CN101063234A - Method for screening SARS coronaviruses main Protease inhibitors from natural product such as Chinese medicinal materials mixture prepared storeroom - Google Patents

Method for screening SARS coronaviruses main Protease inhibitors from natural product such as Chinese medicinal materials mixture prepared storeroom Download PDF

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CN101063234A
CN101063234A CN 200710065119 CN200710065119A CN101063234A CN 101063234 A CN101063234 A CN 101063234A CN 200710065119 CN200710065119 CN 200710065119 CN 200710065119 A CN200710065119 A CN 200710065119A CN 101063234 A CN101063234 A CN 101063234A
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sars coronavirus
sample
main protease
root
coronavirus main
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CN100480440C (en
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饶子和
孙玉娜
娄智勇
向科辉
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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 discloses a method to sieve SARS coronavirus main prolease depressor from natural products mixture optional warehouse, which comprises the following steps: touching crystal of SARS coronavirus main prolease with 'sample with inhibited active'; getting electron density of small molecule depressant combined with SARS coronavirus main prolease; appraising the small molecule depressant based on electron density; getting fine three-dimensional structure of SARS coronavirus main prolease and small molecule depressant; getting small molecule depressant Sichuan scented sea vegetable ala factor. This Sichuan scented sea vegetable ala factor can inhibit coronavirus main prolease activator effectively, which can cure or prevent SARS coronavirus and infectivity gastrointestinal disease virus.

Description

Therefrom screen the novel method of sars coronavirus main proteinase inhibitor in the medicines natural products mixture alternate library
Technical field
The present invention relates to the screening method of medicine, more specifically, relate to the crystalline structure based on the sars coronavirus main protease, the novel method of therefrom screening the inhibitor of sars coronavirus main protease in the medicines natural products mixture alternate library reaches the inhibitor of the sars coronavirus main protease of finding based on this method.
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.et al.Lancet of coronavirus genus, 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, people Coronaviruses such as S.G., 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 etc.; The 3rd cohort has AIBV, Avian InfectiousBronchitis Virus (Spaan such as (avian infectious bronchitis viruses), W.J.M.and 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) pp1a and pp1ab (Ziebuhr, Snijder et al.2000; Thiel, Herold et al.2001; Anand, Yang et al.2005; Ziebuhr 2005), they only just can make virus finish normal transcription, copy function (Ziebuhr, Snijder et al.2000 after the proteolytic cleavage of encoding viral is slit into independent subunit; Anand, Yang et al.2005; Bartlam, Yang et al.2005).Sars coronavirus major protein enzyme (mainprotease 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.
Natural product claims secondary metabolite (Secondary Metabolites) again, 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 DJ, GraggGM, the Snader KM.Nat Prod Rep of tool potentiality in the current medicament research and development process, 2000,17 (3): 215-234; Lee K-H.J NatProd.2004,67 (2): 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.Especially the research that derives from the natural product of Chinese medicine etc. for natural product is necessary, because the history that Chinese medicine was used in China in existing thousands of years, be the treasure-house of the medicine micromolecular compound of a greatness, 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.
Disclosing among the CN1600308A of Chen Shuxia (2005.03.30) and from numerous Chinese medicine, filtered out unique effective constituent of low toxicity simply---sophocarpine monomer (sophocarpine is called for short SC), it is used for anti-SARS virus is to produce effect.But it is not in fact the screening to Chinese medicine integral body, but according to common ground based on SARS virus and CBV existence, and the sophocarpine monomer has result of treatment for CBV and derive and draw, and wherein do not set forth this monomeric mechanism of action.
A kind of middle pharmaceutically active ingredient and biological activity determination method thereof that sars coronavirus infects that suppress disclosed among the CN1602853A of Xu Xiaojie etc. (2005.04.06), be specifically related to from plant gall isolated inhibition sars coronavirus and infect the effective constituent of host cell, this composition at be the key structure territory S2 albumen of coronavirus.The CN1803168A of Huang Jianping (2006.07.19) discloses the Chinese medicine of a kind of treatment severe acute respiratory syndrome (SARS), and this Chinese medicine derives from the effective proved recipe of clinical treatment ARDS, according to above-mentioned SARS etiology and pathogenesis and rules of treatment plus-minus prescription, by Leaf of Indigowoad (dry product) 15-30g, give birth to mast 5-15g, Japanese Honeysuckle 15-30g, Bombyx Batryticatus (parched) 10-15g, radix scrophulariae 10-15g, Tree Peony Bark 10-15g, Radix Salviae Miltiorrhizae 15-30g, Radix Panacis Quinquefolii 5-10g, Radix Glycyrrhizae 15-30g, White Mulberry Root-bark 10-30g, Semen Lepidii (Semen Descurainiae) 15-30g, Radix Stephaniae Tetrandrae 5-10g, Radix Et Rhizoma Rhei 5-10g (back is down) totally ten three flavor Chinese medicinal materialss forms, has clearing heat and relieving dyspnea, detoxifying and descending turbid, blood cooling and ecchymoses removing is promoting blood circulation and removing blood stasis, sharp water clearing damp, internal organs is led in purging the lung of pathogenic fire, and the effect of supplementing QI and nourishing YIN is mainly used in SARS and breathes heavily and suppress phase treatment, go into decoction oral, one day two doses, once take 600 milliliters of soups, drank once every 1-2 hour.The mechanism of action of various Chinese medicines is not made any elaboration in the document.
At present also not based on the crystalline structure of sars coronavirus main protease, from natural drug systems such as Chinese medicine (as medicines natural products mixture alternate library) in screen the sars coronavirus main proteinase inhibitor technology.The invention provides a kind of from derive from natural products mixture alternate sample such as Chinese medicine the screening target combine with the sars coronavirus main protease, and the method for the micromolecular inhibitor that binding ability is best, and explained its inhibition mechanism to the sars coronavirus main protease from atomic level.
Summary of the invention
The invention provides a kind of method of from natural products mixture alternate library, screening the sars coronavirus main proteinase inhibitor, this method comprises steps A: the alternative sample in the mensuration natural products mixture alternate library is to the inhibition activity of sars coronavirus main protease, acquisition has the active sample of inhibition to the sars coronavirus main protease, these samples are called " the active sample of inhibition is arranged ", the crystal of sars coronavirus main protease is contacted with " the active sample of inhibition is arranged ", obtain the crystal of sars coronavirus main protease and micromolecular inhibitor complex body.The crystal of sars coronavirus main protease and " have suppress active sample " as required, can contact with single " the active sample of inhibition is arranged " respectively when contact, also can " the active sample of inhibition be arranged " with blended and contact.
Behind the crystal that obtains sars coronavirus main protease and micromolecular inhibitor complex body, carry out step B again: the X-ray diffraction in crystals data of the complex body that obtains among collection and the analytical procedure A obtain the electron density with sars coronavirus main protease bonded micromolecular inhibitor.
After the electron density of acquisition and sars coronavirus main protease bonded micromolecular inhibitor, carry out step C again: based on the electron density of main protease bonded micromolecular inhibitor that obtain and sars coronavirus among the step B, identify and sars coronavirus main protease bonded micromolecular inhibitor, obtain the fine three dimensional structure of micromolecular inhibitor and sars coronavirus main protease complex body.
After step C, carry out step D again: measure the inhibition activity of micromolecular inhibitor to the sars coronavirus main protease.
Being used for screening the natural products mixture alternate library of sars coronavirus main proteinase inhibitor among the present invention, can be to be raw material with any natural product, the alternative sample for preparing with any method.
The present invention preferably is used to screen the natural products mixture alternate library of sars coronavirus main proteinase inhibitor with the preparation of following primary raw materials: comprise traditional medicinal material, by the Chinese medicines such as mixing medicinal material that the medicinal material kind and the consumption of prescriptions of traditional Chinese medicine regulation are formed, also do not carry out plant and animal species, marine organisms, microorganism of medicinal exploitation or the like.
Because the contained composition of middle medicines natural products crude extract is quite complicated, therefore must the crude extract of natural product be optimized, purpose is to remove interfering factors such as albumen, polysaccharide etc. in sars coronavirus main protease active determination in vitro and the crystal immersion test, be retained as property of medicine small molecules and make it as far as possible to concentrate preferably, improve relative concentration, be applicable to that with preparation with sars coronavirus main protease crystalline structure be the alternative storehouse that inhibitor screening is carried out on the basis.The invention provides and to realize that this purpose prepares the technical scheme in alternative storehouse, its ultimate principle is: after primary raw materials is extracted, extract uses the solvent such as the ethyl acetate of opposed polarity to extract earlier, water after the extraction is handled with the isolating method of macroporous resin again, can guarantee to remove albumen like this, interfering factorss such as polysaccharide, can utilize the difference of two kinds of different separation mechanisms again, make small molecules focus on the different section of polarity, obtain containing moderate molecular weight thus and to can be used in sars coronavirus main protease crystalline structure be the natural products mixture alternate library that inhibitor screening is carried out on the basis.
Preparation provided by the invention is used to screen the method for sars coronavirus main proteinase inhibitor natural products mixture alternate library, specifically comprises the steps:
(a), get a certain amount of primary raw materials, use the ethanolic soln refluxing extraction, united extraction liquid, concentrate medicinal extract;
(b), take by weighing the medicinal extract of gained in a certain amount of step (a), suspend with distilled water, use organic solvent extraction, separate organic solvent phase and water; Merge water, standby; Merge each organic solvent extraction thing, steam the organic solvent that removes wherein, obtain dried paste organic solvent phase sample, standby;
(c), steam to remove a small amount of organic solvent of dissolved in the water of the aqueous portion after the extraction in the step (b), separate with macroporous resin column chromatography, water and ethanolic soln are the moving phase wash-out successively; Discard the washing part, steam the solvent that removes ethanolic soln wash-out part, the shape sample that gets dry extract is standby;
(d), the dried paste sample that makes in dried paste organic solvent phase sample that makes in the above-mentioned steps (b) and the step (c) is 2 alternative samples that are derived from a kind of primary raw materials.
The preferred method of preparation natural products mixture alternate library provided by the invention is: the consumption mass ratio of ethanolic soln and primary raw materials is 6 in the step (a): 1-12: 1; Used ethanolic soln concentration is 70%-95%;
Medicinal extract is 1 with the mass ratio that suspends with distilled water in the step (b): 5-1: 15, and extraction is ethyl acetate or chloroform with organic solvent, its volume is 6 with the volume ratio that suspends with distilled water: 1-10: 1;
Used ethanolic soln concentration is 50% in the step (c).
Among the above-mentioned preparation method, preferred primary raw materials is a Chinese medicine, and preferred primary raw materials is: Herba Houttuyniae, Wild Chrysanthemum, Leaf of Indigowoad, reed rhizome, Japanese Honeysuckle, the capsule of weeping forsythia, the root of large-flowered skullcap, the wind-weed, folium ilicis chinensis, Herba Andrographis, Herba Commelinae, cape jasmine, blackberry lily, Radix Sophorae Tonkinensis, Lasiosphaera fenzlii, radix scrophulariae, Root-bark of Chinese Wolfberry, root, henry rabdosia leaf.
In addition, method of from natural products mixture alternate library, screening the sars coronavirus main proteinase inhibitor provided by the invention, more preferably in steps A: sars coronavirus main protease crystal and " have suppress active sample " are when contact, and the method for using is the crystal immersion process of crystal immersion or cocrystallization.
In one embodiment, the present invention has obtained the crystal of sars coronavirus main protease-root third plain complex body.
In another embodiment, the present invention's screening has obtained micromolecular inhibitor root third element of sars coronavirus main protease.
In another embodiment, the invention provides root and can be used for the treatment of or prevent coronavirus infections such as sars coronavirus, Transmissible gastroenteritis virus or avian infectious bronchitis virus.
In another embodiment, root third element provided by the invention can effectively suppress the coronavirus proteolytic activity, can be used for the treatment of or prevent coronavirus infections such as sars coronavirus, Transmissible gastroenteritis virus or avian infectious bronchitis virus.
In another embodiment, the invention provides and effectively to suppress the active root ethyl acetate extraction of sars coronavirus main protease sample, henry rabdosia leaf ethyl acetate extraction sample, root of large-flowered skullcap ethyl acetate extraction sample, Herba Houttuyniae ethyl acetate extraction sample, Radix Sophorae Tonkinensis ethyl acetate extraction sample, cape jasmine ethyl acetate extraction sample, 7 kinds of Chinese medicine samples of wind-weed ethyl acetate extraction sample and preparation method thereof.
The present invention chooses the known main protease of crystalline structure in the sars coronavirus, and natural product mixture samples such as China's traditional Chinese medicine are carried out active ingredient screening, is to remove to study Chinese materia medica, natural product and biology from a kind of new angle.Its flesh and blood can comprise two aspects: the first, and the expression of target protein, purifying and crystallization, this belongs to the research category of biomacromolecule; The second, in making in the medicines natural products mixture sample natural product small molecules of non-isolated existence freely with target protein---combining of being at war with property of sars coronavirus main protease, this belongs to the particularly category of natural product chemistry research of chemistry.
The method of inhibitor screening of the present invention is based on sars coronavirus main protease crystalline structure, therefrom screen in the medicines natural products mixture alternate library, can seek specific inhibitor fast and accurately, improve the accuracy of screening the sars coronavirus main protease.Method of the present invention is simple relatively, cost is lower, but screening efficiency height, quick, accurate, and can from blend sample, obtain the best inhibitor small molecules of binding ability.
Particularly, compare with existing method, the advantage of the screening method of inhibitor of the present invention is as follows:
(1) at first, this method has been saved loaded down with trivial details Separation of Natural Products purification step, significantly reduce workload, and the middle medicines natural products mixture alternate library of present method in sample be suitable for carrying out the active determination in vitro and the X-ray crystallography test of sars coronavirus main protease, greatly improved screening efficiency and hit rate.
(2) use the crystal of sars coronavirus main protease as bait, soak activated natural product mixture sample, can be having inhibiting micromolecular inhibitor from blend sample, to separate to the sars coronavirus main protease, obtain the fine three dimensional structure of sars coronavirus main protease and micromolecular inhibitor bonded complex body, and from the inhibition mechanism of this micromolecular inhibitor of atomic level explaination to the sars coronavirus main protease.。
(3) use the inventive method, by using the sars coronavirus main protease to soak the natural product mixture sample, can from the natural product mixture sample, find the small molecules best (competition bonded principle) with sars coronavirus main protease binding ability, optimized choice micromolecular inhibitor.
Description of drawings
Fig. 1 Wild Chrysanthemum of the present invention, reed rhizome, Japanese Honeysuckle, the capsule of weeping forsythia, folium ilicis chinensis, Herba Commelinae, cape jasmine, blackberry lily, Lasiosphaera fenzlii, radix scrophulariae, the water 50% ethanol elution sample of Root-bark of Chinese Wolfberry is to the inhibition activity curve figure of sars coronavirus main protease.Sample concentration: 100 μ g/mL
Fig. 2 Herba Houttuyniae of the present invention, Leaf of Indigowoad, the root of large-flowered skullcap, the wind-weed, Herba Andrographis, the water 50% ethanol elution sample of Radix Sophorae Tonkinensis is to the inhibition activity curve figure of sars coronavirus main protease.Sample concentration: 100 μ g/mL
Fig. 3 Japanese Honeysuckle of the present invention, the root of large-flowered skullcap, Herba Andrographis, folium ilicis chinensis, Herba Commelinae, Lasiosphaera fenzlii, radix scrophulariae, reed rhizome, Leaf of Indigowoad, the ethyl acetate extraction sample of Root-bark of Chinese Wolfberry is to the inhibition activity curve figure of sars coronavirus main protease.Sample concentration: 100 μ g/mL
Fig. 4 Herba Houttuyniae of the present invention, Wild Chrysanthemum, the capsule of weeping forsythia, the wind-weed, cape jasmine, blackberry lily, the ethyl acetate extraction sample of Radix Sophorae Tonkinensis is to the inhibition activity curve figure of sars coronavirus main protease.Sample concentration: 100 μ g/mL
The ethyl acetate extraction sample of Fig. 5 Herba Houttuyniae of the present invention, Radix Sophorae Tonkinensis, cape jasmine, the wind-weed, the root of large-flowered skullcap is to the inhibition activity curve figure of sars coronavirus main protease.Sample concentration: 10 μ g/mL
Fig. 6 henry rabdosia leaf ethyl acetate extraction of the present invention sample, water 50% ethanol elution sample are to the inhibition activity curve figure of sars coronavirus main protease.Sample concentration: 100 μ g/mL
Fig. 7 henry rabdosia leaf ethyl acetate extraction of the present invention sample, water 50% ethanol elution sample are to the inhibition activity curve figure of sars coronavirus main protease.Sample concentration: 10 μ g/mL
Fig. 8 root ethyl acetate extraction of the present invention sample, water 50% ethanol elution sample are to the inhibition activity curve figure of sars coronavirus main protease.Sample concentration: 100 μ g/mL
Fig. 9 root ethyl acetate extraction of the present invention sample, water 50% ethanol elution sample are to the inhibition activity curve figure of sars coronavirus main protease.Sample concentration: 10 μ g/mL
Near electron density comparison diagram Figure 10 sars coronavirus main protease of the present invention avtive spot.A is that the electron density of sars coronavirus main protease parent crystal avtive spot shows; B is that the electron density of the crystal avtive spot after sars coronavirus main protease and root ethyl acetate extraction sample soak shows; C is that the electron density of the crystal avtive spot after sars coronavirus main protease and root third plain the immersion shows.Electron density shown in the figure, blueness is the 2fofc electron density, is 1.0sigma; Cyan and green are 1fofc difference electron density, are 2.5sigma
The micromolecular electron density of the existence in Figure 11 root acetic acid ethyl ester extract of the present invention sample and sars coronavirus main protease avtive spot Cys145 residue electron density form the demonstration figure of covalent linkage.Protein molecule represents that with white ribbon figure the Cys145 residue of avtive spot is represented with ball-and-stick model; Blue electron density is represented the electron density of Cys145, and red electron density is represented unknown micromolecular electron density.
Mass spectrum before and after Figure 12 sars coronavirus main protease of the present invention and the root extract ethyl acetate extraction sample effect.The mass spectra peak of blue expression sars coronavirus main protease itself; Mass spectra peak behind red expression sars coronavirus and the root ethyl acetate extraction sample effect.
Root third element of Figure 13 20 μ M is to the inhibition activity curve figure of sars coronavirus main protease
Root third element of Figure 14 2 μ M is to the inhibition activity curve figure of sars coronavirus main protease
Root third element of Figure 15 20 μ M is to the inhibition activity curve figure of TGEV main protease
Root third element of Figure 16 2 μ M is to the inhibition activity curve figure of TGEV main protease
Root third element of Figure 17 100 μ M is to the inhibition activity curve figure of AIBV main protease
Root third element of Figure 18 10 μ M is to the inhibition activity curve figure of AIBV main protease
The present invention provide specific embodiments of the invention in order to explain in further detail below in conjunction with accompanying drawing.
Embodiment
Embodiment 1
1.SARS the expression of coronavirus proteolytic, purifying and crystal growth
According to document (Yang H etal.Proc Natl Acad Sci.2003 Nov; 100 (23): 13190-5) carry out expression, purifying and the crystal growth of sars coronavirus main protease (aminoacid sequence is seen the sequence table part), obtain the crystal of the sars coronavirus main protease of diffraction resolution higher (being higher than 2.6 ).Particularly:
The expression vector establishment of a.SARS coronavirus proteolytic
Concrete steps comprise:
(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 '
(1.2) through the gene fragment of pcr amplification after BamHI and the two enzyme enzymes of XhoI are cut, reclaiming size with agarose gel electrophoresis is fragment about 1kb.
(1.3) will reclaim fragment and be connected, and 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 with the T carrier.
(1.4) a plurality of mono-clonals of picking from the flat board are inoculated in the test tube (add acillin in this LB solution, making its final concentration is 100mg/L) of the LB that about 5mL is housed overnight incubation respectively.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, reclaim the big or small target gene fragment that is about about 1kb with sepharose then with BamHI and XhoI enzyme.
(1.5) destination carrier pGEX-4T-1 (available from Pharmacia company) is cut with BamHI and XhoI enzyme, reclaim the fragment that enzyme is cut with sepharose then.
(1.6) fragment that (1.4) and (1.5) are obtained is connected that (target gene segment 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 Escherichia coli 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.
The expression and purification of b.SARS coronavirus proteolytic
Comprise step:
(2.1) with the bacterial strain of the pGEX-4T-1 carrier Transformed E scherichia coli BL21 (DE3) that contains encoding SARS coronavirus proteolytic gene that obtains among the above-mentioned steps a, and with LB flat board (containing the 100mg/L penbritin) screening positive clone.
(2.2) at picking positive colony on the LB flat board described in (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 ℃;
(2.3) the centrifugal 10-15min collecting cell of 5000-8000rpm, ice-bath ultrasonic is broken bacterium 20-30 minute then; Collect supernatant liquor behind the broken centrifugal 20-40min of bacterium liquid 13000rpm-15000rpm.
(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, collect the sars coronavirus main protease afterwards at 4 ℃ of enzymes.
(2.5) albumen that (2.4) are obtained uses Mono Q (GE company) anion-exchange chromatography to carry out purifying again
The crystal growth of the main protease of c.SARS coronavirus
The crystal growth of the main protease of sars coronavirus is to adopt hanging drop gas phase diffusion method, under 289-297K (K is temperature unit Kelvin, 16 ℃～24 ℃ of centigradetemperatures), carries out with tissue culturing plate.Crystal growth test kit (Crystal Screening Kit I ﹠amp with Hampton Research company; II, PEG/IONKit, PEG 6000 Grid Kit and (NH 4) 2SO 4Kit) the primary crystallization condition is screened.In the drop under the C3 (20%PEG 6000,0.1M MES (pH 6.0)) of the PEG6000 Grid Kit condition, red nodular precipitation is arranged, and crystallite occurs at sedimentary edge.Under this crystallization condition, we have attempted the damping fluid (as carrying out the pH value from 5.5 to 6.5) of different pH values, PEG (as carrying out from PEG 3350 to PEG 10000), different protein concentration (carrying out between from 2mg/ml to 30mg/ml) and the various additive of different polymerization degree (Hampton Research company ground additive test kit) waits and optimize crystal.At last, PEG 6000,3% dimethyl sulfoxide (DMSO) (DMSO) 1.8～12%, 1mM DTT, (pH 5.0～PH6.0) and protein concentration have obtained the diffraction resolution crystal of higher (being higher than 2.6 ) to 100mM MES damping fluid when being 5～12mg/ml.
2. the preparation of middle medicines natural products mixture alternate library
Be derived from the preparation of the natural products mixture alternate sample of root (I.pharicus (Prain) Murata)
(1) preparation root extract medicinal extract:
Get the root 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;
(2) make root ethyl acetate extraction sample:
Take by weighing this medicinal extract 10.5 grams, suspend, in 1000 milliliters of separating funnels of impouring, use ethyl acetate extraction with 85 milliliters of distilled waters.Each 850 milliliters of ethyl acetate of extraction, shake well leaves standstill layering in 6 hours.Coextraction 6 times merges each time acetic acid ethyl ester extract.Obtain ethyl acetate phase and water after the extraction.Acetic acid ethyl ester extract steams with EYELA N1001 type Rotary Evaporators and desolventizes, and the shape root ethyl acetate extraction sample that gets dry extract is standby;
(3) make root water macroporous resin 50% ethanol elution sample:
Aqueous portion after the extraction steams the middle dissolved amount of ethyl acetate that dewaters with EYELA N1001 type Rotary Evaporators, (macroporous resin is with 170 milliliters with the separation of HP-20 type macroporous resin column chromatography, glass column specification 30 * 300mm), water, 50% ethanol are the moving phase wash-out successively.Water elution 8 times, 250 milliliters of each wash-outs discard the washing part.50% ethanol elution 5 times, 300 milliliters of each wash-outs.Partly use EYELA N1001 type Rotary Evaporators to steam 50% ethanol elution and desolventize, the shape root water macroporous resin 50% ethanol elution sample that gets dry extract, standby;
(4) acquisition is derived from the natural products mixture alternate sample of root:
The root water macroporous resin 50% ethanol elution sample that the root ethyl acetate extraction sample that above-mentioned steps (2) makes, step (3) make has been formed 2 the alternative samples (the alternative sample of Zhi Bei each all contains multiple natural product micromolecular compound according to the method described above, therefore is called the natural products mixture alternate sample) that are derived from the primary raw materials root.
Be derived from the preparation of the natural products mixture alternate sample of other primary raw materials:
Because the antipyretic in the Chinese medicine often has antibiotic, antivirus action, and also contain this type of medicine mostly in three prescriptions that State Administration of Traditional Chinese Medicine announces during the SARS and (consist of Herba Houttuyniae 15 grams, Wild Chrysanthemum 6 grams, oriental wormwood 15 grams, eupatorium 10 grams, tsaoko 3 grams such as prescription one medicine.Its effect is clearing heat and detoxicating, dampness removingization is turbid, and wherein Herba Houttuyniae, Wild Chrysanthemum are antipyretic; Two medicines of writing out a prescription consist of taraxacum 15 grams, Flower of Chinese Globeflower 6 grams, Leaf of Indigowoad 10 grams, the root of kudzu vine 10 grams, Su Ye 6 grams.Its effect is clearing heat and detoxicating, the wind that looses is evil thoroughly, and wherein taraxacum, Flower of Chinese Globeflower, Leaf of Indigowoad are antipyretic; Three medicines of writing out a prescription consist of reed rhizome 15 grams, Japanese Honeysuckle 10 grams, the capsule of weeping forsythia 10 grams, peppermint 6 grams, Radix Glycyrrhizae 5 grams.Its effect relieving superficies by cooling, dispelling wind are evil thoroughly, and wherein reed rhizome, Japanese Honeysuckle, the capsule of weeping forsythia are antipyretic).So start with from these medicines earlier.Look into Chinese materia medica monograph (Huang Zhaosheng chief editor, People's Health Publisher, August in 2002 the 1st edition) this type of medicine commonly used and have 44 kinds, wherein return lung channel for 17 kinds, for: Herba Houttuyniae, Wild Chrysanthemum, Leaf of Indigowoad, reed rhizome, Japanese Honeysuckle, the capsule of weeping forsythia, the root of large-flowered skullcap, the wind-weed, folium ilicis chinensis, Herba Andrographis, Herba Commelinae, cape jasmine, blackberry lily, Radix Sophorae Tonkinensis, Lasiosphaera fenzlii, radix scrophulariae, Root-bark of Chinese Wolfberry (plant provides Kunming, Chinese Academy of Sciences Yunnan) is classified the primary raw materials of the medicines natural products mixture alternate library that is used for carrying out the screening of sars coronavirus main proteinase inhibitor as.
Other has henry rabdosia leaf (plant provides Kunming, Chinese Academy of Sciences Yunnan) is China's active drug that is used for treating multiple inflammation among the people, and from this platymiscium, isolate recently and multiplely be proved to be diterpenes composition, so also classify the primary raw materials that is used for carrying out the medicines natural products mixture alternate library that the sars coronavirus main proteinase inhibitor screens as with physiologically active such as antiviral, antitumor.
36 natural products mixture alternate samples that are derived from above 18 kinds of primary raw materialss are according to the above-mentioned preparation method's preparation that is derived from the natural products mixture alternate sample of root (I.pharicus (Prain) Murata).
Zhi Bei 38 natural products mixture alternate samples have been formed the medicines natural products mixture alternate library that is used for carrying out the screening of sars coronavirus main proteinase inhibitor according to the method described above.
3. the natural products mixture alternate sample suppresses determination of activity to the sars coronavirus main protease
The determination of activity of sars coronavirus main protease is to use fluorogenic substrate MCA-AVLQSGFR-Lys (Dnp)-Lys-NH 2(purity is greater than 95%, the biochemical company limited of Shanghai gill) is finished.The aminoacid sequence of this fluorogenic substrate derives from the N end of sars coronavirus main protease from shearing sequence.
The instrument that is used for fluorescent strength determining is that (Finland), exciting light and radiative wavelength are respectively 320nm and 405nm to Fluoraskan Ascent luminoscope for ThermoLabsystems, Helsinki.
The sample that is used for carrying out in the medicines natural products mixture alternate library of sars coronavirus main proteinase inhibitor screening according to the present invention preparation is called the natural products mixture alternate sample.The natural products mixture alternate sample of 38 dried pastes of preparation in the step 2 is dissolved in to make its final concentration in the dimethyl sulfoxide (DMSO) (DMSO) be 50mg/mL, makes the DMSO solute of 38 natural products mixture alternate samples respectively.
At buffered soln (50mM Tris-HCl (pH 7.3), 1mM EDTA (containing or do not contain DTT)) adds sars coronavirus main protease (final concentration 0.5 μ M) in, the DMSO solute (as the DMSO solute of root ethyl acetate extraction sample) that adds the natural products mixture alternate sample makes its final concentration be: 100 μ g/mL, concentration of substrate is 20 μ M, 298K placed after 10 minutes, add fluorescent mark substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH2, final concentration 20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 320nm and 405nm, and temperature keeps 298K, writes down the first order fluorescence reading per 2 seconds.
Contrast: do not add alternative sample, all the other conditions are identical.The results are shown among Fig. 1-Fig. 9.
Fig. 1-Fig. 4 and Fig. 6, Fig. 8 are that 38 natural products mixture alternate samples suppress active measurement result to the sars coronavirus main protease, and wherein the concentration of each alternative sample is 100 μ g/mL.
By above Fig. 1-Fig. 9 as can be seen, wherein most of alternative sample all has the activity of inhibition to the sars coronavirus main protease, wherein suppressing activity has 7 preferably, is respectively the acetic acid ethyl ester extract sample of the root of large-flowered skullcap, Herba Houttuyniae, Radix Sophorae Tonkinensis, cape jasmine, the wind-weed, root, henry rabdosia leaf.
Fig. 5, Fig. 7 and Fig. 9 are above 7 and suppress activity preferably: during concentration dilution to the 10 μ g/mL of the ethyl acetate extraction sample of the root of large-flowered skullcap, Herba Houttuyniae, Radix Sophorae Tonkinensis, cape jasmine, the wind-weed, root, henry rabdosia leaf the sars coronavirus main protease is suppressed the determination of activity result, can find, when the acetic acid ethyl ester extract concentration of the root of large-flowered skullcap, Herba Houttuyniae, Radix Sophorae Tonkinensis, cape jasmine, the wind-weed, root, henry rabdosia leaf is 10 μ g/mL, the sars coronavirus main protease is still had stronger inhibition activity.
This just illustrates may contain the activity that certain composition can effectively suppress the sars coronavirus main protease in the ethyl acetate extraction sample of these 7 kinds of Chinese medicines, illustrate that also these 7 kinds of Chinese medicines might become the active drug of treatment sars coronavirus.In order to understand them to the inhibiting definite molecule mechanism of sars coronavirus main protease, the crystal that we can select the ethyl acetate extraction sample of these 7 kinds of Chinese medicines to carry out the sars coronavirus main protease soaks or the cocrystallization experiment.
4, sars coronavirus main protease crystal soaks root ethyl acetate extraction sample
Owing to each sample in the medicines natural products mixture alternate library that is used for the screening of sars coronavirus main proteinase inhibitor all contains many kinds of natural product small molecules (therefore being called the natural products mixture alternate sample), if adopt the method for cocrystallization, influence to sars coronavirus main protease crystallizing system is bigger, it is good to obtain the diffraction quality, the crystal of the sars coronavirus main protease of abundant amount, even possibly can't growing crystal, the therefore main method that adopts crystal to soak obtains the crystal of sars coronavirus main protease and micromolecular inhibitor complex body.
In order to guarantee on the basis of crystal mass as much as possible, improve when soaking the concentration of micromolecular compound in the root ethyl acetate sample, can adopt following two kinds of methods to prepare the crystal soak solution of sars coronavirus main protease.
Method one, the DMSO solute of root ethyl acetate extraction sample (according to step 3 preparation) is diluted to sars coronavirus main protease crystal growth pond liquid (1.8～12% PEG 6000 for 10 times, 3% dimethyl sulfoxide (DMSO) (DMSO), 1mM DTT, in the 100mM MES damping fluid (pH 5.0～PH6.0)), the 13000-15000rpm high speed centrifugation, get supernatant liquor afterwards, get soak solution 1, standby.
Method two, the root ethyl acetate extraction sample (step 2 preparation) of dried paste directly is dissolved in sars coronavirus main protease crystal growth pond liquid (1.8～12% PEG 6000,3% dimethyl sulfoxide (DMSO) (DMSO), 1mM DTT, in the 100mM MES damping fluid (pH 5.0～PH6.0)), the 13000-15000rpm high speed centrifugation, get supernatant liquor afterwards, get soak solution 2, standby.
When guaranteeing that crystal soaks, be unlikely to make the micromolecular compound excessive concentration in the root ethyl acetate extraction sample influence crystalline diffraction quality, so soak solution 1 and 2 is diluted to 10 times of original liquid concentration respectively, standby.
During immersion, utilize instruments such as nylon crystal rings, the crystal of the good sars coronavirus main protease of growing is taken out from crystallizing pond liquid, add in soak solution 1 and 2 respectively and in corresponding 10 times of diluents, soaked 2-48 hour.
5. the Collection and analysis of crystalline diffraction data
On the basis of step 4, use the crystal that possesses better diffracting power (resolving power is preferably greater than 2.6 ), carry out the Collection and analysis of X ray diffracting data.
When carrying out data gathering, at first use the nylon crystal rings of Hampton Research company, from soak solution 1 and soak solution 2, obtain the crystal that soaks certain hour (being generally 2-48 hour); And use the cooling system of Rigaku company or the cooling system of Oxford Cyrosystem company rapidly, and crystal is refrigerated to-150 ℃ in the cryogenic nitrogen air-flow that above-described two kinds of refrigeration systems produce--180 ℃; Make X ray pass through crystal, use the precession method to collect X ray diffracting data.
Whether select the measured crystal of diffraction matter (resolving power is more preferably greater than 2.6 ) to carry out data gathering, processing, detecting has micromolecular combination.The statistics of data gathering such as following table 1:
Complex body data gathering statistics after table 1:SARS coronavirus proteolytic parent, sars coronavirus main protease and root acetic acid ethyl ester extract sample soak
Statistical parameter
Sars coronavirus main protease parent crystal Crystal (complex body) after sars coronavirus main protease and root ethyl acetate extraction sample soak
The space group cell parameter (, °) all point diffraction independence point diffraction redundancy I/ σ (I) Rmerge (%) integrity degrees (%) of resolution ratio () C2 a＝108.4 b＝81.8 c＝53.6 β＝104.7 50-1.50(1.55-1.50) 219,294 63,241 3.5(2.5) 30.4(2.0) 3.8(28.6) 87.7(40.6) C2 a＝107.8 b＝82.5 c＝52.9 β＝104.9 50.0-3.0(3.1-3.0) 63,908 9.039 7.1(6.4) 13.9(3.1) 8.2(67.5) 99.9(100.0)
By the sars coronavirus main protease parent of observation equal angular and the avtive spot electron density map (Figure 10) (blueness is the electron density of protein itself) of complex body, can find, can observe the electron density of nonprotein itself (cyan is represented) clearly at the avtive spot of complex body, prompting has micromolecular compound and is combined on the protein molecule.What show among Figure 10 a is near the electron density of avtive spot of sars coronavirus main protease precursor structure, and Figure 10 b is that sars coronavirus main protease and root ethyl acetate extraction sample soak near the electron density of back crystal avtive spot.
Examine the electron density (as shown in figure 10) at this place, we can find to be present in unknown micromolecular electron density in the root acetic acid ethyl ester extract sample and the electron density of the sars coronavirus main protease avtive spot Cys145 of place is closely linked, point out that we this should be that the γ sulphur atom of small molecules and Cys145 has formed covalent linkage, thereby suppressed the activity (as shown in figure 11) of sars coronavirus main protease.
6. in conjunction with micromolecular analysis and evaluation
We with sars coronavirus main protease (concentration is the sars coronavirus main protease aqueous solution of 12mg/ml) and root ethyl acetate extraction sample according to mass concentration ratio 1: 1-1: 4 mixed, and 4 ℃-16 ℃ effects 6-18 hour down.With the sample after the effect, 13,000-15, the 000rpm high speed centrifugation discards post precipitation and is ready for use on mass spectroscopy.Mass spectrum adopts MALDI (matrix-assistedlaser-desorption ionization) spectrum that sample is analyzed, and the result is referring to Figure 12.
We can find as a result from mass spectral, and after adding root acetic acid ethyl ester extract sample, tangible skew has appearred in molecular weight, and this mainly is because the small molecules bonded reason in protein molecule and the root acetic acid ethyl ester extract sample.Can calculate (deducting lower mass spectra peak) by the mass spectrum result and find that bonded small molecules molecular weight is about 390Da with higher mass spectrum peak value.
By analyzing existing root composition, we find, on this molecular weight level, mainly have compound as follows:
Figure A20071006511900211
We can find in the main component of root, in this molecular weight ranges, main component is based on diterpene-kind compound, comprise Sichuan-Tibet scented tea first, second, third, fourth, penta, own plain (PseurataA, PseurataB, PseurataC, PseurataD, PseurataE, PseurataF above corresponding respectively), Isodomedin and Dihydropseurata) etc.Owing to be combined in the small molecules at sars coronavirus main protease active pocket place and the γ sulphur atom of Cys145 has formed covalent linkage, knowing this small molecules by inference is Michael acceptor micromolecular, has α, β conjugation unsaturated double-bond again.
Based on the analysis of molecular weight and chemical environment, reach a conclusion: the bonded small molecules is root third element or Isodomedin.
For further checking, we have obtained the pure compound of root third element and Isodomedin from Kunming, Chinese Academy of Sciences Yunnan plant, prepare the crystal of root third element and Isodomedin and sars coronavirus main protease complex body according to above-described method, and carried out data gathering (data statistics the results are shown in table 2).By observing electron density, we have only found similar electron density at the crystal of the root third plain sars coronavirus main protease that soaked, shown in Figure 10 .c.Therefore can determine that the inhibitor small molecules of finding that combines with the sars coronavirus main protease should be root third element from root ethyl acetate extraction sample.
Crystal avtive spot at root acetic acid ethyl ester extract sample and the root third plain sars coronavirus main protease that soaked, unknown electron density is difference to some extent also, mainly be because the quality of data difference of different crystal, and the relative concentration of root third element is sure lower than pure product in root acetic acid ethyl ester extract sample, therefore may occupation rate also descend to some extent.
Therefore, can determine that the bonded small molecules is root third element.
The complex body data gathering statistics of table 2:SARS coronavirus proteolytic parent, sars coronavirus main protease and root third element
Statistical parameter Sars coronavirus main protease and related complexes
Sars coronavirus main protease parent Sars coronavirus main protease and the root third plain complex body
The space group cell parameter (, °) all point diffraction independence point diffraction redundancy I/ σ (I) Rmerge (%) integrity degrees (%) of resolution ratio () C2 a＝108.4 b＝81.8 c＝53.6 β＝104.7 50-1.50(1.55-1.50) 219,294 63,241 3.5(2.5) 30.4(2.0) 3.8(28.6) 87.7(40.6) C2 a＝107.3 b＝83.3 c＝53.3 β＝105.2 50.0-2.4(2.5-2.4) 176,898 19,456 9.1(5.3) 15.2(4.1) 5.4(56.6) 99.9(100.0)
Thus, we have obtained the fine three dimensional structure of sars coronavirus main protease and the root third plain complex body.
Here, we can see that novel method of the present invention compares with traditional inhibitor screening method, can great raising be arranged to screening efficiency.According to our method, (1) only need screening once just can be from the root acetic acid ethyl ester extract quick discovery can with sars coronavirus main protease bonded small molecules, (2) this small molecules is a small molecules best with the binding ability of sars coronavirus main protease in the root acetic acid ethyl ester extract, and (3) need not to prepare numerous pure product compounds when inhibitor screening; And if utilize traditional method, not only need before screening, separation and Extraction prepare at least 8 kinds of compounds, and need carry out 8 times determination of activity at least and carry out at least 8 crystal immersion tests etc., just might find the micromolecular compound that binding ability is best.
7, root third element suppresses active mensuration to the main protease of sars coronavirus
We use pure product compound small molecules (root third element), have measured its inhibition activity to the sars coronavirus main protease.Root third element suppresses active mensuration to the sars coronavirus main protease to carry out according to following steps: at buffered soln (50mM Tris-HCl (pH 7.3), 1mM EDTA (containing or do not contain DTT)) adds sars coronavirus main protease (final concentration 0.5 μ M) in, (final concentration is root third element: 20 μ M), concentration of substrate is 20 μ M, 298K placed after 10 minutes, add fluorescent mark substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH2, final concentration 20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 320nm and 405nm, and temperature keeps 298K, writes down first order fluorescence reading (the results are shown in Figure 13) per 2 seconds.Change the concentration of root third element then, measure its inhibition activity (the results are shown in Figure 14) the sars coronavirus main protease at 2 μ M.Contrast: do not add alternative sample, all the other conditions are identical.Determination of activity the results are shown in Figure 13-Figure 14.Can find that from Figure 13-Figure 14 root third element of 20 μ M has stronger inhibition activity to the sars coronavirus main protease, when its concentration is 2 μ M, the sars coronavirus main protease still be had the activity of inhibition.This root third element that just illustrates that Chinese medicine root the inside is contained can effectively suppress the activity of sars coronavirus main protease, and the active drug that root third have may become the treatment sars coronavirus also is described.
8, root third element suppresses active mensuration to the main protease of transmissible gastro-enteritis virus (TGEV)
In damping fluid (20mM Tris-HCl pH 7.0,1mM DTT), add described proteolytic enzyme (0.5 μ M), root third element (20 μ M), 298K placed after 10 minutes, added fluorescent mark substrate (MCA-AVLQSGFRL (DNP) L-NH2,20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 320nm and 405nm, and temperature keeps 298K, writes down the first order fluorescence reading per 2 seconds.Change inhibitor concentration then, under 2 μ M, measure and suppress active.Contrast: do not add inhibitor, all the other conditions are identical.The results are shown among Figure 15-Figure 16.The expression and purification reference of TGEV: Conservation of substratespecificities among coronavirus main proteases.J.Gen.Virol.2002; 83 (Pt 3): 595-9.
9. root third element suppresses active mensuration to the main protease of avian infectious bronchitis virus (AIBV)
In damping fluid (20mM Tris-HCl pH 7.0,1mM DTT), add described proteolytic enzyme (1 μ M), root third element (100 μ M), 298K placed after 10 minutes, added fluorescent mark substrate (MCA-AVLQSGFRL (DNP) L-NH2,20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 320nm and 405nm, and temperature keeps 298K, writes down the first order fluorescence reading per 2 seconds.Change inhibitor concentration then, under 10 μ M, measure and suppress active.Contrast: do not add inhibitor, all the other conditions are identical.The results are shown among Figure 17-Figure 18.
The expression and purification reference of AIBV: Preliminary crystallographic analysis of avianinfectious bronchitis virus main protease.Acta.Cryst.F.2007; 63 (Pt1): 24-6.
By Figure 13-Figure 18 as can be seen, root third element all has the activity of inhibition to the main protease of SARS, TGEV, AIBV, and wherein the inhibition activity to the sars coronavirus main protease is the strongest.Because TGEV belongs to first cohort of coronavirus genus, sars coronavirus belongs to second cohort of coronavirus genus, and AIBV belongs to the 3rd cohort of coronavirus genus.Therefore, can infer that root third element all has the main protease of whole coronavirus family suppresses active.
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 the applying date, 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〉Nankai University, Tsing-Hua University, Institute of Biophysics, Academia Sinica
＜120〉novel method of screening sars coronavirus main proteinase inhibitor in the medicines natural products mixture alternate library therefrom
<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 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

Claims (15)

1, the method for screening sars coronavirus main proteinase inhibitor from natural products mixture alternate library, it is characterized in that comprising steps A: the alternative sample in the mensuration natural products mixture alternate library is to the inhibition activity of sars coronavirus main protease, and obtaining has inhibition active " the active sample of inhibition is arranged " to the sars coronavirus main protease; The crystal of sars coronavirus main protease is contacted with " the active sample of inhibition is arranged ", obtain the crystal of sars coronavirus main protease and micromolecular inhibitor complex body.
2, method according to claim 1, it is characterized in that also comprising step B after the steps A: the X-ray diffraction in crystals data of the complex body that obtains among collection and the analytical procedure A obtain the electron density with sars coronavirus main protease bonded micromolecular inhibitor.
3, method according to claim 2, it is characterized in that also comprising step C after the step B: based on the electron density of main protease bonded micromolecular inhibitor that obtain and sars coronavirus among the step B, identify and sars coronavirus main protease bonded micromolecular inhibitor, obtain the fine three dimensional structure of micromolecular inhibitor and sars coronavirus main protease complex body.
4, method according to claim 3 is characterized in that also comprising step D after step C: measure the inhibition activity of micromolecular inhibitor to the sars coronavirus main protease.
5, method according to claim 4 is characterized in that the alternative sample in the wherein said natural products mixture alternate library, is to prepare with the following method:
(a), get a certain amount of primary raw materials, use the ethanolic soln refluxing extraction, united extraction liquid, concentrate medicinal extract;
(b), take by weighing the medicinal extract of gained in a certain amount of step (a), suspend with distilled water, use organic solvent extraction, separate organic solvent phase and water; Merge water, standby; Merge each organic solvent extraction thing, steam the organic solvent that removes wherein, obtain dried paste organic solvent phase sample, standby;
(c), steam to remove a small amount of organic solvent of dissolved in the water of the aqueous portion after the extraction in the step (b), separate with macroporous resin column chromatography, water and ethanolic soln are the moving phase wash-out successively; Discard the washing part, steam the solvent that removes ethanolic soln wash-out part, the shape sample that gets dry extract is standby;
(d), the dried paste sample that makes in dried paste organic solvent phase sample that makes in the above-mentioned steps (b) and the step (c) is 2 alternative samples that are derived from a kind of primary raw materials.
6, method according to claim 5 is characterized in that:
The consumption mass ratio of ethanolic soln and primary raw materials is 6 in the step (a): 1-12: 1; Used ethanolic soln concentration is 70%-95%;
Medicinal extract is 1 with the mass ratio that suspends with distilled water in the step (b): 5-1: 15, and extraction is ethyl acetate or chloroform with organic solvent, its volume is 6 with the volume ratio that suspends with distilled water: 1-10: 1;
Used ethanolic soln concentration is 50% in the step (c).
7, according to the described method of each claim among the claim 1-6, it is characterized in that in steps A: sars coronavirus main protease crystal and " have suppress active sample " are when contact, and the method for using is the crystal immersion process of crystal immersion or cocrystallization.
8, according to the method described in the claim 7, it is characterized in that in steps A: the crystal of sars coronavirus main protease and " have suppress active sample " are when contacting, can be respectively contact with single " the active sample of inhibition is arranged ", also can with contacting that blended " has the active sample of inhibition ".
9, according to Claim 8 in each described method, the alternative sample source in the wherein said natural products mixture alternate library is from Chinese medicine.
10, according to each described method in the claim 9, the alternative sample source in the wherein said natural products mixture alternate library is from following primary raw materials: Herba Houttuyniae, Wild Chrysanthemum, Leaf of Indigowoad, reed rhizome, Japanese Honeysuckle, the capsule of weeping forsythia, the root of large-flowered skullcap, the wind-weed, folium ilicis chinensis, Herba Andrographis, Herba Commelinae, cape jasmine, blackberry lily, Radix Sophorae Tonkinensis, Lasiosphaera fenzlii, radix scrophulariae, Root-bark of Chinese Wolfberry, root, henry rabdosia leaf.
11, according to the crystal of the complex body of each method acquisition among the claim 1-10, it is the crystal of sars coronavirus main protease-root third plain complex body.
12, according to each described method among the claim 1-10, the wherein said micromolecular inhibitor that filters out is root third element.
13, the purposes of root in the medicine of preparation treatment coronavirus infection.
14, the purposes of root third element in the medicine of preparation treatment or prevention of infections by coronaviruses.
15, according to claim 13 or 14 described purposes, wherein said coronavirus is sars coronavirus, transmissible gastro-enteritis virus or avian infectious bronchitis virus.
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CN102247341A (en) * 2007-12-14 2011-11-23 清华大学 Diterpenes natural product inhibitor for main proteases of coronaviruses, such as SARS (Severe Acute Respiratory Syndrome) and the like as well as screening method thereof
CN101497568B (en) * 2009-01-09 2012-08-08 上海交通大学医学院 Use of Sichuang-Tibet amethystoidin A in preventing and treating acute lymphocyte leukaemia and cervical cancer
CN101701245B (en) * 2009-10-21 2013-06-19 中国科学院生物物理研究所 Method for separating SARS coronavirus main proteinase inhibitor from traditional Chinese medicine
CN101921823A (en) * 2010-05-05 2010-12-22 中国科学院生物物理研究所 Method for screening SARS corona virus major protease inhibitor from traditional Chinese medicine and screened SARS corona virus major protease inhibitor
CN101921823B (en) * 2010-05-05 2013-01-02 中国科学院生物物理研究所 Method for screening SARS corona virus major protease inhibitor from traditional Chinese medicine and screened SARS corona virus major protease inhibitor
CN103184264A (en) * 2011-12-29 2013-07-03 天津市国际生物医药联合研究院 HIV type I protease inhibitor screened out from crude extract of Berberis nummularia Bge, and application thereof
CN103184264B (en) * 2011-12-29 2017-05-17 天津市国际生物医药联合研究院 HIV type I protease inhibitor screened out from crude extract of Berberis nummularia Bge, and application thereof
CN104592349A (en) * 2015-02-15 2015-05-06 天津国际生物医药联合研究院 Small molecule inhibitor of coronavirus main proteases as well as preparation method and application of small molecule inhibitor
CN115305245A (en) * 2022-04-29 2022-11-08 山东达因海洋生物制药股份有限公司 Preparation method of small molecule compound and novel coronavirus main protease protein cocrystal

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