CN101921823A - 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 PDFInfo
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- 230000003612 virological effect Effects 0.000 description 1
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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
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 vitro inhibition determination of activity based on the sars coronavirus main protease, 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 be coronavirus genus a kind of virus (Peiris J.Lancet, 2003,361,1319-1325).Coronavirus is a positive chain RNA virus.Coronavirus genus is under the jurisdiction of coronaviridae.In 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 three cohorts (group) again: wherein first cohort has TGEV, Porcine Transmissible Gastroenteritis Virus (transmissible gastro-enteritis virus) etc.; Second cohort has sars coronavirus, MHV (murine hepatitis virus) etc.; The 3rd cohort has AIBV, Avian Infectious Bronchitis Virus (Spaan such as (avian infectious bronchitis viruses), 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 replicative enzyme 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 resist sars coronavirus infecting effectively so to human body.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.Though till today, do not infect again, do not get rid of the possibility that SARS and mutation thereof are staged a comeback.Existing precedent when SARS virus infected in 2003 with Chinese herb on the prevention or treatment.China's traditional Chinese medicine is the important source of a large amount of natural products, and natural product has diversity structure and bioactive diversity.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).In the ascendant to the research of the natural product in conventional medicaments such as Chinese medicine, marine organisms and the microbial metabolism now, all can have every year the compound of a large amount of novel structures to be found provides out, these compounds be synthetic method the medicine that can't realize and the important source of lead compound, in the discovery of new drug and lead compound, play an important role.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 based on the vitro inhibition determination of activity of sars coronavirus main protease, for example screen the application report of sars coronavirus main proteinase inhibitor the crotons from rushing down lower class Chinese medicine.The present invention uses the vitro enzyme screening method alive of sars coronavirus main protease, provide a kind of from Chinese medicine for example the crotons screening sars coronavirus main proteinase inhibitor suitable as 6, the method for 9 suitable-18 diolefinic acids (isolinoleicacid) and derivative thereof.
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 the sars coronavirus main protease vitro inhibition activity from the multiple extract of single medicinal material; B. select to have the active a kind of extract of best vitro inhibition; 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, selection has the isolation and selection that the active a kind of component of best vitro inhibition enters next round, the active component of best vitro inhibition that has that obtains until described isolation and selection 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 active method of sars coronavirus main protease vitro inhibition 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). measure the sars coronavirus main protease vitro inhibition activity of test substance.
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 an 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 embodiment preferred, 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 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, standby; 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 to remove dissolved amount of ethyl acetate in the water of 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 embodiment preferred, 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 and is used for the treatment of and/or prevent application in the medicine of sars coronavirus infection in preparation.
In a preferred embodiment, described sars coronavirus main proteinase inhibitor is 6 suitable, 9 suitable-18 diolefinic acids (isolinoleic acid).
In a preferred embodiment, described sars coronavirus main proteinase inhibitor is suitable by 6,6 suitable-octadecenic acids (petroselinic acid) that 9 suitable-18 diolefinic acids (isolinoleic acid) are derived, 9 suitable-octadecenic acids (oleic acid), 9 suitable-octadecenic acid sodium salts (sodium oleate), 9 anti--octadecenic acids (elaidic acid), 11 suitable-octadecenic acids (cis-vaccenic acid), 13 suitable-docosenoic acids (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 and is used for the treatment of and/or prevents application in the medicine that sars coronavirus infects in preparation:
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, 6 suitable-octadecenic acids, 9 suitable-octadecenic acids, 9 suitable-octadecenic acid sodium salts, 9 anti--octadecenic acids, 11 suitable-octadecenic acids, 13 suitable-docosenoic acids, 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.
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 the 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 active measuring method of described sars coronavirus main protease vitro inhibition 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). measure the sars coronavirus main protease vitro inhibition activity of test substance; 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 the sars coronavirus main protease vitro inhibition activity of test substance by the method that includes but not limited to colorimetric, fluorescence reading or chemiluminescence readings; 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 the simplification 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 write down 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. as benzene, toluene, diformazan; 2. fat hydrocarbon solvent: as pentane, hexane, octane etc.; 3. alicyclic hydrocarbon type solvent: as hexanaphthene, pimelinketone, toluene pimelinketone etc.; 4. halogenated hydrocarbon solvent: as chlorobenzene, dichlorobenzene, methylene dichloride etc.; 5. alcoholic solvent: as methyl alcohol, ethanol, Virahol etc.; 6. ether solvent: 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: as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether etc.; 10. other organic solvents: 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 (as 500 grams), with 95% ethanolic soln (as 4500 milliliters) refluxing extraction 3 times, united extraction liquid, concentrate 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 (as 10 grams), suspend with distilled water (as 150 milliliters), with ethyl acetate (as 650 milliliters) extraction 3 times, separating ethyl acetate phase and water; Merge water, standby; 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 dissolved amount of ethyl acetate in the water that removes 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 (as 250 milliliters) wash-out for example 8 times, 95% ethanol (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 to study Chinese materia medica, natural product chemistry and biology from a kind of new angle, is that the various cross disciplines of utilization are to modern valuable trial of Chinese materia medica.Because contained composition is quite complicated 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 property of medicine small molecules and make it as far as possible to concentrate preferably, improve relative concentration.The present invention not only provides and can realize this purpose technology of preparing scheme, and screening is had the active sample of inhibition, separates, has identified that wherein micromolecular inhibitor 6 is suitable, 9 suitable-18 diolefinic acids with various chromatograms, spectral method.By buying with 6 suitable, 9 suitable-18 diolefinic acid structural similitudies, the fatty acid compound that the double key number amount is different with the position, and their inhibition activity is verified, our several strong inhibitor of having got back with the enzyme screening of living.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 has the inhibition activity to the sars coronavirus main protease probably.
At present, be the basic drug screening of carrying out based on sars coronavirus main protease crystalline structure, the micromolecular inhibitor of being found 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 natural product in the conventional Chinese medicine greatly, its toxicity and metabolism absorption characteristic have incomparable superiority than other synthetic compounds such as polypeptide classes, are splendid medicine that may go on the market or potential prodrugs.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 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: 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 (6 is suitable, 9 suitable-18 diolefinic acids); 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, 9 suitable-octadecenic acid sodium salts, 6 suitable, 9 is suitable, 12 suitable-18 trienic acids, 9 suitable, 12 is suitable, 15 suitable-18 trienic acids, 6 suitable, 9 suitable-18 diolefinic acids, 9 suitable, 12 suitable-18 diolefinic acids, 9 suitable-octadecenic acids, 9 anti--octadecenic acids, 11 suitable-octadecenic acids, 6 suitable-octadecenic acids, 13 suitable-docosenoic acids.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 big 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, transform colibacillus (Escherichia coli) DH5 α competent cell with connecting product then, 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, the test tube that is inoculated in the LB that about 5mL is housed respectively (adds penbritin in this LB solution, making its final concentration is 100mg/L) in, overnight incubation, use plasmid extraction kit (vast Imtech Type B plasmid is the rapid extraction test kit in a small amount) to extract plasmid then, and cut with BamHI and XhoI enzyme, reclaim size with sepharose then 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, reclaims the fragment that enzyme is cut with sepharose then;
1.1.6 the fragment that (1.1.4) and (1.1.5) are obtained is connected, and (target gene fragment after enzyme is cut back to close and destination carrier fragment are according to mole number 3: 1-6: 1 ratio mixing, requirement according to Takara DNA Ligation was reacted 30 minutes-18 hours at 16 ℃), transform colibacillus DH5 α competent cell, be coated in LB flat board (containing the 100mg/L penbritin) and go up overnight incubation.With the positive colony that screens, be used for identifying and order-checking.
Sequencing result shows that 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 with the bacterial strain of the pGEX-4T-1 carrier transformed into escherichia coli BL21 (DE3) that contains encoding SARS coronavirus proteolytic gene that obtains in the above-mentioned steps 1.1, and with LB flat board (containing the 100mg/L penbritin) screening positive clone;
1.2.2 at picking positive colony on the LB flat board described in (1.2.1) (containing the mono-clonal that grows out on the LB flat board of penbritin), overnight incubation changes the LB substratum (containing the 100mg/L penbritin) of 1L then 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, ice-bath ultrasonic is broken bacterium 20-30 minute then; 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 the human rhinovirus 3C proteolytic enzyme about 2ml 0.1mg/ml at last, cut 12-20 hour at 4 ℃ of enzymes, collect the sars coronavirus main protease afterwards;
1.2.5 use Mono Q (GE company) anion-exchange chromatography to carry out purifying again the sars coronavirus main protease that (1.2.4) obtains.
Get the crotons medicinal material of 500 gram drying and crushing,, extract 3 times with 4500 milliliter of 95% alcohol reflux, each 2 hours, united extraction liquid, concentrated 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 layering in 6 hours, coextraction 3 times, separating ethyl acetate phase and water, merge water, standby, 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), standby;
Aqueous portion after the extraction steams the middle dissolved amount of ethyl acetate 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, standby.
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, add crotons ethyl acetate extraction sample, water macroporous resin 95% ethanol elution sample and 95% total ethanol extract in this proteic buffered soln respectively in different containing then, 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). measure the sars coronavirus main protease vitro inhibition activity of test substance, wherein control curve is the mensuration curve that only adds albumen and substrate, do not add each segmentation sample of crotons, and the low more explanation restraining effect of curve is strong more.
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) separating, is the moving phase wash-out with chloroform-methanol (1: 1), receives altogether to have merged 5 parts, 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, add the a1-a5 sample respectively in different containing in this proteic buffered soln then, 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). measure the sars coronavirus main protease vitro inhibition activity of test substance, wherein control curve is the mensuration curve that only adds albumen and substrate, do not add the a1-a5 sample, and the low more explanation restraining effect of curve is strong more.
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, add b1, b2 sample in this proteic buffered soln respectively in different containing then, 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). measure the sars coronavirus main protease vitro inhibition activity of test substance, wherein control curve is the mensuration curve that only adds albumen and substrate, do not add b1 and b2 sample, and the low more explanation restraining effect of curve is strong more.
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: 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 being correlated with of 2 hydrogen and 3 carbon proves that fully the position of two pairs of keys is 6 and 9 (see figure 4)s.Its NMR data and 6 suitable, 9 suitable-18 diolefinic 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, therefore confirm that compound 1 is 6 suitable, 9 suitable-18 diolefinic acids (isolinoleic acid).
(6 is suitable to the above-mentioned compound that obtains 1 for we, 9 suitable-18 diolefinic acids) carried out the vitro inhibition determination of activity of sars coronavirus main protease, mensuration is carried out according to 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 fluorescent mark substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH rapidly
2, final concentration 16 μ M).Excitation wavelength and emission wavelength are respectively 320nm and 405nm, temperature remains on 298K, write down first order fluorescence reading (the results are shown in Fig. 5) (control curve is for only adding albumen and substrate, not adding 6 suitablely, and the mensuration curve of 9 suitable-18 diolefinic acids, the low more explanation restraining effect of curve is strong more) per 2 seconds.6 is suitable as shown in Figure 5, and 9 suitable-18 diolefinic acids have the good restraining activity, are in the crotons sars coronavirus main protease to be played inhibiting principal constituent.
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: 6 suitable-octadecenic acids (Shanghai Sigma-aldrich company), 9 suitable-octadecenic acids (Tianjin Alfa-aesar company), 9 suitable-octadecenic acid sodium salts (Shanghai Sigma-aldrich company), 9 anti--octadecenic acids (Tianjin Alfa-aesar company), 11 suitable-octadecenic acids (Shanghai Sigma-aldrich company), 13 suitable-docosenoic acids (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:
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.Mensuration is carried out according to 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, contain in difference and to add 9 suitable-octadecenic acid sodium salts in this proteic buffered soln respectively, 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, 9 suitable-octadecenic acids, 9 anti--octadecenic acids, 11 suitable-octadecenic acids, 6 suitable-octadecenic acids, 13 suitable-docosenoic acids (final concentration is 50 μ M) add fluorescent mark substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH rapidly
2, final concentration 16 μ M).Excitation wavelength and emission wavelength are respectively 320nm and 405nm, and temperature remains on 298K, write down the first order fluorescence reading per 2 seconds.Control curve for only add albumen and substrate, mensuration curve when not adding any inhibitor, the low more explanation restraining effect of curve is strong more.
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 specific activity 6 suitable, 9 suitable-18 diolefinic acids are 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 influences the dissolving properties of entire 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 have 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 structure and representativeness, and the compound that therefore has this general formula should fall within protection scope of the present invention.
Among the related herein 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 (for example buy, preparation etc.) voluntarily acquisition easily.
Should be appreciated that under situation without departing from the spirit and scope of the present invention those of ordinary skill in the art can make various changes and improvements to it in form and details, 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 (19)
1. a method of screening the sars coronavirus main proteinase inhibitor comprises the steps:
A. measure sars coronavirus main protease vitro inhibition activity from the multiple extract of single medicinal material;
B. select to have the active a kind of extract of best vitro inhibition; 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, selection has the isolation and selection that the active a kind of component of best vitro inhibition enters next round, the active component of best vitro inhibition that has that obtains until described isolation and selection is a kind of compound, with this compound as the sars coronavirus main proteinase inhibitor.
2. method according to claim 1, wherein said steps A comprises the steps:
(1). in buffered soln, add the sars coronavirus main protease;
(2). add substrate and test substance in buffered soln; With
(3). measure the sars coronavirus main protease vitro inhibition activity of test substance.
3. method according to claim 1, wherein the separation described in the step C is selected from one or more following methods: supercritical extraction, organic solvent extraction, chromatographic separation, electrophoretic separation and membrane sepn.
4. method according to claim 3, wherein said extract is an extractive with organic solvent.
5. method according to claim 4, wherein said organic solvent are 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.
6. method according to claim 4, wherein said organic solvent is selected from alcoholic solvent and esters solvent.
7. according to each described method among the claim 1-6, wherein said single medicinal material is crotons (fructus ofCroton tiglium L.).
8. method according to claim 7, wherein said multiple extract are crotons 95% total ethanol extract, crotons ethyl acetate extract and crotons water macroporous resin 95% ethanol extraction.
9. method according to claim 8, the extracting method of wherein said crotons 95% total ethanol extract, crotons ethyl acetate extract and crotons water macroporous resin 95% ethanol extraction comprises the steps:
(a). with crotons with 95% ethanolic soln refluxing extraction, united extraction liquid, concentrate 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, standby; 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; With
(c). steam to remove dissolved amount of ethyl acetate in the water of 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.
10. according to the method for claim 9, wherein said substrate is the fluorescent mark substrate.
11. according to the method for claim 10, wherein said fluorescent mark substrate is MCA-AVLQSGFRL (DNP) L-NH
2
12. the sars coronavirus main proteinase inhibitor that each method screening obtains according to claim 1-11 is used for the treatment of and/or prevent application in the medicine of sars coronavirus infection in preparation.
13. according to the application of claim 12, wherein said sars coronavirus main proteinase inhibitor is the compound that meets following unsaturated fatty acids general structure:
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.
14. according to the application of claim 12, wherein said sars coronavirus main proteinase inhibitor is 6 suitable, 9 suitable-18 diolefinic acids (isolinoleic acid).
15. application according to claim 12, wherein said sars coronavirus main proteinase inhibitor is for having with 6 suitable, the unsaturated fatty acids acid compound of 9 suitable-18 diolefinic acid similar: 6 suitable-octadecenic acids, 9 suitable-octadecenic acids, 9 suitable-octadecenic acid sodium salts, 9 anti--octadecenic acids, 11 suitable-octadecenic acids, 13 suitable-docosenoic acids, 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.
16. one kind is used for the treatment of and/or prevents the pharmaceutical composition that sars coronavirus infects, and comprises each method screening obtains according to claim 1-11 the sars coronavirus main proteinase inhibitor and the pharmaceutically acceptable carrier of treatment significant quantity.
17. according to the pharmaceutical composition of claim 16, wherein said sars coronavirus main proteinase inhibitor is unsaturated fatty acids compound and the pharmaceutically acceptable carrier with following general structure:
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.
18. according to the pharmaceutical composition of claim 16, wherein said sars coronavirus main proteinase inhibitor is: 6 is suitable, 9 suitable-18 diolefinic acids (isolinoleic acid).
19. pharmaceutical composition according to claim 16, wherein said sars coronavirus main proteinase inhibitor is with 6 suitable, the unsaturated fatty acids acid compound of 9 suitable-18 diolefinic acid similar: 6 suitable-octadecenic acids, 9 suitable-octadecenic acids, 9 suitable-octadecenic acid sodium salts, 9 anti--octadecenic acids, 11 suitable-octadecenic acids, 13 suitable-docosenoic acids, 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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