同;
X
e选自下述氨基酸:Ser、Asn、Gln、Glu、Asp、Lys、Arg、His、Tyr、Trp、Met和Cys,各X
e相同或不同;
E为Glu;
K为Lys;
R
1表示胆固醇、类固醇、鞘氨醇和脂肪酸(例如C
6-24脂肪酸);
L
1选自甘氨酸、β-丙氨酸(β-Ala)、γ-氨基丁酸(GABA)、6-氨基己酸(6-Aca)和NH
2-(CH
2CH
2-O)
n-CH
2CH
2-COOH,其中,n为选自1-25的整数;
m选自2、3、4、5、6、7、8、9和10。
在某些优选的实施方案中,X
a为可与I型包膜病毒融合蛋白跨膜亚基NHR区域发生疏水相互作用的氨基酸残基。
在某些优选的实施方案中,X
a选自Ala、Val、Leu、Ile、Pro、Phe、Tyr、Trp和Met,各X
a相同或不同。
在某些优选的实施方案中,X
a选自Ala、Val、Leu、Ile、Phe和Tyr,各X
a相同或不同。
在某些优选的实施方案中,X
a为Ile。
在某些优选的实施方案中,X
d为可与I型包膜病毒融合蛋白跨膜亚基NHR区域发生疏水相互作用的氨基酸残基。
在某些优选的实施方案中,X
d选自Ala、Val、Leu、Ile、Pro、Phe、Tyr、Trp和Met,各X
d相同或不同。
在某些优选的实施方案中,X
d选自Ala、Val、Leu、Ile、Phe和Tyr,各X
d相同或不同。
在某些优选的实施方案中,X
d为Ile。
在某些优选的实施方案中,X
e为可与I型包膜病毒融合蛋白跨膜亚基NHR区域发生极性相互作用的氨基酸残基。
在某些优选的实施方案中,X
e选自Ser、Gln、Glu、Lys、His、Tyr和Trp,各X
e相同或不同。
在某些优选的实施方案中,X
e为Gln。
在某些优选的实施方案中,R
1选自C
6-24饱和脂肪酸。
在某些优选的实施方案中,R
1选自辛酸、癸酸、月桂酸、肉豆蔻酸和棕榈酸。
在某些优选的实施方案中,L
1选自β-丙氨酸(β-Ala)、6-氨基己酸(6-Aca)和NH
2-(CH
2CH
2-O)
n-CH
2CH
2-COOH,其中,n=1、2、3、4、5、7、9、11或23。
在某些优选的实施方案中,L
1选自β-丙氨酸(β-Ala)、6-氨基己酸(6-Aca)和NH
2-(CH
2CH
2-O)
7-CH
2CH
2-COOH。
在某些优选的实施方案中,m选自3、4、5、6、7和8。
在某些优选的实施方案中,m选自4、5和6。
在某些优选的实施方案中,m为5。
在某些优选的实施方案中,所述式(I)化合物选自:
AEEASKK AEEASKK AEEASKK AEEASKK AEEASKK-a-K(C
16),
VEEVSKK VEEVSKK VEEVSKK VEEVSKK VEEVSKK-a-K(C
16),
FEEFSKK FEEFSKK FEEFSKK FEEFSKK FEEFSKK-a-K(C
16),
YEEYSKK YEEYSKK YEEYSKK YEEYSKK YEEYSKK-a-K(C
16),
LEELSKK LEELSKK LEELSKK LEELSKK LEELSKK-a-K(C
16),
IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-a-K(C
16),
IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-a-K(C
16),
IEEIWKK IEEIWKK IEEIWKK IEEIWKK IEEIWKK-a-K(C
16),
IEEIHKK IEEIHKK IEEIHKK IEEIHKK IEEIHKK-a-K(C
16),
IEEIKKK IEEIKKK IEEIKKK IEEIKKK IEEIKKK-a-K(C
16),
IEEIEKK IEEIEKK IEEIEKK IEEIEKK IEEIEKK-a-K(C
16),
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C
16),
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C
14),
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C
12),
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C
10),
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C
8),
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-z-K(C
16),
IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-p-K(C
16),
IEEIQKK LEEIQKK IEEIQKK IEEIQKK IEELQKK-a-K(C
16)和
IEEISKK IEEIQKK IEEISKK IEEIQKK IEEISKK-a-K(C
16);
其中,a为β-Ala,z为6-Aca,p为NH
2-(CH
2CH
2-O)
7-CH
2CH
2-COOH,C
8为辛酸,C
10为癸酸,C
12为月桂酸,C
14为肉豆蔻酸,C
16为棕榈酸。
在本申请的某些优选实施方案中,本发明的化合物与式(I)化合物具有至少90%同一性,优选至少91%同一性、至少92%同一性、至少93%同一性、至少94%同一性、至少95%同一性、至少96%同一性、至少97%同一性、至少98%同一性、或至少99%同一性。
在另一个方面,本申请提供一种药物组合物,其含有前文所述式(I)化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,以及一种或多种药学上可接受的载体或赋形剂。
当口服给药时,本申请的药物组合物可制成片剂、缓释片、控释片、锭剂、硬或软胶囊、水性或油混悬剂、乳剂、可分散的散剂或颗粒剂、糖浆剂或酏剂、滴丸、微丸或口服溶液。用于口服使用的药物组合物还可以含有例如一种或多种着色剂、甜味剂、矫味剂和/或防腐剂。
用于片剂的适宜的赋形剂包括,惰性稀释剂例如乳糖、碳酸钠、磷酸钙或碳酸钙;崩解剂例如玉米淀粉和藻酸;粘合剂例如淀粉;润滑剂例如硬脂酸镁、硬脂酸或滑石粉;防腐剂例如对羟基苯甲酸乙酯或丙酯;和抗氧剂,例如抗坏血酸等。片剂可以是未包衣的,也可以采用包衣以改变其崩解作用以及活性成分在胃肠道内的后续吸收作用,或改进其稳定性和/或外观,在任意情况中,均可使用该领域熟知的常规包衣剂和方法。
用于硬胶囊的适宜的赋形剂包括,惰性固体稀释剂例如碳酸钙、磷酸钙或高岭土等。用于软胶囊的适宜的赋形剂包括水或油例如花生油、液体石蜡或橄榄油等。
水性混悬剂一般含有微粉形式的活性成分和一种或多种分散剂、湿润剂或助悬剂,所述助悬剂例如为羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、藻酸钠、聚乙烯-吡咯烷酮、西黄蓍胶和阿拉伯胶等;分散剂或湿润剂,例如卵磷脂或烯基氧化物与脂肪酸的缩合物(例如聚氧乙烯硬脂酸酯),或环氧乙烷与长链脂肪醇的缩合产物,例如十七氧化亚乙基鲸蜡醇,或环氧乙烷与衍生自脂肪酸与己糖醇的偏酯的缩合产物,例如聚氧化乙烯山梨糖醇一油酸酯,或环氧乙烷与衍生自脂肪酸和己糖醇酐的偏酯的缩合产物,例如聚乙烯脱水山梨糖醇一油酸酯。水性混悬剂还可含有一种或多种防腐剂(例如对羟基苯甲酸乙酯或丙酯)、抗氧剂(例如抗坏血酸)、着色剂、矫味剂、和/或甜味剂(例如蔗糖,糖精和天冬酰苯丙氨酸甲酯)等。
可通过将活性成分悬浮在植物油(例如花生油、橄榄油、芝麻油或椰子油)或矿物油(例如液体石蜡)中来配制油性混悬剂。油性混悬剂也可含有增稠剂例如蜂蜡、固体石蜡或鲸蜡醇。可以加入如上所述的甜味剂和矫味剂以提升口服制剂的口感。所述药物组合物可以通过加入抗氧剂例如抗坏血酸来防腐。
本申请的药物组合物也可以采用水包油的乳剂的形式。油相可以是植物油,例如 橄榄油或花生油,或者矿物油,例如液体石蜡或者它们的混合物。适当的乳化剂可以是例如,天然树胶例如阿拉伯胶或西黄芪胶,天然磷脂例如大豆卵磷脂,和衍生自脂肪酸和己糖醇酐的酯或偏酯(例如脱水山梨糖醇一油酸酯),以及所述偏酯与环氧乙烷的缩合产物,例如聚氧化乙烯脱水山梨糖醇一油酸酯。乳剂也可含有甜味剂、矫味剂和防腐剂等。
糖浆剂和酏剂可与甜味剂(例如甘油、丙二醇、山梨糖醇、阿司帕坦或蔗糖)配制,也可含有缓和剂、防腐剂、矫味剂和/或着色剂等。
当非肠道给药(例如经静脉内、皮下或肌肉内给药)时,所述药物组合物可制成灭菌的水性或油性溶液、无菌粉末、脂质体、乳剂、微乳剂、纳米乳剂或微囊。
所述药物组合物还可以是注射用无菌水性或油性混悬剂的形式,其可以按照已知方法利用一种或多种适宜的分散剂、湿润剂和/或助悬剂来配制,这些试剂如上所述。无菌注射制剂也可以是在稀释剂或溶剂中的注射用无菌水性或油性混悬剂,所述稀释剂或溶剂无毒且肠胃可接受,例如在1,3-丁二醇中的溶液。
有关制剂的其他信息可参考Comprehensive Medicinal Chemistry的第5卷,25.2章(Corwin Hanschl;Chairman of Editorial Board),PergamonPress1990。
可以根据被治疗宿主和具体给药途径的不同,来确定与一种或多种赋形剂混合以制备单一剂量形式的活性成分的量。例如,用于对人口服给药的制剂一般含有例如0.5mg-2g的活性成分以及适当的和常规量的赋形剂(约占组合物总重的5-98%)。单位制剂中一般约含有1mg-500mg的活性成分。有关给药途径和给药方案的进一步信息可参考Comprehensive Medicinal Chemistry的第5卷,25.3章(Corwin Hanschl;Chairman of Editorial Board),Pergamon Press 1990。
供治疗或预防目的的药物组合物的给药量,应根据病症的性质和严重性、动物或患者的年龄和性别和给药途径等进行调整。
在基于治疗或预防使用所述药物组合物时,一般是以日剂量在例如1mg-100mg/kg体重的范围内给药,如有需要可以分剂量给药。通常,以肠胃外途径给药时采用较低剂量,例如经静脉内给药时,一般采用例如1mg-10mg/kg体重范围内的剂量。
在另一个方面,本申请提供前文所述式(I)化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物在制备抑制病毒与靶细胞膜融合的药物中的用途。
在某些优选的实施方案中,所述病毒为包膜病毒。
在某些优选的实施方案中,所述病毒为I型包膜病毒。
在某些优选的实施方案中,所述病毒为MERS-CoV或HIV(例如HIV-1)。
在某些优选的实施方案中,所述靶细胞为细胞系或来自受试者的细胞。
在另一个方面,本申请提供前文所述式(I)化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐在体外抑制病毒与靶细胞膜融合中的用途。
在某些优选的实施方案中,所述病毒为包膜病毒。
在某些优选的实施方案中,所述病毒为I型包膜病毒。
在某些优选的实施方案中,所述病毒为MERS-CoV或HIV(例如HIV-1)。
在某些优选的实施方案中,所述靶细胞为细胞系或来自受试者的细胞。
在另一个方面,本申请提供前文所述式(I)化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物在制备预防或治疗与病毒感染相关的疾病的药物或抗病毒药物中的用途。
在某些优选的实施方案中,所述病毒为包膜病毒。
在某些优选的实施方案中,所述病毒为I型包膜病毒。
在某些优选的实施方案中,所述病毒为MERS-CoV或HIV(例如HIV-1)。
在某些优选的实施方案中,所述与病毒感染相关的疾病选自艾滋病和中东呼吸综合征。
在另一个方面,本申请提供前文所述式(I)化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物,其用于抑制病毒与靶细胞膜融合。
在某些优选的实施方案中,所述病毒为包膜病毒。
在某些优选的实施方案中,所述病毒为I型包膜病毒。
在某些优选的实施方案中,所述病毒为MERS-CoV或HIV(例如HIV-1)。
在某些优选的实施方案中,所述靶细胞为细胞系或来自受试者的细胞。
在某些优选的实施方案中,所述化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物用于体内方法中。
在另一个方面,本申请提供前文所述式(I)化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物,其用于预防或治疗与病毒感染相关的疾病。
在某些优选的实施方案中,所述病毒为包膜病毒。
在某些优选的实施方案中,所述病毒为I型包膜病毒。
在某些优选的实施方案中,所述病毒为MERS-CoV或HIV(例如HIV-1)。
在某些优选的实施方案中,所述与病毒感染相关的疾病选自艾滋病和中东呼吸综合征。
在另一个方面,本申请提供一种抑制病毒与靶细胞融合的方法,其包括向所述靶细胞施用有效量的前文所述式(I)化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物的步骤。
在某些优选的实施方案中,所述病毒为包膜病毒。
在某些优选的实施方案中,所述病毒为I型包膜病毒。
在某些优选的实施方案中,所述病毒为MERS-CoV或HIV(例如HIV-1)。
在某些优选的实施方案中,所述靶细胞为细胞系或来自受试者的细胞。
在某些优选的实施方案中,所述方法在体内进行。
在某些优选的实施方案中,所述方法在体外进行。
在另一个方面,本申请提供一种预防或治疗与病毒感染相关的疾病的方法,其包括向有此需要的受试者施用有效量的前文所述式(I)化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物的步骤。
在某些优选的实施方案中,所述与病毒感染相关的疾病选自艾滋病和中东呼吸综合征。
在另一个方面,本申请提供一种抗病毒的方法,其包括向有此需要的受试者施用有效量的前文所述式(I)化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或药物组合物的步骤。
在某些优选的实施方案中,所述病毒为包膜病毒。
在某些优选的实施方案中,所述病毒为I型包膜病毒。
在某些优选的实施方案中,所述病毒为MERS-CoV或HIV(例如HIV-1)。
在本申请中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。
如本文中所使用的,术语“脂肪酸”是指一端含有一个羧基的脂肪族碳链。根据碳链的饱和度可分为饱和脂肪酸、单不饱和脂肪酸和多不饱和脂肪酸,其中,饱和脂肪酸具有C
xH
2x+1COOH的结构通式。根据碳链的长度又可分为短链脂肪酸(碳链上的碳原子数小于6)、中链脂肪酸(碳链上的碳原子数为6-12)和长链脂肪酸(碳链上的碳原子数大于12)。
如本文中所使用的,术语“疏水性氨基酸”主要包括酪氨酸、色氨酸、苯丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、甲硫氨酸和丙氨酸。
如本文中所使用的,术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如,两个DNA分子的每一个中的某个位置都被腺嘌呤占据,或两个多肽的每一个中的某个位置都被赖氨酸占据),那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如,如果两个序列的10个位置中有6个匹配,那么这两个序列具有60%的同一性。例如,DNA序列CTGACT和CAGGTT共有50%的同一性(总共6个位置中有3个位置匹配)。通常,在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用,例如,可通过计算机程序例如Align程序(DNAstar,Inc)方便地进行的Needleman等人(1970)J.Mol.Biol.48:443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.,4:11-17(1988))的算法,使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外,可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法,使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定 两个氨基酸序列之间的百分数同一性。
如本文中所使用的,术语“受试者”是指动物,特别是哺乳动物,优选人。
如本文中所使用的,术语“有效量”是指,足以获得或至少部分获得期望的效果的量。例如,预防有效量是指,足以预防,阻止,或延迟疾病的发生的量;治疗有效量是指,足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如,对于治疗用途有效的量将取决于待治疗的疾病的严重度,患者自己的免疫系统的总体状态,患者的一般情况例如年龄、体重和性别,药物的施用方式,以及同时施用的其他治疗等等。
本文中所用“pAAV-IRES-EGFP-MERS-S质粒”是指表达MERS-CoV S2蛋白的pAAV-IRES-EGFP报告基因质粒。
图1显示了N端缀合了绿色荧光标签NBD的化合物(记为NBD-IIQ)与靶细胞相互作用的激光共聚焦照片;其中,图A为NBD-IIQ与Huh-7细胞相互作用的激光共聚焦照片,图B为NBD-IIQ与TZM-b1细胞相互作用的激光共聚焦照片。
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。
在本申请中使用的缩写具有下面的含义:
Ala(Alanine,A):丙氨酸
Arg(Arginine,R):精氨酸
Asn(Asparagine,N):天冬酰胺
Asp(Aspartic acid,D):天冬氨酸
CHR(C-terminal heptad repeat):C端重复序列
Cys(Cysteine,C):半胱氨酸
DCM(Dichloromethane):二氯甲烷
DMF(N,N-Dimethyl formamide):二甲基甲酰胺
EBOV:埃博拉病毒
Fmoc(Fluorenylmethoxycarbonyl):芴甲氧羰基
Gln(Glutamine,Q):谷氨酰胺
Glu(Glutamic acid,E):谷氨酸
Gly(Glycine,G):甘氨酸
HBTU:2-(1H-1-羟基苯并三唑)-1,1,3,3-四甲基脲六氟磷酸
His(Histidine,H):组氨酸
HIV:人体免疫缺陷病毒
HOBt(1-Hydroxylbenzotriazole anhydrous):1-羟基苯并三氮唑
HPLC(High performance liquid chromatography):高效液相色谱
Ile(Isoleucine,I):异亮氨酸
Leu(Leucine,L):亮氨酸
Lys(Lysine,K):赖氨酸
MALDI-TOF-MS:基质辅助激光解吸电离飞行时间质谱
MERS-CoV:中东呼吸综合征冠状病毒
Met(Methionine,M):甲硫氨酸
NHR(N-terminal heptad repeat):N端重复序列
NMP(N-Methyl pyrrolidone):N-甲基吡咯烷酮
Phe(Phenylalanine,F):苯丙氨酸
Pro(Proline,P):脯氨酸
Ser(Serine,S):丝氨酸
TFA(trifluoroacetic acid):三氟乙酸
Thr(Threonine,T):苏氨酸
Trp(Tryptophan,W):色氨酸
Tyr(Tyrosine,Y):酪氨酸
Val(Valine,V):缬氨酸
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以及Fmoc保护的天然氨基酸或D型的非天然氨基酸为上海吉 尔生化公司以及北京欧凯纳斯科技有限公司产品。N-甲基吡咯烷酮(NMP)、三氟乙酸(TFA)为北京百灵威科技有限公司产品。DMF、DCM为国药集团化学试剂有限公司产品。色谱纯乙腈为Fisher公司产品。其他试剂如无说明均为国产分析纯产品。
实施例1:多肽1-20的制备
多肽合成采用标准的Fmoc固相方法。选用Rink Amide树脂,肽链由C端向N端延长。缩合剂为HBTU/HOBt/DIEA。脱保护剂为哌啶/DMF溶液。裂解液为三氟乙酸(TFA),粗肽水溶解后冻干保存。用中压液相色谱法或高压液相色谱法(HPLC)分离纯化,纯肽含量大于90%。基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)确定肽序列分子量。
利用CEM微波多肽合成仪合成肽序列。
合成条件如下:
保护氨基酸:0.2M保护氨基酸的DMF溶液,
缩合试剂:0.45M HBTU/HOBt的DMF溶液,
活化碱:2M DIEA的NMP溶液,
脱保护试剂: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0.23g(0.1mmol)置于CEM微波多肽合成仪反应器中,然后将保护氨基酸,缩合试剂,活化碱,脱保护试剂,封闭试剂按上述浓度配置好后,用CEM微波全自动多肽合成仪进行合成。完成后肽树脂转移至多肽固相合成反应器中,分别用DMF、无水甲醇、DCM各洗涤两遍,室温真空干燥,得肽树脂1.25g。
肽树脂[含有Lys(Dde)特殊氨基酸)]加少许DCM溶胀树脂20min,抽干。配制脱除Dde保护基试剂:2ml水合肼溶于40ml DMF中,体积比20:1。将脱保护试剂加入树脂中,室温搅拌3min,抽干,重复4次,脱除反应毕。称取3equiv的饱和脂肪酸(辛酸、癸酸、月桂酸、肉蔻酸或棕榈酸)、0.11g HBTU(3equiv)和40mg HOBt(3equiv)溶于6ml DMF,加入反应器,再加入320μl DIEA(6equiv),室温搅拌反应1h。反应毕,抽干反应液,DMF、无水甲醇、DCM交替洗涤树脂两次,乙醚再洗去残留饱和脂肪酸,真空干燥,得肽树脂1.31g。
裂解液(体积百分比):三氟乙酸:间甲酚:苯甲硫醚:水=8.5:0.5:0.5:0.5。
肽树脂的裂解:称取微波合成仪合成好的肽树脂1.31g,放入250ml茄形瓶中,冰浴。按1克肽树脂加入10ml的量配制裂解液。TFA需预先冰浴降温30min或者预先存放于冰箱中使用;将配好的裂解液加入到冰浴条件下的肽树脂中,慢速电磁搅拌,树脂变橙红色,冰浴条件下反应30min,然后撤掉冰浴室温下继续搅拌反应150min,反应完成,剧烈搅拌下加入预先在4℃冷却的冰乙醚200ml,析出白色沉淀,继续搅拌30min后静置30min;用G4砂芯漏斗滤出析出物,用冰乙醚反复洗涤滤饼3遍,晾干。加入双蒸水30ml,乙腈10ml使固体充分溶解,抽滤,滤液冻干得粗肽1.04g。
粗肽的纯化:粗肽用中压或高压色谱进行纯化。色谱柱为C8柱,洗脱剂为乙腈,水及少量TFA。具体操作步骤:称取粗肽1.00g,加水20ml,乙腈5ml使固体溶解,离心10min(3000转/分钟)取上清液,上清液经0.23μm无菌滤膜过滤后上样。色谱柱预先用20%乙腈/水/0.1%TFA溶液200ml平衡。上样后继续用20%乙腈/水/0.1%TFA溶液200ml冲洗,高效液相检测洗脱液成分。根据液相检测结果逐渐升高乙腈含量,直至所纯化的多肽主峰被洗脱出来。合并洗脱液,旋转蒸发除去全部乙腈溶剂,冻干纯肽,HPLC检测含量大于90%,MALDI-TOF确证分子量。各多肽序列如表1所示:
表1 多肽序列信息
其中,a:β-Ala;z:6-Aca;p:NH
2-(CH
2CH
2-O)
7-CH
2CH
2-COOH;C
8:辛酸,C
10:癸酸,C
12:月桂酸,C
14:肉豆蔻酸,C
16:棕榈酸。
另外,用作阳性对照的多肽化合物21和22序列信息如下,其中,化合物21为特异性抗MERS-CoV多肽HR2P,化合物22为特异性抗HIV-1多肽T20:
SEQ ID NO:21 SLTQINTTLLDLEYEMKKLEEVVKKLEESYIDLKEL
SEQ ID NO:22 YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
实施例2:多肽的圆二色谱分析
将实施例1分离得到的多肽溶解于PBS(pH 7.4)中并稀释至终浓度10μM进行测试。使用MOS-450圆二色谱仪对多肽溶液进行波谱扫描,设置参数:扫描波长180-280nm,扫描光程1mm,扫描单位1nm,每个样品扫描重复次数3次。具备典型α螺旋结构的多肽在CD谱图上表现为在208nm和222nm处的负峰和195nm处的正峰,α螺旋度则是以在222nm处的摩尔椭圆率为-33000(deg·cm
2·dmol
-1)为100%α螺旋度来计算多肽的螺旋含量百分比。
按照上述方法,各多肽圆二色谱结果见下面表2。
表2 圆二色谱测定多肽的α螺旋含量
实施例3:多肽抑制MERS-CoV S2蛋白介导的细胞-细胞融合活性评价
细胞:293T细胞(来源于ATCC(Manassas,VA,USA))
实验方法:
1)第一天:消化293T细胞并制备细胞悬液,6孔板中每孔中加入2~4×10
5个细胞。
2)第二天:培养12~24h后,待密度在80%左右进行转染,其中一孔转染 pAAV-IRES-EGFP-MERS-S质粒(该组细胞记为293T/MERS/EGFP细胞)用于融合实验;一孔转染pAAV-IRES-EGFP质粒(该组细胞记为293T/EGFP细胞)用作阴性对照。
3)转染8~12h后,更换新鲜DMEM细胞培养液。
4)第四天:转染36~48h后细胞能够表达较强的荧光,可进行后续融合抑制试验。
5)融合前6~12h,消化Huh-7细胞(来源于中国科学院细胞库),制备细胞悬液,每孔中加入4~5×10
4个细胞。37℃培养备用。
6)配制梯度稀释的多肽,每孔55μL反应体系。
7)将293T/MERS/EGFP细胞,以及293T/EGFP细胞用EDTA进行消化,制备单个细胞悬液。
8)药物板中每孔加入10
4个293T/MERS/EGFP细胞。同时设置无药物的培养孔做阳性对照(293T/MERS/EGFP+Huh-7),293T/EGFP细胞孔做阴性对照(293T/EGFP+Huh-7)。终体积控制在110μL。
9)室温孵育30min,使药物与细胞充分作用。
10)抽弃Huh-7细胞的培养基,取100μL药物与细胞的混合液加入到Huh-7细胞之上。
11)24h观察融合情况,待阳性对照组(293T/MERS/EGFP+Huh-7)细胞有明显的融合,加入等体积的4%多聚甲酸固定,终止融合。
12)荧光显微镜或者高内涵筛选系统进行拍照分析。计算多肽对细胞融合的抑制率,并使用CalcuSyn软件计算多肽的IC
50值。
按照上述方法,活性测试结果见下面的表3。
实施例3:化合物抑制HIV-1 Env蛋白介导的细胞-细胞融合活性评价
染色转移法检测HIV-1介导的细胞-细胞融合:HIV-1
IIIB感染的H9细胞(H9/HIV-1
IIIB)被荧光试剂Calcein-AM(Molecular Probes,Inc.,Eugene,OR)标记,然后于96孔板中37℃加入或不加受试化合物与MT-2细胞(比率=1:10)共培养2h。测试化合物从250μg/ml浓度两倍梯度稀释。融合及未融合的Calcein标记HIV-1感染的细胞用反向荧光显微镜(Zeiss,Germany)计数。计算IC
50值。
按照上述方法,活性测试结果见下面的表3。
表3 多肽抑制MERS-CoV S蛋白或HIV-1 Env蛋白介导的细胞融合活性
注:21号肽为特异性抗MERS-CoV多肽HR2P;22号肽为特异性抗HIV-1多肽T20
由表3活性结果可知,本申请的化合物可有效抑制MERS-CoV S蛋白或HIV-1 Env蛋白介导的细胞融合,其中化合物12-18抑制MERS-CoV融合活性与阳性对照特异性抗MERS-CoV多肽HR2P(化合物21)相当;且其抑制HIV-1融合活性与阳性对照特异性抗HIV-1多肽T20(化合物22)相当。
实施例4:化合物抑制MERS-CoV假病毒活性测试
1.MERS-CoV假病毒的包装
(1)转染前16h消化293T细胞,在10cm的组织培养皿中铺板(2×10
6/dish)。
(2)转染前2h给细胞更换预温的新鲜DMEM培养基。
(3)采用磷酸钙转染试剂共转染编码MERS-CoV S蛋白真核表达质粒pcDNA3.1-MERS-S,以及编码缺失Env、表达luciferase的HIV-1基因组质粒(pNL4-3.luc.RE),每种质粒转染20μg。
(4)转染8~10h后,更换10mL含10%FBS和1%青霉素链霉素的新鲜DMEM培养液。
(5)72h后收集含有MERS-CoV假病毒的转染上清液。
(6)4000rpm离心4min去除细胞碎片,0.45μm无菌过滤器过滤,分装并储存在-80℃备用。
2.MERS-CoV假病毒滴度检测
(1)提前12h消化Huh-7细胞,使用DMEM制备单个细胞悬液并调整细胞浓度。
(2)96孔板中每孔中加入10
4个细胞。37℃5%CO
2培养12h备用。
(3)使用DMEM培养基在96孔板中2倍梯度稀释假病毒。
(4)去除96孔板中Huh-7的细胞培养上清,加入100μL稀释后的病毒稀释液。
(5)37℃5%CO
2培养12h后,去除病毒液,更换200μL新鲜DMEM培养基。
(6)加入病毒72h后抽弃上清,测定萤光值。
(7)选择萤光值大于空白对照1000倍的病毒稀释度进行后续假病毒中和实验。
3.萤火虫酶(luciferase)检测
(1)使用ddH
2O稀释5×细胞裂解液至1×工作浓度,平衡至室温。
(2)小心吸弃96孔板中的待测细胞的培养基,使用PBS洗细胞一次,动作轻柔以防止细胞脱落,并尽可能将PBS去除。
(3)每孔中加入1×细胞裂解液20μL,之后置涡旋混匀仪上震荡混匀30~60min 直至细胞完全裂解。
(4)抽取20μL裂解液至96孔不透明白色酶标板中。
(5)每孔中加入100μL萤火虫酶检测试剂。
(6)多功能酶标仪中检测萤光。
4.多肽活性检测
(1)使用DMSO溶解多肽并测定多肽浓度。
(2)实验前16h消化靶细胞Huh-7,制备细胞悬液,调整细胞浓度后每孔中加入10
4个细胞。
(3)96孔板中使用含10%FBS的DMEM培养液2倍梯度稀释多肽药物多肽,每孔60μL;注意要缓慢加入多肽,边加入边搅拌,防止因短时间内加入大量多肽导致多肽析出。
(4)根据之前测定的MERS-CoV假病毒滴度进行稀释,在药物稀释板中每孔加入60μL稀释后的假病毒。
(5)室温作用30min,使药物与病毒充分作用。
(6)之后抽弃细胞的培养上清,每孔中加入药物与病毒混合液100μL。
(7)37℃培养12h后更换含10%FBS的新鲜DMEM培养基。
(8)继续培养,72h后测定luciferase。
(9)根据萤光值与药物浓度的对应关系,计算制作抑制率曲线,并计算药物的半数有效量IC
50。
按照上述方法,活性测试结果见下面的表4。
实施例5:化合物抑制HIV-1假病毒活性测试
(1)将化合物及对照抑制剂倍比稀释,加入96孔细胞培养板中,50μL/孔;
(2)将浓度为250ng p24/ml的假病毒加入96孔细胞培养板中,50μL/孔,与各浓度的抑制剂共孵育30min;
(3)将1×10
5/ml,100μL/孔的TZM-b1细胞(来自美国NIH AIDS Research and Reference Reagent Program)加入上述培养板中,37℃ 5%CO
2培养过夜;
(4)感染24h后换上新鲜DMEM培养基;
(5)感染72h后,弃掉培养基,用1×PBS清洗TZM-b1细胞;
(6)加入荧光素酶试剂盒中专用1×裂解液,70μL/孔,振荡器上裂解1h;
(7)取裂解后的细胞转移至测定荧光吸收的96孔白板中,50μL/孔,加入荧光素酶底物,50μL/孔,轻轻拍打混匀测定相应的荧光吸收值。
按照上述方法,活性测试结果见下面的表4。
表4 多肽抑制MERS-CoV或HIV-1假病毒活性
实施例7:激光共聚焦显微镜成像实验研究多肽与细胞膜相互作用
(1)复苏/冻存细胞:将细胞冻存管从液氮中取出,37℃水浴迅速升温,取出1ml细胞冻存液,加至15ml离心管,并加入1ml培养基,离心(800rpm,10min),除去培养基,重新加入1ml新鲜培养基,并轻吹使细胞均匀悬浮,将细胞悬浮液全部转移至含有15ml培养基的75cm
2培养瓶中,在37℃、5%CO
2下培养。消化细胞并计数后,离心,弃上清,加冻存液轻吹使细胞均匀悬浮(10
6个/ml),分装至冻存管(1ml/管),分别置于4℃(30min)、-20℃(2h)、-80℃(12h)、-196℃/液氮中保存。
(2)传代培养:取出细胞培养瓶,倒去培养基,加入2ml消化液,轻晃使其在细胞表面平铺均匀,倒去消化液,重新加入2ml消化液(胰酶/EDTA),铺匀,37℃消化2min,加入4ml培养基终止消化,取出所有液体,离心,弃上清,加4ml培养基并轻吹使细胞均匀悬浮,取10μl计数,取4-5×10
5细胞置于培养瓶中传代培养。
(3)染色液制备:精确称取DiD或者DiI,用DMSO配制浓度2mM的储存液(注:未使用的储存液分装储存在-20℃,避免反复冻融)。用PBS缓冲液稀释储存液,配制浓度为2μM的工作液。
(4)染色实验:a.分别取MERS-CoV融合活性测试所用靶细胞Huh-7细胞和HIV-1融合活性测试所用TZM-b1细胞悬浮液稀释至10
5个/mL,铺入NEST 15mm共聚焦培养皿中,培养24h;b.加入染色工作液重悬细胞,37℃孵育细胞20min;c.孵育结束,按1500rpm离心5min,倾倒上清液,PBS清洗两次,再次缓慢加入37℃预热的生长培养液重悬细胞,重复此步骤两次以上。
(5)细胞孵育及固定:a.第一组:向Huh-7细胞培养皿中加入经在N端缀合绿色荧光标签NBD的IIQ样品(即表1中序列12),命名为NBD-IIQ,37℃孵育1h后倾倒上清液,PBS清洗两次,使用3%多聚甲醛固定样本,4℃避光保存,待共聚焦显微镜观察。b.第二组:向TZM-b1细胞培养皿中加入经NBD-IIQ,37℃孵育1h后倾倒上清液,PBS清洗两次,使用3%多聚甲醛固定样本,4℃避光保存,待共聚焦显微镜观察。
(6)共聚焦显微镜观察:在Carl Zeiss LSM510meta激光共聚焦显微镜下观察、拍照,60倍油镜观察,NBD、DiI、DiD激发波长分别为466nm、550nm、644nm。结果如图1所示。
如图1所示,NBD-IIQ分别与两种细胞经足够时间孵育后再由PBS清洗,仍然能够观察到多肽分布于细胞表面,说明经C16脂肪酸修饰的多肽能够与细胞膜作用,附着于细胞膜。
尽管本发明的具体实施方式已经得到了详细的阐述,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。本发明的全部范围有所附权利要求及其任何等同物给出。
Claims (16)
- 式(I)所示化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,Ac-[X aEEX dX eKK] m-L 1-K(R 1)-NH 2(I)其中,X a表示疏水性氨基酸,各X a相同或不同;X d表示疏水性氨基酸,各X d相同或不同;X e选自下述氨基酸:Ser、Asn、Gln、Glu、Asp、Lys、Arg、His、Tyr、Trp、Met和Cys,各X e相同或不同;E为Glu;K为Lys;R 1选自胆固醇、类固醇、鞘氨醇和脂肪酸;L 1选自甘氨酸、β-丙氨酸(β-Ala)、γ-氨基丁酸(GABA)、6-氨基己酸(6-Aca)和NH 2-(CH 2CH 2-O) n-CH 2CH 2-COOH,其中,n为选自1-25的整数;m选自2、3、4、5、6、7、8、9和10。
- 权利要求1的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,其中X a选自Ala、Val、Leu、Ile、Pro、Phe、Tyr、Trp和Met,各X a相同或不同;优选地,X a选自Ala、Val、Leu、Ile、Phe和Tyr,各X a相同或不同;优选地,X a为Ile。
- 权利要求1或2的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,其中X d选自Ala、Val、Leu、Ile、Pro、Phe、Tyr、Trp和Met,各X d相同或不同;优选地,X d选自Ala、Val、Leu、Ile、Phe和Tyr,各X d相同或不同;优选地,X d为Ile。
- 权利要求1-3任一项的化合物或与其具有至少80%同一性的化合物、其立体异 构体或药学上可接受的盐,其中X e选自Ser、Gln、Glu、Lys、His、Tyr和Trp,各X e相同或不同;优选地,X e为Gln。
- 权利要求1-4任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,其中R 1选自C 6-24饱和脂肪酸;优选地,R 1选自辛酸、癸酸、月桂酸、肉豆蔻酸和棕榈酸。
- 权利要求1-5任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,其中L 1选自β-丙氨酸(β-Ala)、6-氨基己酸(6-Aca)和NH 2-(CH 2CH 2-O) n-CH 2CH 2-COOH,其中,n=1、2、3、4、5、7、9、11或23;优选地,L 1选自β-丙氨酸(β-Ala)、6-氨基己酸(6-Aca)和NH 2-(CH 2CH 2-O) 7-CH 2CH 2-COOH。
- 权利要求1-6任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,其中所述化合物选自:AEEASKK AEEASKK AEEASKK AEEASKK AEEASKK-a-K(C 16),VEEVSKK VEEVSKK VEEVSKK VEEVSKK VEEVSKK-a-K(C 16),FEEFSKK FEEFSKK FEEFSKK FEEFSKK FEEFSKK-a-K(C 16),YEEYSKK YEEYSKK YEEYSKK YEEYSKK YEEYSKK-a-K(C 16),LEELSKK LEELSKK LEELSKK LEELSKK LEELSKK-a-K(C 16),IEEISKK IEEISKK IEEISKK IEEISKK IEEISKK-a-K(C 16),IEEIYKK IEEIYKK IEEIYKK IEEIYKK IEEIYKK-a-K(C 16),IEEIWKK IEEIWKK IEEIWKK IEEIWKK IEEIWKK-a-K(C 16),IEEIHKK IEEIHKK IEEIHKK IEEIHKK IEEIHKK-a-K(C 16),IEEIKKK IEEIKKK IEEIKKK IEEIKKK IEEIKKK-a-K(C 16),IEEIEKK IEEIEKK IEEIEKK IEEIEKK IEEIEKK-a-K(C 16),IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 16),IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 14),IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 12),IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 10),IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-a-K(C 8),IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-z-K(C 16),IEEIQKK IEEIQKK IEEIQKK IEEIQKK IEEIQKK-p-K(C 16),IEEIQKK LEEIQKK IEEIQKK IEEIQKK IEELQKK-a-K(C 16)和IEEISKK IEEIQKK IEEISKK IEEIQKK IEEISKK-a-K(C 16);其中,a为β-Ala,z为6-Aca,p为NH 2-(CH 2CH 2-O) 7-CH 2CH 2-COOH,C 8为辛酸,C 10为癸酸,C 12为月桂酸,C 14为肉豆蔻酸,C 16为棕榈酸。
- 药物组合物,其含有权利要求1-7任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐,以及一种或多种药学上可接受的载体或赋形剂。
- 权利要求1-7任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物在制备抑制病毒与靶细胞膜融合的药物中的用途;优选地,所述病毒为包膜病毒;优选地,所述病毒为I型包膜病毒;优选地,所述病毒为MERS-CoV或HIV(例如HIV-1)。
- 权利要求1-7任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物在制备预防或治疗与病毒感染相关的疾病的药物或抗病毒药物中的用途;优选地,所述病毒为包膜病毒;优选地,所述病毒为I型包膜病毒;优选地,所述病毒为MERS-CoV或HIV(例如HIV-1);优选地,所述与病毒感染相关的疾病选自艾滋病和中东呼吸综合征。
- 权利要求1-7任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物在体外抑制病毒与靶细胞膜融合中的用途;优选地,所述病毒为包膜病毒;优选地,所述病毒为I型包膜病毒;优选地,所述病毒为MERS-CoV或HIV(例如HIV-1)。
- 权利要求1-7任一项的化合物或与其具有至少80%同一性的化合物、其立体异 构体或药学上可接受的盐或权利要求8的药物组合物,其用于抑制病毒与靶细胞膜融合的药物中的用途;优选地,所述病毒为包膜病毒;优选地,所述病毒为I型包膜病毒;优选地,所述病毒为MERS-CoV或HIV(例如HIV-1),优选地,所述化合物、其立体异构体、药学上可接受的盐或药物组合物用于体内方法中。
- 权利要求1-7任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物在制备预防或治疗与病毒感染相关的疾病的药物或抗病毒药物中的用途;优选地,所述病毒为包膜病毒;优选地,所述病毒为I型包膜病毒;优选地,所述病毒为MERS-CoV或HIV(例如HIV-1);优选地,所述与病毒感染相关的疾病选自艾滋病和中东呼吸综合征。
- 一种抑制病毒与靶细胞融合的方法,其包括向所述靶细胞施用有效量的权利要求1-7任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物的步骤;优选地,所述病毒为包膜病毒;优选地,所述病毒为I型包膜病毒;优选地,所述病毒为MERS-CoV或HIV(例如HIV-1);优选地,所述靶细胞为细胞系或来自受试者的细胞;优选地,所述方法在体内进行;优选地,所述方法在体外进行。
- 一种预防或治疗与病毒感染相关的疾病的方法,其包括向有此需要的受试者施用有效量的权利要求1-7任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物的步骤;优选地,所述与病毒感染相关的疾病选自艾滋病和中东呼吸综合征。
- 一种抗病毒的方法,其包括向有此需要的受试者施用有效量的权利要求1-7任一项的化合物或与其具有至少80%同一性的化合物、其立体异构体或药学上可接受的盐或权利要求8的药物组合物的步骤;优选地,所述病毒为包膜病毒;优选地,所述病毒为I型包膜病毒;优选地,所述病毒为MERS-CoV或HIV(例如HIV-1)。
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WO2022023380A3 (en) * | 2020-07-27 | 2022-03-10 | King's College London | Method for identifying an agent modulating cell fusion or syncytia formation and|compositions for treating coronaviruses |
CN114957403A (zh) * | 2021-02-24 | 2022-08-30 | 成都奥达生物科技有限公司 | 一种广谱冠状病毒膜融合抑制剂及其药物用途 |
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CN108659105B (zh) * | 2018-05-23 | 2021-11-09 | 中国人民解放军军事科学院军事医学研究院 | 抗病毒多肽及其药物组合物和用途 |
CN110128510B (zh) * | 2019-05-22 | 2021-02-26 | 中国人民解放军军事科学院军事医学研究院 | MERS-CoV融合抑制剂 |
CN111675752B (zh) * | 2020-03-16 | 2023-07-07 | 成都奥达生物科技有限公司 | 一种冠状病毒膜融合抑制剂及其药物用途 |
CN111643656B (zh) * | 2020-05-14 | 2022-07-19 | 悦康药业集团股份有限公司 | 广谱冠状病毒膜融合抑制剂及其抗艾滋病病毒的应用 |
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