CN110128510A - MERS-CoV fusion inhibitor - Google Patents

MERS-CoV fusion inhibitor Download PDF

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CN110128510A
CN110128510A CN201910427530.7A CN201910427530A CN110128510A CN 110128510 A CN110128510 A CN 110128510A CN 201910427530 A CN201910427530 A CN 201910427530A CN 110128510 A CN110128510 A CN 110128510A
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ile
seq
gln
leu
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CN110128510B (en
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王潮
李悦
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Institute of Pharmacology and Toxicology of AMMS
Academy of Military Medical Sciences AMMS of PLA
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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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
    • 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
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    • C12N2740/10011—Retroviridae
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention belongs to biomedicine fields, are related to Formulas I compound represented, its stereoisomer, its mixture or its pharmaceutically acceptable salt.The invention further relates to the purposes of the compound, its stereoisomer, mixture or pharmaceutically acceptable salt in the drug of preparation treatment and/or prevention MERS-CoV infection or disease relevant to MERS-CoV infection (such as Middle East respiration syndrome).(Z-Ile-Glu-X1‑Ile‑Val‑Lys‑Lys‑Ile‑X2‑X3‑Ile‑Glu‑Arg‑Ala‑Ile‑Glu‑Ala‑Gln‑Gln‑X4‑Leu‑Leu‑Gln‑Leu‑Thr‑Val‑Trp‑X5‑Ile‑Lys‑Gln‑Leu‑Gln‑Ala‑Arg‑Ile‑Leu‑B)n(I)。

Description

MERS-CoV fusion inhibitor
Technical field
The invention belongs to biomedicine field, be related to the polypeptide compound of anti-MERS-CoV infection, its stereoisomer, its Mixture or its pharmaceutically acceptable salt.The invention further relates to the polypeptide compound, its stereoisomer, mixture or Pharmaceutically acceptable salt is in preparation treatment and/or prevention MERS-CoV infection or disease (example relevant to MERS-CoV infection Such as Middle East respiration syndrome) drug in purposes.
Background technique
2012, one kind being referred to as the novel coronavirus of Middle East respiration syndrome coronavirus (MERS-CoV) in the Middle East It is regional popular.The virus can cause the disease of SARS sample, lead to the failure of multiple organ dysfunction, and about 40% disease is caused in crowd Dead rate.Since MERS-CoV has the ability of human-to-human transmission, thus there is a possibility that extensive popular in crowd, it is therefore, anxious MERS-CoV need to be furtherd investigate as early as possible, to find its drug design target spot and develop corresponding anti-MERS-CoV drug, Clinical treatment and prevention MERS-CoV for patient are potentially very popular possibility.
MERS-CoV fusion protein is mainly made of S1 subunit and S2 subunit.Wherein, S1 subunit is responsible for and host cell table The CD26 receptor-specific in face, which identifies, to be combined；S2 subunit is responsible for merging between mediate retroviral and target cell membrane.S2 subunit again may be used It is further divided into fusogenic peptide (fusion peptide, FP), seven repetitive sequence of N-terminal (N-terminal heptad Repeat, NHR) and the functional areas seven repetitive sequence of C-terminal (C-terminal heptad repeat, CHR).S1 subunit with After CD26 receptor combines, S2 subunit occurred conformation changes, its FP is inserted into host cell membrane, while inducing the area NHR and CHR Domain interacts to form six bursts of α helical bundle (6HB) structures, so that mediate retroviral and host cell membrane merge.Fusion inhibitor It is a kind of novel antiviral drug that viral interference enters target cell.It is crucial stable intermediate in above-mentioned fusion process by preventing The formation of body 6HB, so that blocking virus and host cell membrane merge.Early-stage study shows additional derived from the virus region CHR Polypeptide fragment (C peptide), make its close NHR formed tripolymer surface " groove " and therewith formation heterologous 6HB, Ke Yiyou Effect blocks the formation of endogenous 6HB, and then blocking virus and target cell are merged.However, at present for being derived from MERS-CoV The research of the N peptides fusion inhibitor in the region NHR is simultaneously immature, therefore the polypeptide that development can efficiently inhibit MERS-CoV to merge Compound is very necessary.
Summary of the invention
I type enveloped virus (such as HIV, SARS-CoV, MERS-CoV etc.) known in the art is in fusion process, fusion Albumen will form 6HB higher structure, but be formed by 6HB structure in every kind of virus, and NHR sequence and CHR sequence exist Primary structure specificity, the i.e. polypeptide fragment derived from certain virus amalgamation protein CHR can only be specifically in conjunction with the viruses The NHR of NHR rather than other viruses.This, which causes this field to generally believe, can efficiently inhibit the fusion inhibitor of virus a kind of not The fusion (Nat.Commun.2014,5,3067) of other viruses can effectively be inhibited, such as derived from SARS-CoV fusion protein Peptide C P-1 cannot effectively inhibit the fusion of MERS-CoV, and the polypeptide HR2P derived from MERS-CoV fusion protein can not be effective Inhibit the invasion of SARS-CoV.It is unexpectedly, however that the present inventor is by many experiments and gropes repeatedly, is based on HIV-1 The alpha helical region NHR polypeptide sequence construct can efficiently inhibit MERS-CoV merge inhibitor, and thus provide treatment and The solution for preventing MERS-CoV infection.
Polypeptide compound
Therefore, in one aspect, the present invention provides Formulas I compound represented, its stereoisomer, its mixture or Its pharmaceutically acceptable salt:
(Z-Ile-Glu-X1-Ile-Val-Lys-Lys-Ile-X2-X3-Ile-Glu-Arg-Ala-Ile-Glu-Ala- Gln-Gln-X4-Leu-Leu-Gln-Leu-Thr-Val-Trp-X5-Ile-Lys-Gln-Leu-Gln-Ala-Arg-Ile-Leu- B)n(I)；
Wherein, n=1 or 3, also,
As n=1, Formulas I indicates monomer；
As n=3, Formulas I indicates the tripolymer that three monomers are formed by covalent cross-linking two-by-two；
X1、X2、X3、X4It is each independently selected from L-type amino acid；
X5For the amino acid residue that hydrophobic interaction can occur with MERS-CoV CHR, it is selected from hydrophobic amino Acid；
Z represents N-terminal group, is-NH2Or the group that amino obtains after the modification of amido protecting group；
B represents C-terminal group, the group obtained after carboxy protective group is modified for-COOH or carboxyl.
In certain embodiments, three monomers for forming tripolymer are mutually the same.
Compound of formula I of the invention is N peptide derivant, therefore is also referred to as N peptide in the present invention.As n=1, the present invention Formulas I compound represented be known as Formulas I monomer；As n=3, Formulas I compound represented of the invention is known as Formulas I tripolymer.Formula I monomer can be assembled to form tripolymer naturally in the solution.In order to increase the stability and activity of tripolymer, the present invention further exists Covalent cross-linking is increased between this three polypeptides, form more stable and there is higher active tripolymer compound, i.e. Formulas I Tripolymer.
In certain embodiments, the covalent cross-linking is the X in a Formulas I monomer14th lysine afterwards (Lys) with another Formulas I monomer in X3Covalent bond, preferably amido bond (- NC are formed between the 2nd glutamic acid (Glu) afterwards (O)-).In certain embodiments, the covalent cross-linking is the X in a Formulas I monomer1The side of 4th lysine afterwards X in chain amino and another Formulas I monomer3Amido bond (- NC (O) -) is formed between the side chain carboxyl group of the 2nd glutamic acid afterwards.? In certain embodiments, an amido bond can be formed between two Formulas I monomers, therefore in the tripolymer of formation, three Formulas I Three amido bonds can be formed between the polypeptide of monomer, thus achieve the purpose that stable tripolymer.
In the present invention, as statement " XnWhen the amino acid of which position afterwards ", XnIt is not counted in wherein, that is, XnAdjacent ammonia afterwards Base acid residue is Xn1st amino acid afterwards.
In certain embodiments, X1、X2、X3、X4It is each independently selected from glycine (Gly), glutamic acid (Glu), paddy ammonia Amide (Gln), alanine (Ala), leucine (Leu), isoleucine (Ile), aspartic acid (Asp), asparagine (Asn), Valine (Val), lysine (Lys), serine (Ser), threonine (Thr), tyrosine (Tyr), arginine (Arg), group ammonia Sour (His) and tryptophan (Trp).
In certain embodiments, X1Selected from glycine (Gly) and glutamic acid (Glu).
In certain embodiments, X2Selected from histidine (His) and asparagine (Asn).
In certain embodiments, X3Selected from histidine (His) and asparagine (Asn).
In certain embodiments, X4Selected from histidine (His) and lysine (Lys).
In certain embodiments, X5Selected from L-type hydrophobic amino acid.In certain embodiments, X5Selected from glycine (Gly), valine (Val), isoleucine (Ile), arginine (Arg) and glutamic acid (Glu).
In certain embodiments, X1Selected from glycine (Gly) and glutamic acid (Glu)；X2Selected from histidine (His) and day Winter amide (Asn)；X3Selected from histidine (His) and asparagine (Asn)；X4Selected from histidine (His) and lysine (Lys)；X5 Selected from glycine (Gly), valine (Val), isoleucine (Ile), arginine (Arg) and glutamic acid (Glu).
In the present invention, the N-terminal amino acid of Formulas I compound represented can optionally include amido protecting group, C End amino acid can optionally include carboxy protective group.Therefore, the N-terminal of Formulas I compound represented can be freely Or acylated, C-terminal is free, amidation or esterification.
In the present invention, the Z in Formulas I and B refers respectively to the N-terminal group and C-terminal group of polypeptide.When especially not dated When, the N-terminal of the compound of formula I (i.e. polypeptide) is NH2, C-terminal COOH.When special instruction N-terminal or C-terminal group When, refer to blocking group in NH2Or the group obtained after being modified on the basis of COOH is (that is, amino is through amino protecting group The group that the group or carboxyl obtained after group's modification obtains after carboxy protective group is modified), such as when N-terminal is acetyl When base (Ac), the group of the compound N end is AcNH, is expressed as Ac in the sequence, when C-terminal is amide groups, the chemical combination The group of object C-terminal is CONH2, it is expressed as NH in the sequence2。
In certain embodiments, Z represents the primary amine functional group of N-terminal amino acid, and the primary amine functional group is free Or replaced by amido protecting group.In certain embodiments, Z can be NH2Or amino is repaired through amido protecting group The group obtained after decorations, such as C1-6Alkylamidoalkyl, such as AcNH bpy- β A etc..In certain embodiments, the ammonia Base blocking group is selected from Ac (acetyl group), Fmoc (fluorenylmethyloxycarbonyl), trifluoroacetyl group, allyloxy carbonyl, Dde [i.e. 1- (4,4- dimethyl -2,6- dioxocyclohexylidene) ethyl] or Npys (i.e. 3- nitro -2- pyridine sulfenyl).
In certain embodiments, Z can be-NH2、-NR1C(O)R2、C1-6Alkyl, C1-6Alkoxy or-L-R3, wherein
R1And R2Independently selected from H and alkyl,
L is junction fragment, is selected from dicarboxylic acids (such as ethanedioic acid, malonic acid, succinic acid etc.), N-terminal is derived carboxylic Base and with have hydroxyl group R3At ester,
R3The group of cross-linking reaction can be reacted and not influenced with carboxyl for R ' OH, PEG, cholesterol etc., wherein R ' is alkane Base.
In certain embodiments, B represents the carboxyl functional group of C-terminal amino acid, and the carboxyl functional group is free Or replaced by carboxy protective group.In certain embodiments, B can be COOH or carboxyl through carboxy protective group The group obtained after modification, such as amide (- CONH2) or ester (- COOR), such as methyl esters, ethyl ester, benzyl ester, amide groups, hydrazide group Deng.In certain embodiments, the carboxy protective group is selected from methyl esters, ethyl ester, benzyl ester, amide groups, hydrazide group etc..
In certain embodiments, B can be-COOH or-C (O) NR4R5, wherein R4And R5Independently selected from H and alkane Base, preferably R4And R5It is simultaneously H.
In certain embodiments, the amino acid residue that Formulas I compound represented is included optionally includes blocking group. Blocking group, their relative reactivity and their inert conditions of holding are known to the skilled in the art, and can be referred to Such as Atherton B.and Sheppard R.C., " Solid Phase Peptide Synthesis:A practical approach",(1989),IRL Oxford University Press。
In certain embodiments, X3The 2nd glutamic acid optionally includes blocking group, such as OAll (O- allyl afterwards Base).
In some embodiments, the blocking group strategy used in the present invention is as follows: amino group is protected by Boc, carboxylic Base group is protected by Bzl, cHx or All, and tyrosine side chain is protected by 2-BrZ or Bzl, histidine side chains by Tos or Bom radical protection, aspartic acid and glutamate side chain are protected by Bzl, cHx or All, glutamine and the non-plus side of asparagine Chain protecting field uses, and the non-plus side chain protecting field of methionine uses, and arginine side chain is protected by Tos, and Trp side chain passes through For or Mts is protected and lysine side-chain passes through ClZ, Fmoc or Alloc and protects.
In other embodiments, the protectiveness group strategy used in the present invention is as follows: amino group is protected by Fmoc Shield, carboxylic group by tBu, All or Trt ester protect, tyrosine side chain by tBu protection, histidine side chains pass through Trt or Mtt radical protection, aspartic acid and glutamate side chain are protected by tBu or All, and glutamine and asparagine pass through side chain Trt radical protection, the non-plus side chain protecting field of methionine use, and arginine side chain is protected by Pmc or Pbf, Trp side chain By Boc protection or it is unprotected use, and lysine side-chain passes through Boc, Trt or Alloc protection.
The example of above-mentioned protective group and other protective groups, their introducing and removing, being capable of bibliography [Atherton B.and Sheppard R.C.,"Solid Phase Peptide Synthesis:A practical approach",(1989),IRL Oxford University Press]。
In certain exemplary implementation schemes, Formulas I compound represented is selected from:
IEGIVKKINNIERAIEAQQHLLQLTVWGIKQLQARIL (SEQ ID NO:1)；
(IEGIVKKINNIERAIEAQQHLLQLTVWGIKQLQARIL)3(SEQ ID NO:1)3；
IEGIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL (SEQ ID NO:2)；
(IEGIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL)3(SEQ ID NO:2)3；
IEEIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL (SEQ ID NO:3)；
(IEEIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL)3(SEQ ID NO:3)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWGIKQLQARIL (SEQ ID NO:4)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWGIKQLQARIL)3(SEQ ID NO:4)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWEIKQLQARIL (SEQ ID NO:5)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWEIKQLQARIL)3(SEQ ID NO:5)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWRIKQLQARIL (SEQ ID NO:6)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWRIKQLQARIL)3(SEQ ID NO:6)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWVIKQLQARIL (SEQ ID NO:7)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWVIKQLQARIL)3(SEQ ID NO:7)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWIIKQLQARIL (SEQ ID NO:8)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWIIKQLQARIL)3(SEQ ID NO:8)3；
Wherein, the N-terminal of each compound is free or acylated, and C-terminal is free, amidation or esterification 's.Herein, when compound is tripolymer, its N-terminal and C-terminal are stated, refers to form the monomer sequence of the tripolymer The N-terminal and C-terminal of column, and formed three sequence monomers of the tripolymer N-terminal and C-terminal it is mutually the same.
In certain embodiments, the N-terminal amino acid of each compound optionally includes amido protecting group, C End amino acid optionally includes carboxy protective group.
In certain embodiments, in above-mentioned each tripolymer, the 7th lysine from N-terminal of monomer sequence with Another sequence monomer forms amido bond between the 12nd glutamic acid from N-terminal, so that covalent cross-linking forms trimerization two-by-two Body.
In certain exemplary embodiments, Formulas I compound represented is selected from:
In certain embodiments, in above compound 2,4,6,8,10,12,14,16, monomer sequence from N end That has held the 7th lysine and another sequence monomer forms amido bond between the 12nd glutamic acid from N-terminal, thus two-by-two Covalent cross-linking forms tripolymer.
Polypeptide compound of the invention is not limited by its producing method, for example, it can pass through gene engineering method (weight Group technology) it generates, it can also be generated by chemical synthesis process.
In certain embodiments, Formulas I monomer of the invention can be synthesized using the Fmoc solid phase method of standard, be selected Rink Amide resin, peptide chain are extended from C-terminal to N-terminal.HBTU/HoBt/DIEA can be used in condensing agent.Piperazine can be used in deprotection agent Pyridine/DMF solution.It can use CS Bio Peptide synthesizer synthesis peptide sequence, the N-terminal acetic anhydride reagent acetoxylation of last polypeptide Sealing end.Trifluoroacetic acid/dithioglycol/metacresol (TFA/EDT/m-cresol) can be used in decomposition agent, and thick peptide freezes after being dissolved with water It is dry to save.It can be isolated and purified, pure peptide content > 95%, be used with medium pressure liquid chromatography method or high pressure lipuid chromatography (HPLC) (HPLC) MALDI TOF MS (MALDI-TOF-MS) determines peptide sequence molecular weight.
In certain embodiments, the synthesis of Formulas I tripolymer of the invention is with the following method: Solid phase synthesis Formulas I list Body, X in synthesis process3The 2nd glutamate side chain semipermanent protecting group is selected as OAll group afterwards；After Peptide systhesis, Selectively removing OAll protecting group on peptide resin, then to X in Formulas I monomer32nd glutamate side chain carboxyl and benzyl mercaptan afterwards Reaction forms thioesters；After the reaction was completed at ester, peptide resin, purified polypeptide.By it is above-mentioned it is purified after obtain polypeptide dissolution In reaction solution, then it can spontaneously form Trimeric structures, at this time X in a polypeptide monomer14th lysine and another afterwards The benzyl thioesters of one polypeptide monomer Glutamic Acid side chain reacts, and forms the amido bond between polypeptide chain, i.e. isopeptide bond.At this In Trimeric structures, due to X1X in 4th lysine and another Formulas I monomer afterwards3The 2nd glutamic acid is located at more afterwards G and e of peptide, it is all more appropriate on space length and direction in space, advantageously form isopeptide bond, and the amino of remaining position Acid cannot participate in the formation of isopeptide bond since space length and direction in space are improper, that is, the ammonia of remaining position Improper formation without will affect isopeptide bond of the base acid due to space length and direction in space.
In certain embodiments, the pharmaceutically acceptable salt is inorganic acid salt or acylate.Certain preferred Embodiment in, the pharmaceutically acceptable salt be acylate, such as citrate, fumarate, maleate or Tartrate.In certain preferred aspects, the pharmaceutically acceptable salt is inorganic acid salt, such as hydrochloride, sulphur Hydrochlorate, phosphate, mesylate or benzene sulfonate.
In certain embodiments, polypeptide compound of the invention can be as the mixed of stereoisomer or stereoisomer Object is closed to exist；For example, configuration L-, D- or racemic can be had independently of each other by constituting their amino acid.Accordingly, it is possible to It is to obtain the mixture or pure diastereomer or enantiomer of heterogeneous mixture and racemic mixture or diastereomer, take The certainly asymmetric carbon present in the number of asymmetric carbon and isomers or heterogeneous mixture.The preferred structure of the compounds of this invention Pure isomer namely enantiomer or diastereomer.
On the other hand, the present invention provides a kind of isolated nucleic acid molecules, and it includes encode Formulas I as described herein The nucleotide sequence of monomer.
On the other hand, the present invention provides a kind of carriers, and it includes the nucleic acid molecules separated as described above.This hair Bright carrier can be cloning vector, be also possible to expression vector.
On the other hand, the present invention provides the host cells comprising isolated nucleic acid molecules or carrier of the invention. Such host cell includes but is not limited to prokaryotic cell such as Bacillus coli cells and eukaryocyte such as yeast cells, elder brother Worm cell, plant cell and zooblast (such as mammalian cell, such as mouse cell, people's cell etc.).Cell of the invention It can also be cell line, such as 293T cell.
Pharmaceutical composition and medical usage
Polypeptide compound of the invention can be used for external or inhibit MERS-CoV and cell fusion in subject's body, thus For preventing and/or treating MERS-CoV infection or disease relevant to MERS-CoV infection (for example, Middle East respiration syndrome).
Therefore, on the other hand, the present invention provides a kind of pharmaceutical compositions, and it includes Formulas I institutes as described herein Compound, its stereoisomer, its mixture or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier shown And/or excipient.
In certain embodiments, described pharmaceutical composition includes at least one Formulas I compound represented, its alloisomerism Body, its mixture or its pharmaceutically acceptable salt.
Pharmaceutical composition of the invention can be any form known to medical domain.For example, described pharmaceutical composition can To be tablet, pill, suspension, emulsion, solution, gelling agent, capsule, pulvis, granule, elixir, pastille, suppository, injection The forms such as agent (including injection, freeze dried powder), inhalant, spray.Preferred dosage form depends on expected administration mode and controls Treat purposes.
Pharmaceutical composition of the invention may include the Formulas I as described herein of " therapeutically effective amount " or " prevention effective dose " Compound represented, its stereoisomer, its mixture or its pharmaceutically acceptable salt." prevention effective dose " refers to, it is sufficient to Prevention prevents, or postpones the amount of the generation of disease." therapeutically effective amount " refers to, it is sufficient to cure or at least partly prevent to have suffered from The disease of the patient of disease and the amount of its complication.It will be appreciated by those skilled in the art that chemical combination shown in Formulas I as described herein Object, its stereoisomer, its mixture or its pharmaceutically acceptable salt therapeutically effective amount can be become according to following factor Change: the severity of disease to be treated, the overall status of the immune system of patient oneself, the ordinary circumstance of patient such as age, Weight and gender, method of application of drug, and the other treatment being administered simultaneously etc..
On the other hand, the present invention relates to Formulas I compound represented as described herein, its stereoisomer, mixing Object or pharmaceutically acceptable salt are being prepared for MERS-CoV infection or and MERS- to be treated and/or prevented in subject CoV infects the purposes in the drug of relevant disease.
In certain embodiments, the disease relevant to MERS-CoV infection is Middle East respiratory system syndrome.
In certain embodiments, the subject is mammal, such as people.In certain embodiments, it is described by Examination person (such as people) infects with MERS-CoV, alternatively, having the risk of infection MERS-CoV.
On the other hand, feel the present invention provides a kind for the treatment of and/or prevention MERS-CoV infection or with MERS-CoV Contaminate the method for relevant disease comprising apply shown in a effective amount of Formulas I as described herein to subject with this need Compound, its stereoisomer, its mixture or its pharmaceutically acceptable salt or pharmaceutical composition as described herein.
In certain embodiments, the disease relevant to MERS-CoV infection is Middle East respiratory system syndrome.
In certain embodiments, the subject is mammal, such as people.In certain embodiments, it is described by Examination person (such as people) infects with MERS-CoV, alternatively, having the risk of infection MERS-CoV.
On the other hand, the invention further relates to a kind of method for inhibiting MERS-CoV and cell fusion in vitro, packets Include by MERS-CoV and Formulas I compound represented as described herein, its stereoisomer, its mixture or its pharmaceutically may be used The step of salt contact of receiving.In certain embodiments, the method is used for non-treatment purpose.
In the present invention, Formulas I compound represented as described herein, its stereoisomer, its mixture or its pharmacy Upper acceptable salt or pharmaceutical composition can be applied by any suitable method known in the art, including but unlimited In, take orally, oral cavity, sublingual, eyeball, part, parenteral, rectum, in leaf sheath, in endoplasm net slot, in groin, bladder, office Portion's (e.g., pulvis, ointment or drops) or nasal.But for many therapeutical uses, preferred administration route/side Formula is parenteral (such as being injected intravenously, be subcutaneously injected, intraperitoneal injection, intramuscular injection).It should be understood to the one skilled in the art that administration Approach and/or mode will change according to expected purpose.
Term definition
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The normally understood meaning of personnel institute.Also, virology used herein, molecular biology, nucleic acid chemistry, cell culture, life The operating procedures such as object chemistry, cell biology are widely used conventional steps in corresponding field.Meanwhile in order to more preferable geographical The solution present invention, is provided below the definition and explanation of relational language.
Present document relates to 20 writing for conventional amino acid follow common usage.See, for example, Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates, Sunderland, Mass. (1991)), it is incorporated herein by reference.In the present invention, term " polypeptide " and " albumen Matter " has the same meaning and is used interchangeably.And in the present invention, amino acid usually use single-letter well known in the art and Trigram is abridged to indicate.For example, alanine can be indicated with A or Ala.
As used herein, term " alkyl " refers to the linear chain or branched chain monovalent hydrocarbon of saturation, can be and is taken Generation it is (single or multiple) or unsubstituted.Preferably, the alkyl is C1-20Alkyl, more preferable C1-15, more preferable C1-10Alkyl, More preferable C1-8Alkyl, more preferable C1-6Alkyl, more preferable C1-4Alkyl.Particularly preferred alkyl includes such as methyl, ethyl, just Propyl, isopropyl, normal-butyl, sec-butyl, isobutyl group, tert-butyl, n-pentyl, tertiary pentyl, neopentyl, n-hexyl, n-heptyl and N-octyl etc..Suitable substituent group include for example hydroxyl, alkyl, halogen, alkoxy, halogenated alkyl, halogenated alkoxy, amino, Aminoalkyl or naphthenic base.
As used herein, term " C1-C6Alkyl " refers to that the linear chain or branched chain alkane containing 1-6 carbon atom is gone Fall the group obtained after a hydrogen atom, specific example includes but is not limited to: methyl, ethyl, propyl, normal-butyl, n-pentyl, N-hexyl, isopropyl, tert-butyl or isobutyl group.
As used herein, term " alkoxy " refers to, the group formed in a manner of alkyl-O-, specific example Including but not limited to methoxyl group, ethyoxyl, positive propoxy, isopropoxy, tert-butoxy etc..
As used herein, term " C1-6Alkylamidoalkyl " refers to C1-6Alkyl-CO-NH, the C1-6Alkyl refers to Linear chain or branched chain univalent saturated hydrocarbon radical containing 1-6 carbon atom, for example, methyl, ethyl, n-propyl, isopropyl, normal-butyl, Isobutyl group, tert-butyl, amyl, hexyl etc..
As used herein, term " blocking group " indicates to inhibit or forbid the group of undesirable chemical reaction, But the group be designed to activity adequately so as to its do not change the remainder of molecule it is enough mild under conditions of, can From the functional group's fracture discussed.After deprotection, desired product is obtained.Term " amido protecting group " refers to, can be used in Protect the blocking group of amino (for example, polypeptide N-terminal amino).Such blocking group is well known to those skilled in the art, Example includes but is not limited to Boc (tertiary oxygen-butyl carbonyl), Fmoc (fluorenylmethyloxycarbonyl), trifluoroacetyl group, allyloxy carbonyl Base, Dde [i.e. 1- (4,4- dimethyl -2,6- dioxocyclohexylidene) ethyl]
Or Npys (i.e. 3- nitro -2- pyridine sulfenyl) etc..Term " carboxy protective group " refers to, can be used in protecting The blocking group of carboxyl (for example, peptide C terminal carboxyl group).Such blocking group is well known to those skilled in the art, the example Including but not limited to, methyl esters, the tert-butyl ester or benzyl ester etc..
As used herein, term " pharmaceutically acceptable salt " refers to, (i) in compound provided by the present invention The salt that existing acidic functionality (such as-COOH) and appropriate inorganic or organic cation (alkali) are formed, and including but It is not limited to, alkali metal salt, such as sodium salt, sylvite, lithium salts；Alkali salt, such as calcium salt, magnesium salts；Other metal salts, such as aluminium Salt, molysite, zinc salt, mantoquita, nickel salt, cobalt salt etc.；Inorganic base salts, such as ammonium salt；Organic alkali salt, such as t-octyl amine salt, dibenzyl amine Salt, alkylbenzyldimethylasaltsum saltsum, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-METHYL-ALPHA-L-GLUCOSAMINE salt, guanidine salt, diethylamine salt, Triethylamine salt, dicyclohexyl amine salt, N, N '-dibenzyl ethylenediamine salt, chloroprocanine salt, procaine salt, diethanolamine salt, N- benzyl-phenethyl amine salt, piperazine salt, tetramethyl amine salt, three (methylol) aminomethane salt.And (ii) present invention is mentioned Basic functionality present in the compound of confession (such as-NH2) with appropriate inorganic or organic anion (acid) formation salt, And include but is not limited to, halogen acid salt, such as hydrofluoride, hydrochloride, hydrobromate, hydriodate；Inorganic acid salt, such as nitre Hydrochlorate, perchlorate, sulfate, phosphate etc.；Rudimentary alkyl sulfonate, such as mesylate, fluoroform sulphonate, esilate Deng；Arylsulphonate, such as benzene sulfonate, P-TOLUENE SULFO ACID 99's salt；Acylate, such as acetate, malate, fumarate, amber Amber hydrochlorate, citrate, tartrate, oxalates, maleate etc.；Amino-acid salt, such as glycinate, trimethylglycine Salt, arginine salt, ornithine salt, glutamate, aspartate etc..
As used herein, term " pharmaceutically acceptable carrier and/or excipient " refers to, pharmacology and/ Or carrier and/or excipient physiologically compatible with subject and active constituent, be it is well known in the art (see, for example, Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th Ed.Pennsylvania:Mack Publishing Company, 1995), and include but is not limited to: pH adjusting agent, surface Activating agent, ionic strength reinforcing agent maintain the reagent of osmotic pressure, postpone the reagent absorbed, diluent, adjuvant, preservative, stabilization Agent etc..For example, pH adjusting agent includes but is not limited to phosphate buffer.Surfactant include but is not limited to cation, yin from Son or nonionic surface active agent, such as Tween-80.Ionic strength reinforcing agent includes but is not limited to sodium chloride.It maintains to seep The reagent pressed thoroughly includes but is not limited to sugar, NaCl and the like.The reagent that delay absorbs includes but is not limited to Monostearate And gelatin.Diluent includes but is not limited to water, aqueous buffer solution (such as buffered saline), pure and mild polyalcohol (such as glycerol).Adjuvant Including but not limited to aluminium adjuvant (such as aluminium hydroxide), Freund's adjuvant (such as complete Freund's adjuvant) etc..Preservative includes but not It is limited to various antibacterial agents and antifungal agents, such as thimerosal, 2- Phenoxyethanol, metagin, anesin, Phenol, sorbic acid etc..Stabilizer have the normally understood meaning of those skilled in the art, can stablize in drug activity at The expectation divided is active (such as oncolytic activity), including but not limited to sodium glutamate, gelatin, SPGA, carbohydrate (such as sorbierite, sweet dew Alcohol, starch, sucrose, lactose, glucan or glucose), amino acid (such as glutamic acid, glycine), protein (such as drying whey, Albumin or casein) or its catabolite (such as lactalbumin hydrolysate).
As used herein, term " carrier (vector) " refers to, the one kind that can be inserted polynucleotide Nucleic acid delivery vehicle.When carrier can make the albumen of the polynucleotide encoding of insertion obtain expression, carrier is known as expression vector.It carries Body can import host cell by conversion, transduction or transfection, and the inhereditary material element for carrying it obtains in host cell It must express.Carrier is well known to those skilled in the art, including but not limited to: plasmid；Phasmid；Coemid；It is artificially colored Body, such as the artificial chromosome (PAC) of yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or the source P1；Phagocytosis Body such as λ bacteriophage or M13 bacteriophage and animal virus etc..The animal virus that can be used as carrier includes but is not limited to reverse transcriptase Viral (including slow virus), adenovirus, adeno-associated virus, herpesviral (such as herpes simplex virus), poxvirus, baculoviral, Papillomavirus, papova viruses (such as SV40).A kind of element that carrier can be expressed containing various control, including but It is not limited to, promoter sequence, transcriptional initiation sequence, enhancer sequence, selection element and reporter gene.In addition, carrier can also contain There is replication origin.
As used herein, term " host cell " refers to, can be used for importing the cell of carrier comprising but it is unlimited In, such as the prokaryotic cell of Escherichia coli or withered grass bacterium, such as the fungal cell of yeast cells or Aspergillus, such as S2 drosophila cell Or the insect cell of Sf9 etc., or such as fibroblast, Chinese hamster ovary celI, COS cell, NSO cell, HeLa cell, bhk cell, The zooblast of 293 cell of HEK or people's cell etc..
Advantageous effect of the invention
The specificity of the primary structure as present in NHR sequence and CHR sequence, this field, which generally believes, efficiently to be inhibited A kind of fusion inhibitor of I type enveloped virus can not effectively inhibit the fusion of other viruses.However, the present inventor is unexpected Ground constructs the inhibitor that can efficiently inhibit MERS-CoV to merge based on the alpha helical region HIV-1 NHR polypeptide sequence, for The treatment and prevention of MERS-CoV infection have great clinical value, and the research and development to novel MERS-CoV fusion inhibitor Provide new approaches.
Sequence information
In the table 1 that the information of partial sequence of the present invention is provided below.
Table 1: the description of sequence
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is It can be with conventional products that are commercially available.
The abbreviation being used in the present invention has following meaning:
Ala (Alanine, A) alanine
Asn (Asparagine, N) asparagine
Asp (Aspartic acid, D) aspartic acid
DCM (Dichloromethane) dichloroethanes
DMF (N, N-Dimethyl malonate) dimethylformamide
Fmoc (Fluorenylmethoxycarbonyl) fluorenylmethyloxycarbonyl
Gln (Glutamine, Q) glutamine
Glu (Glutamic acid, E) glutamic acid
Gly (Glycine, G) glycine
HBTU 2- (1H-1- hydroxybenzotriazole) -1,1,3,3- tetramethylurea hexafluorophosphoric acid
His (Histidine, H) histidine
HOBt (1-Hydroxylbenzotriazole anhydrous) 1- hydroxy benzo triazole
HR1 (N-terminal heptad repeat, NHR) N-terminal repetitive sequence
HR2 (C-terminal heptad repeat, CHR) C-terminal repetitive sequence
HPLC (High performance liquid chromatography) high performance liquid chromatography
Ile (Isoleucine, I) isoleucine
Leu (Leucine, L) leucine
Lys (Lysine, K) lysine
MERS (the Middle East Middle East respiratory syndrome respiration syndrome
The Middle East MERS-CoV respiration syndrome coronavirus
Met (Methionine, M) methionine
NIle (Norleucine, Z) nor-leucine
Ser (Serine, S) serine
TFA (trifluoroacetic acid) trifluoroacetic acid
Thr (Threonine, T) threonine
Tyr (Tyrosine, Y) tyrosine
Val (Valine, V) valine
Solid-phase synthesized carrier Rink amide resin used in following embodiment is that Tianjin Nankai synthesizes responsibility Co., Ltd product； HBTU, HOBT, DIEA, EDC hydrochloride and fmoc-protected natural amino acid are that Shanghai gill biochemical corp and Chengdu promise are new Technology Co., Ltd product.Trifluoroacetic acid (TFA) is Beijing Bo Mai Science and Technology Ltd. product；DMF, DCM are rich for Beijing Mai Jie Science and Technology Ltd. product；Trifluoroacetic acid aqueous solution is Fisher Products.Other reagents are such as domestic analysis without explanation Net product.
The preparation of 1. polypeptide monomer of preparation example
Compound 1 (SEQ ID NO:9) synthesis uses the Fmoc solid phase method of standard.Select Rink Amide resin, peptide Chain is extended from C-terminal to N-terminal.Condensing agent is HBTU/HOBt/DIEA.Deprotection agent is piperidines/DMF solution.Utilize CS Bio polypeptide Synthesizer synthesizes peptide sequence, and the N-terminal of last polypeptide is blocked with acetic anhydride reagent acetoxylation.Lysate is trifluoroacetic acid/ethylene dithiol Alcohol/metacresol (TFA/EDT/m-cresol), thick peptide are lyophilized after being dissolved with water and save.With medium pressure liquid chromatography method or high pressure liquid phase Chromatography (HPLC) is isolated and purified, pure peptide content > 95%, and uses MALDI TOF MS (MALDI-TOF-MS) peptide sequence molecular weight is determined.
Synthesis condition is as follows:
Protected amino acid: the DMF solution of 0.25M,
Activator: the DMF solution of 0.2M HBTU/HOBt,
Alkali: the DMF solution of 0.4M DIEA is activated,
Deprotection agent: the DMF solution of 20%v/v piperidines,
Closed reagent: the DMF solution of 20%v/v acetic anhydride.
Rink Amide resin 0.53g (0.23mmol) merging CEM microwave Peptide synthesizer is weighed, then by amino acid, Activator, activate alkali, deprotecting regent, after closed reagent is configured by above-mentioned concentration, with the automatic Peptide synthesizer of CS Bio into Row synthesis.It is shunk after peptide resin washs 3 times with DMF after the completion with anhydrous methanol, room temperature in vacuo is dry, obtains peptide resin about 1.95g.
Lysate (percent by volume): trifluoroacetic acid: metacresol: methyl phenyl ethers anisole: water=17:1:1:1.
The cracking of peptide resin: the synthetic peptide resin 1.95g of the automatic Peptide synthesizer of CS Bio is weighed, 500ml eggplant is put into In shape bottle, ice bath, electromagnetic agitation.Lysate is prepared by the amount that 10ml is added in 1 gram of peptide resin.TFA needs preparatory ice bath cooling 30min Or it deposits in refrigerator use in advance；The lysate prepared is added in the peptide resin under condition of ice bath, electromagnetic agitation, is set Rouge becomes orange red, and 30min is reacted under condition of ice bath, then removes ice bath and continues to be stirred to react 200min at room temperature, and reaction is completed, It is vigorously stirred lower addition cold ether 500ml, white precipitate is precipitated, continues to stir 60min；Precipitate is filtered out with G4 sand core funnel, It is washed 3 times, is dried repeatedly with cold ether.10% acetic acid aqueous solution 150ml is added, acetonitrile 5ml dissolves solid sufficiently, it filters, Thick peptide 1.03g is lyophilized to obtain in filtrate.
The purifying of thick peptide: thick peptide is purified with medium-pressure or high pressure chromatography.Chromatographic column is C8 column, and eluant, eluent is acetonitrile, water And a small amount of acetic acid.Concrete operation step: weighing thick peptide 1.00g, and water 20ml, acetonitrile 5ml is added to dissolve solid, is centrifuged 10min (3000 revs/min), take supernatant loading.Chromatographic column uses 15% acetonitrile/water/0.1% glacial acetic acid solution 200ml balance in advance. Continue to be rinsed with 15% acetonitrile/water/0.1% glacial acetic acid solution 200ml after loading, efficient liquid phase detects eluent ingredient.According to Liquid phase testing result gradually rises ethane nitrile content, until purified polypeptide main peak is eluted out.Merge eluent, rotation is steamed Hair takes out most of solvent, and pure polypeptide is lyophilized, and HPLC detection level is greater than 90%, MALDI-TOF and confirms molecular weight.
Reverse phase prepares the secondarily purified N peptide of liquid phase, and the specific method is as follows: by the N peptide of middle pressure after purification, with 2ml acetonitrile and 8ml Pure water dissolution, the loading after the membrane filtration in 0.25 μM of aperture.Reverse phase preparation liquid phase is first balanced each other 5min with 20%B, after loading, The content of B phase is gradually risen according to gradient, until purified polypeptide main peak is eluted out.HPLC detection level is big Eluent in 95% merges, and rotary evaporation removes most of solvent, be lyophilized pure N peptide.
By method same as described above prepare other uncrosslinked monomer N peptides (compound 3,5,7,9,11,13, 15)。
The preparation of 2. polypeptide tripolymer of preparation example
Compound 2 ((SEQ ID NO:9)3) monomer peptide sequence synthesis with preparation example 1, but connect amino acid in resin When, change the E (calculating the 12nd from N-terminal) for needing site to modify into E (OAll, O- allyl).Sequence has synthesized on resin Bi Hou is not cracked, and does chemical modification further below.
On resin, the Side chain protective group OAll of E (OAll) in polypeptide sequence is removed: the polypeptide resin that synthesis is finished, It is transferred to 50ml eggplant-shape bottle, and the 0.26g tetra- that weighs (triphenyl phosphorus palladium) (Pd (PPh3)4), 0.31g5,5- dimedone It is dissolved in 8ml anhydrous tetrahydro furan and methylene chloride mixed solvent (v/v=1/1), is added in eggplant-shape bottle, is protected from light 6h. In polypeptides reactive device, DCM, DMF are washed 3 times, are washed 5 times with 50ml0.5%DIEA/DMF solution later, 50ml0.5% copper Reagent/DMF solution washs 5 times, finally uses DMF, and DCM, MeOH are washed 2 times, drained.
Then on resin, peptide side chain benzyl thioesterification after removing Side chain protective group: in polypeptides reactive device, it is added de- Polypeptide resin after Side chain protective group out, and by 275 μ L benzyl mercaptans, 315mg HOBt, 450mg EDC hydrochloride is dissolved in 5ml It is added in reactor after in DMF/5ml DCM solvent, after reacting 6h and 12h respectively twice, finally uses DMF, DCM, MeOH are washed It washs 2 times, drains.
It is cracked later according to the method for preparation example 1, the method for purifying, obtains pure peptide, HPLC detects purity and is greater than 90%.
The N peptide of thioesters modification after purification is dissolved in reaction dissolvent (20%ACN/30%PBS/50%H2O, concentration is about In 1mg/ml), react 40h at 37 DEG C, HPLC detection reaction is to complete, later in the purifying item to reverse phase preparation efficient liquid phase Under part, purifying obtains target N peptide purity greater than 95%.
Single-stranded N peptide of the invention can form tripolymer (N3 spiral) structure, and in order to increase trimer stability, we are by one 7th lysine of the 12nd glutamic acid of article single-stranded N peptide and another article of single-stranded N peptide is by amido bond covalent cross-linking, thus shape At stable triple-helix structure.
Other N peptide tripolymers (compound 4,6,8,10,12,14,16) is prepared by method same as described above.
The molecular weight of above-mentioned each polypeptide is shown in Table 2.
Table 2: polypeptide molecular weight
Compound Sequence Molecular weight
1 Ac-IEGIVKKINNIERAIEAQQHLLQLTVWGIKQLQARIL-NH2 4302.09
2 (Ac-IEGIVKKINNIERAIEAQQHLLQLTVWGIKQLQARIL-NH2)3 12852.27
3 Ac-IEGIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL-NH2 4311.13
4 (Ac-IEGIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL-NH2)3 12879.39
5 Ac-IEEIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL-NH2 4383.19
6 (Ac-IEEIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL-NH2)3 13095.57
7 Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWGIKQLQARIL-NH2 4429.26
8 (Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWGIKQLQARIL-NH2)3 13233.78
9 Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWEIKQLQARIL-NH2 4501.32
10 (Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWEIKQLQARIL-NH2)3 13449.96
11 Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWRIKQLQARIL-NH2 4528.4
12 (Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWRIKQLQARIL-NH2)3 13531.20
13 Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWVIKQLQARIL-NH2 4471.34
14 (Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWVIKQLQARIL-NH2)3 13360.02
15 Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWIIKQLQARIL-NH2 4485.37
16 (Ac-IEEIVKKIHHIERAIEAQQKLLQLTVWIIKQLQARIL-NH2)3 13402.11
The characterization of the α helicity of 1. polypeptide compound of embodiment
1, the configuration of N peptide solution
The pure peptide of about 1mg is weighed, 700 μ L ddH are dissolved in2It in O, shakes, centrifugation takes after supernatant in nanodrop2000 The concentration of N peptide solution is demarcated on instrument.With buffer PBS (pH=7.2) for diluent, 10 μM of N peptide solution are configured, 500 μ L.
2, the helicity of N peptide solution is tested
Configured N peptide solution is added in cuvette, its spiral absorption value has been measured in circular dichroism instrument ( Deduct blank control absorption value), and according to following formula, it is converted into helicity:
Wherein concentration (c) refers to that the concentration value of N peptide solution, path (L) refer to reference pond length, and residue number (N) refers to measurement N peptide Amide bond number.
The spiral angle value of above-mentioned preparation example polypeptide compound obtained is as shown in the table.
Table 3: polypeptide spiral angle value
The cell-cell fusion activity that 2. polypeptide compound of embodiment inhibits MERS-CoV to mediate evaluates (IC50)
1) first day: digestion 293T simultaneously prepares cell suspension, is added 2~4 × 10 in 6 orifice plates in every hole5A cell.
2) second day: culture 12~for 24 hours after, transfected to density 80% or so, wherein a hole transfect pAAV- IRES-EGFP-MERS-S plasmid (293T/MERS/EGFP) is used for fusion experiment；One hole transfects pAAV-IRES-EGFP (293T/ EGFP) it is used as negative control.
3) after transfecting 8~12h, fresh DMEM cell culture fluid is replaced.
4) the 4th day: cell can express stronger fluorescence after 36~48h of transfection, can carry out subsequent Fusion inhibiting test.
5) preceding 6~12h is merged, Huh-7 cell is digested, cell suspension is prepared, is added 4~5 × 10 in every hole4A cell. 37 DEG C of cultures are spare.
6) polypeptide of gradient dilution, every 55 μ L reaction system of hole are prepared.
7) 293T/MERS/EGFP cell and 293T/EGFP cell are digested with EDTA.It is outstanding to prepare individual cells Liquid.
8) every hole is added 10 in drug plate4A 293T/MERS/EGFP cell.Culture hole without drug is set simultaneously and does sun Property control (293T/MERS/EGFP+Huh-7), 293T/EGFP cell hole does negative control (293T/EGFP+Huh-7).Whole body Product control is in 110 μ L.
9) it is incubated at room temperature 30min, acts on drug sufficiently with cell.
10) take out abandon Huh-7 cell culture medium, take the mixed liquor of 100 μ L drugs and cell be added to Huh-7 cell it On.
11) situation of observation fusion for 24 hours, has to positive controls (293T/MERS/EGFP+Huh-7) cell and significantly melts It closes, 4% isometric poly formic acid is added and fixes, terminate fusion.
12) fluorescence microscope or High content screening system carry out photographic analysis.Calculate inhibition of the polypeptide to cell fusion Rate, and use the IC of CalcuSyn software calculating polypeptide50Value.
The active testing result of each polypeptide compound is as shown in the table.The results show that the polypeptide compound of all tests is equal It shows the significant activity for inhibiting MERS-CoV cell fusion, and has reached nM level, part of compounds is even substantially better than Positive control inhibitor HR2P-M2 derived from the alpha helical region MERS-CoV HR2.
In addition, according to the result of table 4 it is found that the X of Formulas I5It can be any hydrophobic amino acid without influencing peptide The inhibitory activity of object.
Table 4: fusion inhibitory activity
Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.
SEQUENCE LISTING
<110>PLA Academy of Military Sciences's military medical research institute
<120>MERS-CoV fusion inhibitor
<130> IDC190107
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>Formulas I monomer -1
<400> 1
Ile Glu Gly Ile Val Lys Lys Ile Asn Asn Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 2
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<212> PRT
<213>artificial sequence
<220>
<223>Formulas I monomer -2
<400> 2
Ile Glu Gly Ile Val Lys Lys Ile Asn Asn Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 3
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<212> PRT
<213>artificial sequence
<220>
<223>Formulas I monomer -3
<400> 3
Ile Glu Glu Ile Val Lys Lys Ile Asn Asn Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 4
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>Formulas I monomer -4
<400> 4
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 5
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>Formulas I monomer -5
<400> 5
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Glu Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 6
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<212> PRT
<213>artificial sequence
<220>
<223>Formulas I monomer -6
<400> 6
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Arg Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 7
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>Formulas I monomer -7
<400> 7
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Val Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 8
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>Formulas I monomer -8
<400> 8
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Ile Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 9
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>compound 1
<220>
<221> MOD_RES
<222> (1)..(1)
<223> ACETYLATION
<220>
<221> MOD_RES
<222> (37)..(37)
<223> AMIDATION
<400> 9
Ile Glu Gly Ile Val Lys Lys Ile Asn Asn Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
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<220>
<223>compound 3
<220>
<221> MOD_RES
<222> (1)..(1)
<223> ACETYLATION
<220>
<221> MOD_RES
<222> (37)..(37)
<223> AMIDATION
<400> 10
Ile Glu Gly Ile Val Lys Lys Ile Asn Asn Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
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<223>compound 5
<220>
<221> MOD_RES
<222> (1)..(1)
<223> ACETYLATION
<220>
<221> MOD_RES
<222> (37)..(37)
<223> AMIDATION
<400> 11
Ile Glu Glu Ile Val Lys Lys Ile Asn Asn Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 12
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<212> PRT
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<223>compound 7
<220>
<221> MOD_RES
<222> (1)..(1)
<223> ACETYLATION
<220>
<221> MOD_RES
<222> (37)..(37)
<223> AMIDATION
<400> 12
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 13
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>compound 9
<220>
<221> MOD_RES
<222> (1)..(1)
<223> ACETYLATION
<220>
<221> MOD_RES
<222> (37)..(37)
<223> AMIDATION
<400> 13
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Glu Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 14
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>compound 11
<220>
<221> MOD_RES
<222> (1)..(1)
<223> ACETYLATION
<220>
<221> MOD_RES
<222> (37)..(37)
<223> AMIDATION
<400> 14
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Arg Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 15
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>compound 13
<220>
<221> MOD_RES
<222> (1)..(1)
<223> ACETYLATION
<220>
<221> MOD_RES
<222> (37)..(37)
<223> AMIDATION
<400> 15
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Val Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35
<210> 16
<211> 37
<212> PRT
<213>artificial sequence
<220>
<223>compound 15
<220>
<221> MOD_RES
<222> (1)..(1)
<223> ACETYLATION
<220>
<221> MOD_RES
<222> (37)..(37)
<223> AMIDATION
<400> 16
Ile Glu Glu Ile Val Lys Lys Ile His His Ile Glu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln Lys Leu Leu Gln Leu Thr Val Trp Ile Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu
35

Claims (10)

1. Formulas I compound represented, its stereoisomer, its mixture or its pharmaceutically acceptable salt:
(Z-Ile-Glu-X1-Ile-Val-Lys-Lys-Ile-X2-X3-Ile-Glu-Arg-Ala-Ile-Glu-Ala-Gln- Gln-X4-Leu-Leu-Gln-Leu-Thr-Val-Trp-X5-Ile-Lys-Gln-Leu-Gln-Ala-Arg-Ile-Leu-B)n (I)；
Wherein, n=1 or 3, also,
As n=1, Formulas I indicates monomer, and Formulas I compound represented is known as Formulas I monomer；
As n=3, Formulas I indicates the tripolymer that three monomers are formed by covalent cross-linking two-by-two, and Formulas I compound represented is known as Formulas I tripolymer；
X1、X2、X3、X4It is each independently selected from L-type amino acid；
X5Selected from hydrophobic amino acid；
Z represents N-terminal group, is-NH2Or the group that amino obtains after the modification of amido protecting group；
B represents C-terminal group, the group obtained after carboxy protective group is modified for-COOH or carboxyl.
2. Formulas I compound represented described in claim 1, its stereoisomer, its mixture or its is pharmaceutically acceptable Salt, wherein the covalent cross-linking is the X in a Formulas I monomer14th lysine and X in another Formulas I monomer afterwards3 Covalent bond is formed between the 2nd glutamic acid afterwards；
Preferably, the covalent bond is amido bond (- NC (O) -).
3. Formulas I compound represented of any of claims 1 or 2, its stereoisomer, its mixture or its can pharmaceutically connect The salt received, wherein X1、X2、X3、X4It is each independently selected from glycine, glutamic acid, glutamine, alanine, leucine, different bright Propylhomoserin, aspartic acid, asparagine, valine, lysine, serine, threonine, tyrosine, arginine, histidine and color ammonia Acid.
4. the described in any item Formulas I compounds represented of claim 1-3, its stereoisomer, its mixture or its pharmaceutically Acceptable salt, wherein X5Selected from glycine, valine, isoleucine, arginine and glutamic acid.
5. the described in any item Formulas I compounds represented of claim 1-4, its stereoisomer, its mixture or its pharmaceutically Acceptable salt, in which:
X1Selected from glycine and glutamic acid；
X2Selected from histidine and asparagine；
X3Selected from histidine and asparagine；
X4Selected from histidine and lysine；With
X5Selected from glycine, valine, isoleucine, arginine and glutamic acid.
6. the described in any item Formulas I compounds represented of claim 1-5, its stereoisomer, its mixture or its pharmaceutically Acceptable salt, in which:
The amido protecting group is selected from Ac, Fmoc, trifluoroacetyl group, allyloxy carbonyl, Dde or Npys；And/or
The carboxy protective group is selected from methyl esters, ethyl ester, benzyl ester, amide groups or hydrazide group.
7. Formulas I compound represented described in any one of claims 1-6, its stereoisomer, its mixture or its pharmaceutically Acceptable salt, wherein the Formulas I compound represented is selected from:
IEGIVKKINNIERAIEAQQHLLQLTVWGIKQLQARIL (SEQ ID NO:1)；
(IEGIVKKINNIERAIEAQQHLLQLTVWGIKQLQARIL)3(SEQ ID NO:1)3；
IEGIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL (SEQ ID NO:2)；
(IEGIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL)3(SEQ ID NO:2)3；
IEEIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL (SEQ ID NO:3)；
(IEEIVKKINNIERAIEAQQKLLQLTVWGIKQLQARIL)3(SEQ ID NO:3)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWGIKQLQARIL (SEQ ID NO:4)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWGIKQLQARIL)3(SEQ ID NO:4)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWEIKQLQARIL (SEQ ID NO:5)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWEIKQLQARIL)3(SEQ ID NO:5)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWRIKQLQARIL (SEQ ID NO:6)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWRIKQLQARIL)3(SEQ ID NO:6)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWVIKQLQARIL (SEQ ID NO:7)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWVIKQLQARIL)3(SEQ ID NO:7)3；
IEEIVKKIHHIERAIEAQQKLLQLTVWIIKQLQARIL (SEQ ID NO:8)；
(IEEIVKKIHHIERAIEAQQKLLQLTVWIIKQLQARIL)3(SEQ ID NO:8)3；
Wherein, the N-terminal of each compound is free or acylated, and C-terminal is free, amidation or esterification；
Preferably, in above-mentioned each tripolymer, the 7th lysine and another sequence monomer from N-terminal of monomer sequence Form amido bond between the 12nd glutamic acid from N-terminal.
8. pharmaceutical composition, it includes the described in any item Formulas I compounds represented of claim 1-7, its stereoisomer, its Mixture or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier and/or excipient.
9. the described in any item Formulas I compounds represented of claim 1-7, its stereoisomer, mixture can pharmaceutically connect The salt received or pharmaceutical composition according to any one of claims 8, in preparation for MERS- to be treated and/or prevented in subject CoV infects or infects to MERS-CoV the purposes in the drug of relevant disease；
Preferably, the disease relevant to MERS-CoV infection is Middle East respiratory system syndrome；
Preferably, the subject is mammal, such as people.
10. a kind of method for inhibiting MERS-CoV and cell fusion in vitro comprising appoint MERS-CoV and claim 1-7 Formulas I compound represented described in one, its stereoisomer, mixture or pharmaceutically acceptable salt or claim The step of pharmaceutical composition thereof described in 8.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111647048A (en) * 2020-06-22 2020-09-11 中国科学院昆明动物研究所 Application of interference polypeptide in preparing anti-SARS-CoV-2 medicine
US10975126B1 (en) 2020-04-23 2021-04-13 King Faisal University MERS-CoV inhibitor peptides
US12076364B2 (en) 2020-06-22 2024-09-03 Kunming Institute Of Zoology, Chinese Academy Of Sciences Use of interference peptide in preparation of anti-SARS-CoV-2 medicament

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106146624A (en) * 2015-04-28 2016-11-23 中国人民解放军军事医学科学院毒物药物研究所 The HIV-1 inhibitor of the natural N peptides of fixed point covalent cross-linking
WO2018172447A1 (en) * 2017-03-23 2018-09-27 Alpha-O Peptides Ag Self-assembling protein nanoparticles with built-in six-helix bundle proteins
CN108659105A (en) * 2018-05-23 2018-10-16 中国人民解放军军事科学院军事医学研究院 Antiviral polypeptide and its pharmaceutical composition and purposes

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN106146624A (en) * 2015-04-28 2016-11-23 中国人民解放军军事医学科学院毒物药物研究所 The HIV-1 inhibitor of the natural N peptides of fixed point covalent cross-linking
WO2018172447A1 (en) * 2017-03-23 2018-09-27 Alpha-O Peptides Ag Self-assembling protein nanoparticles with built-in six-helix bundle proteins
CN108659105A (en) * 2018-05-23 2018-10-16 中国人民解放军军事科学院军事医学研究院 Antiviral polypeptide and its pharmaceutical composition and purposes

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10975126B1 (en) 2020-04-23 2021-04-13 King Faisal University MERS-CoV inhibitor peptides
CN111647048A (en) * 2020-06-22 2020-09-11 中国科学院昆明动物研究所 Application of interference polypeptide in preparing anti-SARS-CoV-2 medicine
US12076364B2 (en) 2020-06-22 2024-09-03 Kunming Institute Of Zoology, Chinese Academy Of Sciences Use of interference peptide in preparation of anti-SARS-CoV-2 medicament

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