CN101921823B - Method for screening SARS corona virus major protease inhibitor from traditional Chinese medicine and screened SARS corona virus major protease inhibitor - Google Patents

Method for screening SARS corona virus major protease inhibitor from traditional Chinese medicine and screened SARS corona virus major protease inhibitor Download PDF

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CN101921823B
CN101921823B CN 201010170409 CN201010170409A CN101921823B CN 101921823 B CN101921823 B CN 101921823B CN 201010170409 CN201010170409 CN 201010170409 CN 201010170409 A CN201010170409 A CN 201010170409A CN 101921823 B CN101921823 B CN 101921823B
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extract
crotons
acid
sars coronavirus
ethyl acetate
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CN101921823A (en
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饶子和
马明
娄智勇
李雪梅
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Tsinghua University
Institute of Biophysics of CAS
Nankai University
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Tsinghua University
Institute of Biophysics of CAS
Nankai University
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Abstract

The invention provides a method for screen a SARS corona virus major protease inhibitor from a traditional Chinese medicine and a SARS corona virus major protease inhibitor screened by the method. The screening method comprises the following steps of: A. detecting in vitro inhibitory activities against SARS corona virus major protease of extracts from a single traditional Chinese medicine; B. selecting an extract with best in vitro inhibitory activity; and C. separating and selecting the selected extract at least once. The compound screened by using the method has in vitro inhibitory activity against SARS corona virus major protease and is an optimal medicine possibly available in market or a potential prodrug.

Description

The sars coronavirus main proteinase inhibitor that the method for screening sars coronavirus main proteinase inhibitor and screening obtain from Chinese medicine
Technical field
The present invention relates to the method for screening sars coronavirus main proteinase inhibitor from Chinese medicine and the sars coronavirus main proteinase inhibitor that screening obtains.More specifically, the present invention relates to take the vitro inhibition determination of activity of sars coronavirus main protease as the basis, the method of the unsaturated fatty acids acid inhibitor of screening sars coronavirus main protease from Chinese medicine, and a series of unsaturated fatty acids inhibitor of deriving thus.
Background technology
SARS (Severe Acute Respiratory Sydrome) is for causing the abbreviation of severe acute respiratory syndrome (severe acute respiratory syndrome), its pathogenic agent is a kind of virus (Peiris J.Lancet of coronavirus genus, 2003,361,1319-1325).Coronavirus is positive chain RNA virus.Coronavirus genus is under the jurisdiction of coronaviridae.In at present known positive chain RNA virus, their genome is maximum (Siddell, S.G.Coronaviruses, toroviruses, and arteriviruses.in Topley ﹠amp; Wilson ' s Microbiology andMicrobia Infections, 10th edition, Vol.Virology (eds.Mahy, B.W.J.﹠amp; Ter Meulen, V.) 823-856 (Hodder Arnold, London, 2005)).This genus contains 26 kinds of having an appointment; According to their natural reservoir (of bird flu viruses), gene order and serotype relation, this genus can be divided into again three cohorts (group): wherein the first cohort has TGEV, Porcine Transmissible Gastroenteritis Virus (transmissible gastro-enteritis virus) etc.; The second cohort has sars coronavirus, MHV (murine hepatitis virus) etc.; The 3rd cohort has AIBV, (the Spaan such as Avian Infectious Bronchitis Virus (avian infectious bronchitis virus), W J M.Cavanagh, D.Coronaviridae.in Virus taxonomy, VIIIth Report of the ICTV.945-62 (Elsevier-Academic Press., London, 2004)).
The genome encoding of sars coronavirus 2/3 to 3/4 two replicase polyprotein (replicasepolyproteins) ppla and pplab, they only just can make virus finish normal transcription, copy function after the proteolytic cleavage of encoding viral is slit into independent subunit.Sars coronavirus major protein enzyme (main protease is called for short main protease) plays a major role in this process.If can suppress the hydrolytic action of sars coronavirus main protease, will effectively resist sars coronavirus infecting human body so.Therefore, the main protease of sars coronavirus is a main target of anti-SARS drug screening.
The SARS virus of outburst in 2003 infects in the whole world and causes 8096 routine cases, and 774 examples are dead.Although till today, do not infect again, do not get rid of the possibility that SARS and mutation thereof are staged a comeback.Existing precedent with Chinese herb on the prevention or treatment when SARS virus infected in 2003.China's traditional Chinese medicine is the important sources of a large amount of natural products, and natural product has diversity and the bioactive diversity of structure.Natural product and derivative thereof have been brought into play promising effect in disease treatment in the past, also be one of resource (Newman D J, the Gragg G M of tool potentiality in the current medicament research and development process, Snader K M.Nat Prod Rep., 2000,17,215-234; Lee K H.J Nat Prod., 2004,67,273-283).Now in the ascendant to the Natural products research in the conventional medicaments such as Chinese medicine, marine organisms and the microbial metabolism, annual all can have the compound of a large amount of novel structures to be found to provide, these compounds are the medicine that can't realize of synthetic method and the important source of lead compound, play an important role in the discovery of new drug and lead compound.Chinese medicine is the treasure-house of a greatness in the history that China used in existing thousands of years, and the screening of therefore therefrom carrying out important virus or the relevant target protein inhibitor of important diseases in the medicines natural products is necessary.
At present also not take the vitro inhibition determination of activity of sars coronavirus main protease as the basis, from rushing down for example application report of screening sars coronavirus main proteinase inhibitor the crotons of lower class Chinese medicine.The present invention uses the vitro enzyme screening method alive of sars coronavirus main protease, provide a kind of from the Chinese medicine method of screening sars coronavirus main proteinase inhibitor such as isolinoleic acid (isolinoleicacid) and derivative thereof the crotons for example.
Summary of the invention
An object of the present invention is to provide a kind of method of screening the sars coronavirus main proteinase inhibitor, comprise as follows: A. measures from the sars coronavirus main protease vitro inhibition of the multiple extract of single medicinal material active; B. select a kind of extract with best vitro inhibition activity; With C. the selected extract of step B is carried out at least one isolation and selection of taking turns, to every various compound mensuration sars coronavirus main protease vitro inhibition activity of taking turns resulting separation, a kind of component that selection has a best vitro inhibition activity enters the isolation and selection of next round, until the component with best vitro inhibition activity that described isolation and selection obtain is a kind of compound, with this compound as the sars coronavirus main proteinase inhibitor.
In a preferred embodiment, the mensuration in the described steps A comprises the steps: (1) from the method for the sars coronavirus main protease vitro inhibition activity of the multiple extract of single medicinal material. in buffered soln, add the sars coronavirus main protease; (2). add substrate and test substance in buffered soln; (3). the sars coronavirus main protease vitro inhibition of measuring test substance is active.
In another preferred embodiment, one or more the following methods that are selected from are adopted in the separation among the described step C: supercritical extraction, organic solvent extraction, chromatographic separation, electrophoretic separation and membrane sepn.
In another preferred embodiment, described extract is extractive with organic solvent.
In a further preferred embodiment, described organic solvent is selected from aromatic hydrocarbon solvent, fat hydrocarbon solvent, alicyclic hydrocarbon type solvent, halogenated hydrocarbon solvent, alcoholic solvent, ether solvent, esters solvent, ketones solvent, diol, derivatives kind solvent and other organic solvents.
At one further in the preferred embodiment, described organic solvent further is selected from alcoholic solvent and esters solvent.
In another preferred embodiment, described single medicinal material is crotons (fructus of Croton tigliumL.).
In another preferred embodiment, described multiple extract from single medicinal material is crotons 95% ethanol (alcohol concn is in v/v) general extractive, crotons ethyl acetate extract and crotons water macroporous resin 95% ethanol extraction.
In a further preferred embodiment, the preparation method of described crotons 95% total ethanol extract, crotons ethyl acetate extract and crotons water macroporous resin 95% ethanol extraction comprises as follows: (a). get a certain amount of crotons, with 95% ethanolic soln refluxing extraction repeatedly, united extraction liquid, concentrate to get medicinal extract, be crotons 95% total ethanol extract; (b). take by weighing the medicinal extract of gained in a certain amount of step (a), suspend with distilled water, with ethyl acetate extraction repeatedly, separating ethyl acetate phase and water; Merge water, for subsequent use; Merge each acetic acid ethyl ester extract, steam the ethyl acetate of removing wherein, obtain dried paste ethyl acetate phase sample, be the crotons ethyl acetate extract; (c). steam a small amount of ethyl acetate of dissolving in the water except the aqueous portion after the extraction in the step (b), separate with macroporous resin column chromatography, water and 95% ethanolic soln are that wash-out is repeatedly respectively for moving phase successively; Discard the washing part, merge 95% ethanolic soln wash-out part, steam the solvent that removes wherein, the shape sample that gets dry extract is crotons water macroporous resin 95% ethanol extraction.
In a further preferred embodiment, described substrate is the fluorescent mark substrate.
At one further in the preferred embodiment, described fluorescent mark substrate is MCA-AVLQSGFRL (DNP) L-NH 2
Another object of the present invention provides screening obtains according to the described method of above-mentioned any one embodiment sars coronavirus main proteinase inhibitor for the preparation of the application that treats and/or prevents in the medicine that sars coronavirus infects.
In a preferred embodiment, described sars coronavirus main proteinase inhibitor is isolinoleic acid (isolinoleic acid).
In a preferred embodiment, described sars coronavirus main proteinase inhibitor is suitable by 6, the petroselinic acid (petroselinic acid) that 9 suitable-18 diolefinic acids (isolinoleic acid) are derived, Oleate acid (oleic acid), sodium oleate (sodium oleate), elaidic acid (elaidic acid), cis-vaccenic acid (cis-vaccenic acid), erucic acid (erucicacid), 9 is suitable, 12 suitable-18 diolefinic acids (linoleic acid), 9 is suitable, 12 is suitable, 15 suitable-18 trienic acids (linolenic acid) and 6 suitable, 9 is suitable, 12 suitable-18 trienic acids (γ-linolenic acid).
Another purpose of the present invention provides unsaturated fatty acids compound with following general structure for the preparation of the application that treats and/or prevents in the medicine that sars coronavirus infects:
Figure GSA00000112051200051
Wherein, each R is simultaneously or be independently selected from: H ,-OH ,-ONa ,-OK ,-OSO 3H ,-OPO 3H 2,-OAc ,-OCH 3,-OCH 2CH 3,-OCH 2CH 2CH 3,-OCH 2CH 2CH 2CH 3,-Oglu and-Ogal.
In a preferred embodiment, described unsaturated fatty acids compound is 6 suitable, 9 suitable-18 diolefinic acids, petroselinic acid, Oleate acid, sodium oleate, elaidic acid, cis-vaccenic acid, erucic acid, 9 suitable, 12 suitable-18 diolefinic acids, 9 suitable, 12 is suitable, 15 suitable-18 trienic acid and GAMMA-linolenic acids.
A further object of the present invention provides a kind of pharmaceutical composition that is used for the treatment of and/or prevents sars coronavirus to infect, and comprises sars coronavirus main proteinase inhibitor and the pharmaceutically acceptable carrier of the aforesaid method screening acquisition for the treatment of significant quantity.
In the context of the present invention, the measuring method of described sars coronavirus main protease vitro inhibition activity specifically comprises as follows: (1). as containing 50mM Tris-HCl, add the sars coronavirus main protease to buffered soln in the buffered soln of pH 7.3,1mM EDTA; (2). add substrate and test substance in buffered soln; (3). the sars coronavirus main protease vitro inhibition of measuring test substance is active; Wherein said buffered soln can contain or not contain DTT; Substrate includes but not limited to colour developing, fluoresces or chemiluminescent substrate preferred fluorescigenic substrate, for example fluorescently-labeled substrate MCA-AVLQSGFRL (DNP) L-NH 2Can measure by the method that includes but not limited to colorimetric, fluorescence reading or chemiluminescence readings the sars coronavirus main protease vitro inhibition activity of test substance; The concentration that adds sars coronavirus main protease, test substance and substrate in the buffered soln can be determined by those skilled in the art as the case may be, for example the concentration of sars coronavirus main protease can be 0.5 μ M, when test substance is mixture, can be 10-100 μ g/mL, when test substance is single compound, can be 200 μ M, concentration of substrate can be 16 μ M; When the sars coronavirus main protease vitro inhibition of measuring test substance by the fluorescence reading was active, excitation wavelength and emission wavelength can be respectively 320nm and 405nm, and temperature can remain on 298K, can record the first order fluorescence reading per 2 seconds.
In the context of the present invention, one or more the following methods that are selected from are adopted in described separation: supercritical extraction, organic solvent extraction, chromatogram (chromatography) separation, electrophoretic separation and membrane sepn, and wherein supercritical extraction includes but not limited to carbon dioxide upercritical fluid extraction; Organic solvent extraction includes but not limited to alcoholic solvent extraction and esters solvent extraction, for example n-butanol extraction and ethyl acetate extraction; Chromatogram (chromatography) separation includes but not limited to that adsorption chromatography (chromatography) is separated, gel filtration chromatography (chromatography) is separated, ion-exchange chromatography (chromatography) separates, hydrophobic chromatography (chromatography) is separated and separated with HPLC; Electrophoretic separation includes but not limited to paper electrophoresis, film electrophoresis, powder electrophoresis, filament electrophoresis, gel electrophoresis and capillary electrophoresis; Membrane sepn can adopt mineral membrane and organic membrane, and mineral membrane includes but not limited to ceramic membrane and metallic membrane, and organic membrane includes but not limited to cellulose acetate membrane, aromatic polyamide film, poly (ether sulfone) film, poly-fluoropolymer membrane.
In the context of the present invention, described organic solvent includes but not limited to: 1. aromatic hydrocarbon solvent: stupid etc. such as benzene, toluene, diformazan; 2. fat hydrocarbon solvent: such as pentane, hexane, octane etc.; 3. alicyclic hydrocarbon type solvent: such as hexanaphthene, pimelinketone, toluene pimelinketone etc.; 4. halogenated hydrocarbon solvent: such as chlorobenzene, dichlorobenzene, methylene dichloride etc.; 5. alcoholic solvent: such as methyl alcohol, ethanol, Virahol etc.; 6. ether solvent: such as ether, propylene oxide etc.; 7. esters solvent: ritalin, ethyl acetate, propyl acetate etc.; 8. ketones solvent: acetone, espeleton, mibk etc.; 9. diol, derivatives kind solvent: such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether etc.; 10. other organic solvents: such as acetonitrile, pyridine, phenol etc.; Preferred alcohols and/or esters solvent; More preferably ethanol and/or ethyl acetate.
In the context of the present invention, the preparation method of described crotons 95% total ethanol extract, crotons ethyl acetate extract and crotons water macroporous resin 95% ethanol extraction is specific as follows: (a). get the crotons of a certain amount of (such as 500 grams), with 95% ethanolic soln (such as 4500 milliliters) refluxing extraction 3 times, united extraction liquid, concentrate to get medicinal extract, be crotons 95% total ethanol extract; (b). take by weighing the medicinal extract of the middle gained of step (a) of a certain amount of (such as 10 grams), suspend with distilled water (such as 150 milliliters), with ethyl acetate (such as 650 milliliters) extraction 3 times, separating ethyl acetate phase and water; Merge water, for subsequent use; Merge each acetic acid ethyl ester extract, steam the ethyl acetate of removing wherein, obtain dried paste ethyl acetate phase sample, be the crotons ethyl acetate extract; (c). steam a small amount of ethyl acetate of dissolving in the water except the aqueous portion after the extraction in the step (b), separate with macroporous resin column chromatography, water and 95% ethanolic soln are the moving phase wash-out successively, water (such as 250 milliliters) wash-out for example 8 times, 95% ethanol (such as 300 milliliters) wash-out for example 5 times; Discard the washing part, merge 95% ethanolic soln wash-out part, steam the solvent that removes wherein, the shape sample that gets dry extract is crotons water macroporous resin 95% ethanol extraction.
The present invention chooses the known main protease of crystalline structure in the sars coronavirus, China's traditional Chinese medicine crotons are carried out active ingredient screening, being to remove Study of Traditional Chinese Medicine, natural product chemistry and biology from a kind of new angle, is to use various cross disciplines to modern valuable trial of Chinese materia medica.Because contained composition is very complex in the crotons, must use various chromatographic processes that its crude extract is carried out separation and purification, purpose is to remove interfering factors such as albumen, inorganic salt etc. in the sars coronavirus main protease active determination in vitro, be retained as preferably small molecules and make it as far as possible to concentrate of the property of medicine, improve relative concentration.The present invention not only provides and can realize this purpose technology of preparing scheme, and screening is had the sample that suppresses active, separates, identified wherein micromolecular inhibitor isolinoleic acid with various chromatograms, spectral method.By buying and isolinoleic acid structural similitude, fatty acid compound that the double key number amount is different with the position, and live screening to their inhibition activity proved with enzyme, our several strong inhibitor of having got back.We analyze the constructional feature of these inhibitor at last, have obtained having the structural formula of ad hoc structure skeleton, and the compound that meets these structural formulas probably has the inhibition of sars coronavirus main protease active.
At present, be the basic drug screening of carrying out based on sars coronavirus main protease crystalline structure, the micromolecular inhibitor of finding is most to be sars coronavirus main protease substrate analogue and derivative (Dariusz P thereof, Marcin H, Marcin G.Chem.Biol.Drug.Res., 2007,69,269-279; HaitaoY, Wenqing X, Xiaoyu X.PLOS Biology, 2005,3,1742-1752), this class peptide inhibitor not only involves great expense, and oral availability is low, and the transformation period in blood is short, and uniform SARS infection morbidity has been brought difficulty.The inhibitor that the present invention filters out-6 is suitable, 9 suitable-18 diolefinic acids, and the unsaturated fatty acid derivative of deriving thus, do not contain peptide bond in the molecular structure, it still has the vitro inhibition activity when the 25 μ M to the sars coronavirus main protease, consider that they mostly are greatly natural product in the conventional Chinese medicine, its toxicity and metabolism absorption characteristic have incomparable superiority than other synthetic compounds such as polypeptide classes, are the splendid medicine that may go on the market or potential prodrug.This is for further medicinal design and screening provide valuable foundation and reference.
Description of drawings
Fig. 1: the various extracts of crotons of the present invention are to the inhibition activity curve figure of sars coronavirus main protease; Wherein, 1 is control curve, and 2,3,4 are respectively the inhibition curve of crotons water macroporous resin 95% ethanol extraction, crotons 95% total ethanol extract, crotons ethyl acetate extract, and sample concentration is: 100 μ g/mL.
Fig. 2: the inhibition graphic representation of the further separated portion a1-a5 of crotons ethyl acetate extract of the present invention; Wherein, 1 is contrast, and 2,3,4,5,6 are respectively the inhibition curve of a2, a1, a3, a5, a4, and sample concentration is: 10 μ g/mL.
Fig. 3: the inhibition graphic representation of the further separated portion b1 of crotons ethyl acetate extract a4 part of the present invention and b2; Wherein, 1 is contrast, and 2,3 are respectively the inhibition curve of b2, b1, and sample concentration is: 10 μ g/mL.
Fig. 4: the hydrogen that the HMBC collection of illustrative plates of compound 1 is derived and the correlationship between the carbon atom.
Fig. 5: the inhibition graphic representation of compound 1 (isolinoleic acid); Wherein 1 is contrast, and 2 is compound 1, and concentration is: 10 μ g/mL.
Fig. 6: the inhibition graphic representation of various unsaturated fatty acids acid inhibitors.Wherein, 1,2,3,4,5,6,7,8,9,10,11 is respectively control curve, sodium oleate, 6 suitable, 9 is suitable, 12 suitable-18 trienic acids, 9 suitable, 12 is suitable, 15 suitable-18 trienic acids, isolinoleic acid, linoleic acid, Oleate acid, elaidic acid, cis-vaccenic acid, petroselinic acid, erucic acid.Inhibitor concentration is 50 μ M.
Embodiment
The present invention will carry out more specific description according to the following example.Yet protection scope of the present invention is not limited to following embodiment.
Expression, the purifying of embodiment 1.SARS coronavirus proteolytic
According to document (Yang H.Proc Natl Acad Sci., 2003,100,13190-13195) carry out expression, the purifying of sars coronavirus main protease, the aminoacid sequence of this sars coronavirus main protease is as follows:
Ser Gly Phe Arg Lys Met Ala Phe Pro Ser Gly Lys Val Glu Gly Cys
1 5 10 15
Met Val Gln Val Thr Cys Gly Thr Thr Thr Leu Asn Gly Leu Trp Leu
20 25 30
Asp Asp Thr Val Tyr Cys Pro Arg His Val Ile Cys Thr Ala Glu Asp
35 40 45
Met Leu Asn Pro Asn Tyr Glu Asp Leu Leu Ile Arg Lys Ser Asn His
50 55 60
Ser Phe Leu Val Gln Ala Gly Asn Val Gln Leu Arg Val Ile Gly His
65 70 75 80
Ser Met Gln Asn Cys Leu Leu Arg Leu Lys Val Asp Thr Ser Asn Pro
85 90 95
Lys Thr Pro Lys Tyr Lys Phe Val Arg Ile Gln Pro Gly Gln Thr Phe
100 105 110
Ser Val Leu Ala Cys Tyr Asn Gly Ser Pro Ser Gly Val Tyr Gln Cys
115 120 125
Ala Met Arg Pro Asn His Thr Ile Lys Gly Ser Phe Leu Asn Gly Ser
130 135 140
Cys Gly Ser Val Gly Phe Asn Ile Asp Tyr Asp Cys Val Ser Phe Cys
145 150 155 160
Tyr Met His His Met Glu Leu Pro Thr Gly Val His Ala Gly Thr Asp
165 170 175
Leu Glu Gly Lys Phe Tyr Gly Pro Phe Val Asp Arg Gln Thr Ala Gln
180 185 190
Ala Ala Gly Thr Asp Thr Thr Ile Thr Leu Asn Val Leu Ala Trp Leu
195 200 205
Tyr Ala Ala Val Ile Asn Gly Asp Arg Trp Phe Leu Asn Arg Phe Thr
210 215 220
Thr Thr Leu Asn Asp Phe Asn Leu Val Ala Met Lys Tyr Asn Tyr Glu
225 230 235 240
Pro Leu Thr Gln Asp His Val Asp Ile Leu Gly Pro Leu Ser Ala Gln
245 250 255
Thr Gly Ile Ala Val Leu Asp Met Cys Ala Ala Leu Lys Glu Leu Leu
260 265 270
Gln Asn Gly Met Asn Gly Arg Thr Ile Leu Gly Ser Thr Ile Leu Glu
275 280 285
Asp Glu Phe Thr Pro Phe Asp Val Val Arg Gln Cys Ser Gly Val Thr
290 295 300
Phe Gln
305
1.1SARS the expression vector establishment of coronavirus proteolytic
Concrete steps comprise:
1.1.1 utilize the cDNA library of the SARS virus strain that is numbered BJ01 that the large gene center of Beijing China provides, carry out amplification in vitro with round pcr,
Forward primer: 5 '-CGGGATCCAGTGGTTTTAGGAAAATG-3 '
Reverse primer: 5 '-CCGCTCGAGTCATTGGAAGGTAACACCAGA-3 '
1.1.2 after BamHI and the two enzyme enzymes of XhoI were cut, reclaiming size with agarose gel electrophoresis was fragment about 1kb through the gene fragment of pcr amplification;
Be connected with the T carrier 1.1.3 will reclaim fragment, then transform colibacillus (Escherichia coli) DH5 α competent cell with connecting product, and be coated on the LB flat board (containing the 100mg/L penbritin) overnight incubation;
1.1.4 a plurality of mono-clonals of picking from the flat board, be inoculated in respectively and be equipped with approximately that the test tube of the LB of 5mL (adds penbritin in this LB solution, making its final concentration is 100mg/L) in, overnight incubation, then use plasmid extraction kit (vast Imtech Type B plasmid is the rapid extraction test kit in a small amount) to extract plasmid, and cut with BamHI and XhoI enzyme, then reclaim size with sepharose and be about target gene fragment about 1kb;
1.1.5 destination carrier pGEX-4T-1 (available from Pharmacia company) is cut with BamHI and XhoI enzyme, then reclaims the fragment that enzyme is cut with sepharose;
1.1.6 with (1.1.4) be connected 1.1.5) fragment that obtains connects that (the target gene fragment after enzyme is cut back to close and destination carrier fragment are according to mole number 3: 1-6: 1 ratio mixes, requirement according to Takara DNA Ligation was reacted 30 minutes-18 hours at 16 ℃), transform colibacillus DH5 α competent cell, be coated in the upper overnight incubation of LB flat board (containing the 100mg/L penbritin).With the positive colony that screens, for the identification of and the order-checking.
Sequencing result shows, the encoding gene of the main protease of sars coronavirus correctly is cloned in the pGEX-4T-1 carrier.
1.2SARS the expression and purification of coronavirus proteolytic
Comprise step:
1.2.1 the pGEX-4T-1 carrier that contains encoding SARS coronavirus proteolytic gene that obtains in the above-mentioned steps 1.1 is transformed the bacterial strain of e. coli bl21 (DE3), and with LB flat board (containing the 100mg/L penbritin) screening positive clone;
1.2.2 picking positive colony on the LB flat board described in (1.2.1) (mono-clonal that grows out at the LB flat board that contains penbritin), then overnight incubation changes the LB substratum (containing the 100mg/L penbritin) of 1L over to, works as OD 600When reaching 0.6-0.8, add the IPTG about 1mM, cultivated about 12 hours at 16 ℃;
1.2.35000-8000rpm centrifugal 10-15 minute collecting cell, then ice-bath ultrasonic is broken bacterium 20-30 minute; Broken bacterium liquid 13000rpm-15000rpm collected supernatant liquor after centrifugal 20-40 minute;
1.2.4 supernatant liquor is added in the GST affinity column (GE company) of PBS pre-equilibration, remove foreign protein with 20-30 column volume of PBS drip washing, add at last the human rhinovirus's HRV 3CP about 2ml 0.1mg/ml, cut 12-20 hour at 4 ℃ of enzymes, collect afterwards the sars coronavirus main protease;
1.2.5 use again Mono Q (GE company) anion-exchange chromatography to carry out purifying the sars coronavirus main protease that (1.2.4) obtains.
Embodiment 2. preparation crotons extracts
Get the crotons medicinal material of 500 gram drying and crushing, with 4500 milliliter of 95% alcohol reflux, extract 3 times, each 2 hours, united extraction liquid concentrated to get medicinal extract (being crotons 95% total ethanol extracts);
Take by weighing this medicinal extract 10 grams, suspend with 150 milliliters of distilled waters, in 1000 milliliters of separating funnels of impouring, use ethyl acetate extraction, each 650 milliliters of ethyl acetate of extraction, shake well, leave standstill 6 little layereds, coextraction 3 times, separating ethyl acetate phase and water, merge water, for subsequent use, merge each acetic acid ethyl ester extract, acetic acid ethyl ester extract steams with EYELAN1001 type Rotary Evaporators and desolventizes, get stiff paste shape crotons ethyl acetate extraction sample (being the crotons ethyl acetate extracts), for subsequent use;
Aqueous portion after the extraction steams a small amount of ethyl acetate of the middle dissolving that dewaters with EYELA N1001 type Rotary Evaporators, (use 170 milliliters macroporous resin with the separation of HP-20 type macroporous resin column chromatography, glass column specification 30 * 300mm), water successively, 95% ethanol is the moving phase wash-out, wherein water elution is 8 times, 250 milliliters of each wash-outs, discard the washing part, 95% ethanol elution 5 times, 300 milliliters of each wash-outs, merge 95% ethanol elution part, partly using EYELA N1001 type Rotary Evaporators to steam 95% ethanol elution desolventizes, the shape crotons water macroporous resin 95% ethanol elution sample (being crotons water macroporous resin 95% ethanol extraction) that gets dry extract, for subsequent use.
Embodiment 3. screens the sars coronavirus main proteinase inhibitor from the crotons extract
Above-mentioned ethyl acetate extraction sample, water macroporous resin 95% ethanol elution sample and crotons 95% total ethanol extract are carried out sars coronavirus main protease vitro inhibition activity experiment, suppress curve and see Fig. 1.Measuring method specifically comprises as follows: (1). to containing 50mM Tris-HCl, add the sars coronavirus main protease in the buffered soln of pH 7.3,1mM EDTA, final concentration is 0.5 μ M, then add respectively crotons ethyl acetate extraction sample, water macroporous resin 95% ethanol elution sample and 95% total ethanol extract in the different buffered soln that contains this albumen, their concentration is 100 μ g/mL; (2). add substrate MCA-AVLQSGFRL (DNP) L-NH 2In buffered soln, final concentration is 16 μ M; (3). the sars coronavirus main protease vitro inhibition of measuring test substance is active, and wherein control curve is for only adding albumen and substrate, not adding each segmentation sample tests curve of crotons, and the lower explanation restraining effect of curve is stronger.
By the inhibition curve of Fig. 1 as can be known the ethyl acetate extraction sample of crotons partly suppress active best.(manufacturers: Beijing intelligent moral benefit science and technology limited Company) separate, take chloroform-methanol (1: 1) as the moving phase wash-out, reception has merged 5 parts altogether, is a1-a5 with Sephadex LH-20 gel filtration chromatography with ethyl acetate extraction part.These 5 parts are carried out sars coronavirus main protease vitro inhibition activity experiment, and the vitro inhibition activity curve is seen Fig. 2.Measuring method specifically comprises as follows: (1). to containing 50mM Tris-HCl, add the sars coronavirus main protease in the buffered soln of pH 7.3,1mM EDTA, final concentration is 0.5 μ M, then add respectively the a1-a5 sample in the different buffered soln that contains this albumen, their concentration is 10 μ g/mL; (2). add substrate MCA-AVLQSGFRL (DNP) L-NH 2In buffered soln, final concentration is 16 μ M; (3). the sars coronavirus main protease vitro inhibition of measuring test substance is active, and wherein control curve is for only adding albumen and substrate, not adding a1-a5 sample tests curve, and the lower explanation restraining effect of curve is stronger.
The inhibition activity of a4 part is best as shown in Figure 2.The a4 part is prepared with silica gel thin-layer chromatography, use sherwood oil: acetone=be developping agent at 5: 1 is divided into two parts with a4, b1 and b2.These two parts are carried out sars coronavirus main protease vitro inhibition activity experiment, and the vitro inhibition activity curve is seen Fig. 3.Measuring method specifically comprises as follows: (1). to containing 50mM Tris-HCl, add the sars coronavirus main protease in the buffered soln of pH 7.3,1mM EDTA, final concentration is 0.5 μ M, then add respectively b1, b2 sample in the different buffered soln that contains this albumen, their concentration is 10 μ g/mL; (2). add substrate MCA-AVLQSGFRL (DNP) L-NH 2In buffered soln, final concentration is 16 μ M; (3). the sars coronavirus main protease vitro inhibition of measuring test substance is active, and wherein control curve is for only adding albumen and substrate, not adding b1 and b2 sample tests curve, and the lower explanation restraining effect of curve is stronger.
B1 part activity is stronger as shown in Figure 3, and b1 is partly used the HPLC purifying, and (HPLC analyzes and preparation condition is: from 0 minute to 15 minutes, moving phase was methyl alcohol: water=85: 15; From 15 to 45 minutes, proportion of mobile phase is from methyl alcohol: water=gradient was raised to methyl alcohol in 85: 15: water=100: 0, and to detect wavelength and be 210nm, flow velocity is 1ml/min when wherein analyzing, flow velocity is 3ml/min during preparation.The analytical column specification is YMC-Pack ODS-A, AA12S11-1546WT, A-302-10,150 * 4.6mm I.D., S-10 μ m.The preparative column specification is YMC-Pack ODS-A, AA12S11-1510WT, A-322-10,150 * 10mm I.D., S-10 μ m), finally obtain compound 1 (6.5mg).
Compound 1 has been made respectively NMR and GC-MS mensuration, and data are as follows:
Compound 1: colorless oil.GC-MS m/z 352[M+Si (CH 3) 3] +. 1H NMR (chloroform-d, 600MHz) δ 5.30-5.41 (4H, m, H-6,7,9,10), (2.77 2H, t, J=6.8Hz, H-8), 2.35 (2H, t, J=6.9Hz, H-2), 2.05 (4H, q, J=6.9Hz, H-5,11), 1.62 (2H, quintet, J=6.9Hz, H-3), 1.24-1.40 (14H, m, H-4,12,13,14,15,16,17), 0.89 (3H, t, J=6.8Hz, H-18). 13CNMR (chloroform-d, 150MHz) δ 180.5 (C-1), 34.2 (C-2), 24.8 (C-3), 27.3 (C-5), 130.3 (C-6), 128.1 (C-7), 25.7 (C-8), 128.0 (C-9), 130.1 (C-10), 27.2 (C-11), 22.6,29.1-29.7,31.6 (C-4, C-12, C-13, C-14, C-15, C-16, C-17), 14.1 (C-18).In order to determine the position of two keys, carried out again 1H- 1H COSY, HMQC and HMBC experiment.In HMBC experiment, relevant, 5 hydrogen of 2 hydrogen and 4 carbon and 4 carbon be correlated with and the position of two pairs of keys of relevant sufficient proof of 2 hydrogen and 3 carbon at 6 and 9 (seeing Fig. 4).Its NMR data and isolinoleic acid data (Gunstone, F D, Pollard, M R, Scrimgeour, C M, Vedanayagam, H S.Chemistry and Physics of Lipids, 1977,18,115-129.) in full accord, confirm that therefore compound 1 is isolinoleic acid (isolinoleic acid).
Figure GSA00000112051200141
(6 is suitable to compound 1 obtained above for we, 9 suitable-18 diolefinic acids) carried out the vitro inhibition determination of activity of sars coronavirus main protease, measure and carry out in accordance with the following steps: at buffered soln (50mMTris-HCl (pH 7.3), add SARS main protease (final concentration 0.5 μ M) among the 1mM EDTA, 6 is suitable, (final concentration is 9 suitable-18 diolefinic acids: 10 μ g/mL), add rapidly fluorescent mark substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH 2, final concentration 16 μ M).Excitation wavelength and emission wavelength are respectively 320nm and 405nm, temperature remains on 298K, record per 2 seconds first order fluorescence reading (the results are shown in Fig. 5) (control curve for only add albumen and substrate, do not add 6 suitable, the mensuration curve of 9 suitable-18 diolefinic acids, the lower explanation restraining effect of curve is stronger).It is active that isolinoleic acid has good inhibition as shown in Figure 5, is in the crotons sars coronavirus main protease to be played inhibiting principal constituent.
Embodiment 4. has the sars coronavirus main protease and suppresses other unsaturated fatty acidss of activity and the derivation of a few class inhibitor structure general formulas
To separate the inhibitor 6 obtain suitable by analyzing us, the structure of 9 suitable-18 diolefinic acids, we have bought other unsaturated fatty acids: petroselinic acid (Shanghai Sigma-aldrich company), Oleate acid (Tianjin Alfa-aesar company), sodium oleate (Shanghai Sigma-aldrich company), elaidic acid (Tianjin Alfa-aesar company), cis-vaccenic acid (Shanghai Sigma-aldrich company), erucic acid (Shanghai Sigma-aldrich company), 9 is suitable, 12 suitable-18 diolefinic acids (Beijing lark prestige Science and Technology Ltd.), 9 is suitable, 12 is suitable, 15 suitable-18 trienic acids (Beijing lark prestige Science and Technology Ltd.) and 6 suitable, 9 is suitable, and 12 suitable-18 trienic acids (Beijing lark prestige Science and Technology Ltd.) suppress screening active ingredients.Their structure is as follows:
Figure GSA00000112051200151
Above-mentioned nine compounds are carried out the vitro inhibition determination of activity of sars coronavirus main protease, the results are shown in Figure 6.Measure and carry out in accordance with the following steps: at buffered soln (50mM Tris-HCl (pH 7.3), 1mMEDTA (containing or do not contain DTT)) adds SARS main protease (final concentration 0.5 μ M) in, in containing the buffered soln of this albumen, difference adds respectively sodium oleate, 6 is suitable, 9 is suitable, 12 suitable-18 trienic acids, 9 is suitable, 12 is suitable, 15 suitable-18 trienic acids, 6 is suitable, 9 suitable-18 diolefinic acids, 9 is suitable, 12 suitable-18 diolefinic acids, Oleate acid, elaidic acid, cis-vaccenic acid, petroselinic acid, erucic acid (final concentration is 50 μ M) adds rapidly fluorescent mark substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH 2, final concentration 16 μ M).Excitation wavelength and emission wavelength are respectively 320nm and 405nm, and temperature remains on 298K, record the first order fluorescence reading per 2 seconds.Control curve is for only to add albumen and substrate, the mensuration curve when not adding any inhibitor, and the lower explanation restraining effect of curve is stronger.
By Fig. 6 result as can be known, above-mentioned nine unsaturated fatty acids acid compounds all have in various degree restraining effect to the sars coronavirus main protease, have in addition to suppress the specific activity isolinoleic acid stronger.More once their chemical structure can be found, exists number, position, two keys that cis-trans configurations is different in their structures.
The compound that meets following general structure is inhibited to the sars coronavirus main protease:
Wherein, each R is simultaneously or be independently selected from: H ,-OH ,-ONa ,-OK ,-OSO 3H ,-OPO 3H 2,-OAc ,-OCH 3,-OCH 2CH 3,-OCH 2CH 2CH 3,-OCH 2CH 2CH 2CH 3,-Oglu and-Ogal.Because the chemical group of R representative only affects the dissolving properties of whole compound, main inhibition activity is still determined by the chain-like structure of lipid acid, so it is inhibited to the sars coronavirus main protease to meet the compound of this general structure.
Owing to having the compound that the substituent compound of various R may be converted into free hydroxyl group in vivo during metabolism; and the compound of the application screening has diversity and the representativeness of structure, and the compound that therefore has this general formula should fall within protection scope of the present invention.
Herein among the related various experimental articles (including but not limited to: chemical reagent, biological products, cell, organism, instrument etc.), special or be difficult for obtaining for those, Wen Zhongjun has indicated manufacturers, reference or detailed preparation method; Without what specify, be the normal experiment articles for use, in the application before day, can pass through variety of way (such as buy, voluntarily preparation etc.) easily acquisition.
Should be appreciated that those of ordinary skill in the art can make various changes and improvements to it in form and details in the situation that without departing from the spirit and scope of the present invention, and these all are considered to fall into protection scope of the present invention.
Sequence table
<110〉Institute of Biophysics, Academia Sinica, Tsing-Hua University, Nankai University
<120〉the sars coronavirus main proteinase inhibitor that the method for screening sars coronavirus main proteinase inhibitor and screening obtain from Chinese medicine
<160>2
<210>1
<211>306
<212>prt
<213〉sars coronavirus (SARS Coronavirus)
<400>1
Ser Gly Phe Arg Lys Met Ala Phe Pro Ser Gly Lys Val Glu Gly Cys
1 5 10 15
Met Val Gln Val Thr Cys Gly Thr Thr Thr Leu Asn Gly Leu Trp Leu
20 25 30
Asp Asp Thr Val Tyr Cys Pro Arg His Val Ile Cys Thr Ala Glu Asp
35 40 45
Met Leu Asn Pro Asn Tyr Glu Asp Leu Leu Ile Arg Lys Ser Asn His
50 55 60
Ser Phe Leu Val Gln Ala Gly Asn Val Gln Leu Arg Val Ile Gly His
65 70 75 80
Ser Met Gln Asn Cys Leu Leu Arg Leu Lys Val Asp Thr Ser Asn Pro
85 90 95
Lys Thr Pro Lys Tyr Lys Phe Val Arg Ile Gln Pro Gly Gln Thr Phe
100 105 110
Ser Val Leu Ala Cys Tyr Asn Gly Ser Pro Ser Gly Val Tyr Gln Cys
115 120 125
Ala Met Arg Pro Asn His Thr Ile Lys Gly Ser Phe Leu Asn Gly Ser
130 135 140
Cys Gly Ser Val Gly Phe Asn Ile Asp Tyr Asp Cys Val Ser Phe Cys
145 150 155 160
Tyr Met His His Met Glu Leu Pro Thr Gly Val His Ala Gly Thr Asp
165 170 175
Leu Glu Gly Lys Phc Tyr Gly Pro Phe Val Asp Arg Gln Thr Ala Gln
180 185 190
Ala Ala Gly Thr Asp Thr Thr Ile Thr Leu Asn Val Leu Ala Trp Leu
195 200 205
Tyr Ala Ala Val Ile Asn Gly Asp Arg Trp Phe Leu Asn Arg Phe Thr
210 215 220
Thr Thr Leu Asn Asp Phe Asn Leu Val Ala Met Lys Tyr Asn Tyr Glu
225 230 235 240
Pro Leu Thr Gln Asp His Val Asp Ile Leu Gly Pro Leu Ser Ala Gln
245 250 255
Thr Gly Ile Ala Val Leu Asp Met Cys Ala Ala Leu Lys Glu Leu Leu
260 265 270
Gln Asn Gly Met Asn Gly Arg Thr Ile Leu Gly Ser Thr Ile Leu Glu
275 280 285
Asp Glu Phe Thr Pro Phe Asp Val Val Arg Gln Cys Ser Gly Val Thr
290 295 300
Phe Gln
305

Claims (2)

  1. The crotons extract as the sars coronavirus main proteinase inhibitor for the preparation of the application that treats and/or prevents in the medicine that sars coronavirus infects, wherein said crotons extract is crotons 95% total ethanol extract, crotons ethyl acetate extract, and extracting method comprises the steps:
    (a). with crotons with 95% ethanolic soln refluxing extraction, united extraction liquid, the concentrated medicinal extract that to get is crotons 95% total ethanol extract;
    (b). take by weighing the medicinal extract of gained in a certain amount of step (a), suspend with distilled water, with ethyl acetate extraction repeatedly, separating ethyl acetate phase and water; Merge water, for subsequent use; Merge each acetic acid ethyl ester extract, steam the ethyl acetate of removing wherein, obtain stiff paste shape ethyl acetate phase sample, be the crotons ethyl acetate extract.
  2. 2.SARS the coronavirus proteolytic inhibitor is for the preparation of the application that treats and/or prevents in the medicine that sars coronavirus infects, wherein said sars coronavirus main proteinase inhibitor is selected from following lipid acid: 6 is suitable, 9 suitable-18 diolefinic acids, petroselinic acid, Oleate acid, sodium oleate, elaidic acid, cis-vaccenic acid, erucic acid, 9 suitable, 12 suitable-18 diolefinic acids, 9 suitable, 12 is suitable, 15 suitable-18 trienic acids and 6 suitable, 9 is suitable, 12 suitable-18 trienic acids.
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