CN102247341A - 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 - Google Patents
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 Download PDFInfo
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Abstract
The present invention provides a kind of based on the screening of the external inhibitory activity of SARS, TGEV, AIBV main protease of coronaviruses such as and crystal structure, and the method for Diterpenes natural products inhibitor is screened from henry rabdosia leaf. The activity that there is the present invention compound of following general structural formula can effectively inhibit coronavirus proteolytic.
Description
Technical field
The present invention relates to the screening of medicine, more specifically, relate to vitro inhibition screening active ingredients and crystal structure, the method for screening Diterpenes natural product inhibitor from henry rabdosia leaf based on SARS, TGEV, AIBV main protease of coronaviruses such as.
Background technology
SARS(Severe Acute Respiratory Sydrome) for causing the abbreviation of severe acute respiratory syndrome (severe acute respiratory syndrome), its pathogen 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 and Microbia 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 again: wherein first cohort comprises TGEV, Porcine Transmissible Gastroenteritis Virus (transmissible gastro-enteritis virus) etc.; Second cohort comprises sars coronavirus etc.; The 3rd cohort comprises AIBV, Avian Infectious Bronchitis Virus(avian infectious bronchitis virus) (Spaan such as, 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 (replicase polyproteins) 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 after the proteolytic cleavage of encoding viral is slit into independent subunit, duplicate (Ziebuhr, Snijder et al. 2000; Anand, Yang et al. 2005; Bartlam, Yang et al. 2005).Sars coronavirus major protein enzyme (main protease is called for short main protease) plays a major role in this process.If can suppress the hydrolysis of SARS coronavirus proteolytic, 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 multiformity.Natural product and derivant thereof have been brought into play promising effect in disease treatment in the past, also be one of resource (Newman DJ, Gragg GM, 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 Nat Prod.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 chemical 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 a micromolecular compound, 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.
Summary of the invention
An object of the present invention is to provide and a kind ofly can effectively suppress the active Traditional Chinese Herb inhibitor of coronavirus proteolytic.
Another purpose of the present invention provide described Traditional Chinese Herb inhibitor preparation be used for the treatment of or the medicine of prevention of infections by coronaviruses in purposes.
Another object of the present invention provides a kind of method of screening the coronavirus proteolytic inhibitor.
A kind of method of screening the coronavirus proteolytic inhibitor provided by the invention comprises the steps:
A, the crystal of coronavirus proteolytic is contacted immersion with alternative sample, obtain the crystal of coronavirus proteolytic and micromolecular inhibitor complex.
The X-ray diffraction in crystals data of the complex that obtains among B, collection and the analytical procedure A obtain the electron density with the bonded micromolecular inhibitor of coronavirus proteolytic.
C, based on the electron density of that obtain and the bonded micromolecular inhibitor of main protease coronavirus among the step B, identify and the bonded micromolecular inhibitor of coronavirus proteolytic, obtain the fine three dimensional structure of micromolecular inhibitor and coronavirus proteolytic complex.
D, measure the inhibition activity of micromolecular inhibitor to coronavirus proteolytic.
The alternative sample that the present invention is used, preferably preparation with the following method:
(a), get a certain amount of primary raw materials, use the alcoholic solution reflux, extract,, merge extractive liquid,, concentrate extractum;
(b), take by weighing the extractum 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 dissolved a small amount of organic solvent in the water of the aqueous portion after the extraction in the step (b), separate with macroporous resin column chromatography, water and alcoholic solution are the mobile phase eluting successively; Discard the washing part, steam the solvent that removes alcoholic solution eluting 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.
Primary raw materials used among the present invention is preferably henry rabdosia leaf.
Each alternative sample of henry rabdosia leaf of the present invention can be prepared as follows:
Get the henry rabdosia leaf medical material of 500 gram drying and crushing,, extract 3 times with 4500 milliliter of 95% alcohol reflux, each 2 hours, merge extractive liquid,, concentrated extractum.
Take by weighing this extractum 20.5 grams, suspend, in 1000 milliliters of separatory funnels of impouring, use ethyl acetate extraction 3 times with 150 milliliters of distilled waters, the each extraction with 650 milliliters of ethyl acetate, shake well leaves standstill layering in 6-10 hour.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 henry rabdosia leaf ethyl acetate extraction sample that gets dry extract is standby.
Dissolved amount of ethyl acetate during aqueous portion after the extraction dewaters with the steaming of 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 * 300 mm), water, 50% ethanol are the mobile phase eluting successively.Elder generation's water eluting 8 times, 250 milliliters of each eluting discard the washing part.Use 50% ethanol elution 5 times then, 300 milliliters of each eluting merge the eluent that each time 50% ethanol elution obtains.Partly use EYELA N1001 type Rotary Evaporators to steam 50% ethanol elution and desolventize, the shape henry rabdosia leaf water macroporous resin 50% ethanol elution sample that gets dry extract, standby.
Above-mentioned ethyl acetate extraction sample and water macroporous resin 50% ethanol elution sample are henry rabdosia leaf to the active alternative sample of sars coronavirus main protease vitro inhibition.
The X-ray diffraction in crystals data collection can adopt following method with arrangement among the step B:
At first use the nylon crystal rings of Hampton Research company, from the crystal soak, obtain the crystal that soaks certain hour (soak time was controlled between 2-48 hour), and use the cooling system of Rigaku company or the cooling system of Oxford Cyrosystem company rapidly, crystal is refrigerated to subzero 150 ℃-180 ℃ in the cryogenic nitrogen air-flow that above-described two kinds of refrigeration systems produce; Make X ray pass through crystal, use the precession method to collect X ray diffracting data.
Collecting after the crystal of target proteins matter and the X-ray diffraction in crystals data after the natural products mixture alternate sample contacts, need carry out corresponding date processing according to following step:
At first, use the method for date processing commonly used known in the art such as (comprising Mosflm, D*trek etc.) diffraction data handling procedure bag such as HKL2000, previous step is collected the diffraction data that obtains in rapid handle, obtain complete data file;
Secondly, use the data processing methods commonly used known in the art such as various molecular replacements such as Phaser, Molrep (Molecular Replacement) program in CNS or the CCP4 program package, utilize the method for molecular replacement (Molecular Replacement), parent crystal structure with target proteins is an initial model, obtains the fine three dimensional structure of the complex of candidate's target proteins and target molecule;
Once more, the method for date processing commonly used known in the art such as visualization procedure bags such as service routine O, COOT, XtalView is observed the electron density of possible target proteins and inhibitor or part micromolecule complex.If find this moment, at the corresponding avtive spot of target proteins, there is the unknown electron density that does not belong to protein molecule itself, that do not belong to solvent molecule, do not belong to the molecule that exists in the crystallization solution to exist, and the aminoacid of the avtive spot of this electron density and target proteins has generation to comprise rational chemical environments such as hydrogen bond action, covalent effect, then can confirm in this natural products mixture alternate sample have ligand moleculars such as inhibitor micromolecule to combine with target proteins.
Coronavirus among the present invention comprises sars coronavirus, transmissible gastro-enteritis virus or avian infectious bronchitis virus.The present invention also respectively separates the vitro inhibition determination of activity that section (sample that promptly separates the various piece obtain with methods such as extraction, macroporous resins), Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element have carried out TGEV, AIBV main protease of coronaviruses such as to henry rabdosia leaf.
Because contained composition is quite complicated in the henry rabdosia leaf, therefore must be optimized its crude extract, purpose is to remove interference factor 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 micromolecule and make it as far as possible to concentrate preferably, improve its relative concentration, be applicable to that with preparation with sars coronavirus main protease crystal structure be the alternative sample that inhibitor screening is carried out on the basis.The present invention not only provides and can realize that this purpose prepares the technical scheme of alternative sample, and screening is had the active sample of inhibition, separates, identified the wherein structure of micromolecular inhibitor-Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element with various chromatographs, spectral method.
At present, be the basic drug screening of carrying out based on sars coronavirus main protease crystal structure, the micromolecular inhibitor of being found is most to be sars coronavirus main protease substrate analogue and derivant (Dariusz P. thereof, Marcin H., Marcin G., et al. Chem. Biol. Drug. Res., 2007,69:269-279; Haitao Y., Wenqing X., Xiaoyu X., et al. PLOS Biology, 2005,3 (10): 1742-1752), this class peptide inhibitor not only involves great expense, and oral availability is low, and the half-life in blood is short, and uniform SARS infection morbidity has been brought difficulty.The inhibitor that the present invention filters out-Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element, for not containing the Diterpenes natural product of peptide bond, it still demonstrates stronger inhibition activity to the sars coronavirus main protease when the concentration of 20 μ M, is fabulous medicine or potential prodrug.In addition, we find by the fine three dimensional structure of analyzing the plain crystalline composites of sars coronavirus main protease and Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first, can be combined in the avtive spot of sars coronavirus main protease with some diterpene-kind compounds of the plain similar of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first, suppress active thereby produce.This is for further drug design and screening provide valuable foundation and reference.
The present invention finds that Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element is effectively to suppress the active inhibitor of sars coronavirus main protease.
The present invention finds that henry rabdosia leaf can be treated coronavirus such as sars coronavirus, transmissible gastro-enteritis virus or avian infectious bronchitis virus infect.
The present invention finds that Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element can treat or prevent coronavirus such as sars coronavirus, transmissible gastro-enteritis virus or avian infectious bronchitis virus to infect.
The present invention finds that the chemical compound with following general structure (I) can combine and produce inhibitory action with the sars coronavirus main protease.The pharmaceutical composition that the chemical compound of following general structure (I) or the acceptable salt of its pharmacy or solvate and one or more pharmaceutically acceptable carriers are formed can be used for treatment or prevention coronavirus such as sars coronavirus, transmissible gastro-enteritis virus or avian infectious bronchitis virus to be infected.
R=H wherein, OH, OAc, OCH
3, OCH
2CH
3, OCH (CH
3)
2, OXO (wherein OAc represents acetoxyl group, and on behalf of this position, OXO form carbonyl).Consult document, the natural product of having found that meets above-listed general formula has following array structure:
Description of drawings
Fig. 1 is the activity inhibition curve chart of henry rabdosia leaf screening sample of the present invention to the sars coronavirus main protease.Sample concentration: 100
μG/mL.1: contrast (activity curve that does not add the sars coronavirus main protease of any sample) 2: henry rabdosia leaf water macroporous resin 50% ethanol elution sample suppresses curve 3 to the activity of SARS main protease: henry rabdosia leaf total extract sample suppresses curve 4 to the activity of SARS main protease: henry rabdosia leaf ethyl acetate extraction sample suppresses curve to the activity of SARS main protease
Fig. 2 is near the electron density comparison diagram the sars coronavirus main protease avtive spot of the present invention.The left side has shown the electron density of sars coronavirus main protease parent crystal avtive spot; The right has shown the electron density of the crystal avtive spot after sars coronavirus main protease and henry rabdosia leaf ethyl acetate extraction sample soak.Electron density shown in the figure, black are 2
FofcElectron density is 1.0 sigma; Redness is 1
FofcThe difference electron density is 2.5 sigma
Fig. 3 has shown the electron density of the crystal avtive spot after sars coronavirus main protease of the present invention and henry rabdosia leaf acetic acid ethyl ester extract segmentation sample a1 soak.
Fig. 4 has shown the electron density of the crystal avtive spot after sars coronavirus main protease of the present invention and henry rabdosia leaf acetic acid ethyl ester extract segmentation sample a2 soak.
Fig. 5 has shown the electron density of the crystal avtive spot after sars coronavirus main protease of the present invention and henry rabdosia leaf acetic acid ethyl ester extract segmentation sample a3 soak.
Fig. 6 has shown the electron density of the crystal avtive spot after sars coronavirus main protease of the present invention and henry rabdosia leaf acetic acid ethyl ester extract segmentation sample a4 soak.
Fig. 7 has shown the electron density of the crystal avtive spot after sars coronavirus main protease of the present invention and henry rabdosia leaf acetic acid ethyl ester extract segmentation sample a5 soak.
Fig. 8 has shown the electron density of the crystal avtive spot after sars coronavirus main protease of the present invention and henry rabdosia leaf acetic acid ethyl ester extract segmentation sample a6 soak.
Fig. 9 has shown the electron density of the crystal avtive spot after sars coronavirus main protease of the present invention and henry rabdosia leaf acetic acid ethyl ester extract segmentation sample a7 soak.
Figure 10 is the HPLC chromatogram of henry rabdosia leaf acetic acid ethyl ester extract segmentation sample a3 of the present invention and a4 assembling section.
Figure 11 is the ESIMS figure of chemical compound 1 of the present invention.
Figure 12 is a chemical compound 1 of the present invention
1H NMR spectrum.
Figure 13 is a chemical compound 1 of the present invention
13C NMR spectrum.
Figure 14 is the HMQC spectrum of chemical compound 1 of the present invention.
Figure 15 is the HMQC spectrum part sectional drawing of chemical compound 1 of the present invention.
Figure 16 is the HMBC spectrum of chemical compound 1 of the present invention.
Figure 17 is the HMBC spectrum part sectional drawing of chemical compound 1 of the present invention.
Figure 18 is the activity inhibition curve chart of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element of the present invention to the sars coronavirus main protease.1: contrast (activity curve that does not add the sars coronavirus main protease of any sample); 2:20
μThe Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element of M suppresses curve to the activity of sars coronavirus main protease; 3:200
μThe Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element of M suppresses curve to the activity of sars coronavirus main protease.
Figure 19 has shown sars coronavirus main protease of the present invention and chemical compound 1(Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element) electron density of crystal avtive spot after soaking.Electron density shown in the figure, black are 2
FofcElectron density is 1.0sigma; Redness is 1
FofcThe difference electron density is 2.5 sigma.
Figure 20 is the figure that Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element of the present invention and sars coronavirus main protease avtive spot Cys145 residue form covalent bond.Protein molecule represents that with white ribbon figure the plain covalent bond that forms of the Cys145 residue of avtive spot and Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first is represented with ball-and-stick model; The electron density of blue expression Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element.
Figure 21 Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first of the present invention element suppresses curve chart to the activity of the main protease of transmissible gastro-enteritis virus (TGEV).1: contrast (activity curve that does not add the TGEV main protease of any sample); 2:100
μThe Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element of M suppresses curve to the activity of TGEV main protease.
Figure 22 Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first of the present invention element suppresses curve chart to the activity of the main protease of avian infectious bronchitis virus (AIBV).1: contrast (activity curve that does not add the AIBV main protease of any sample); 2:100
μThe Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element of M suppresses curve to the activity of AIBV main protease.
Figure 23 is the binding site figure of sars coronavirus main protease of the present invention and Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element.Wherein the carbon atom of plain 17 of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first forms covalent bonds with 145 cysteine of sars coronavirus main protease, 141 leucines of hydrogen on plain 12 carbon atoms of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first and sars coronavirus main protease and 144 serine formation hydrogen bond.
The present invention provide specific embodiments of the invention in order to explain in further detail below in conjunction with accompanying drawing.Should be appreciated that following embodiment is only used for explaining the present invention, and not should be understood to limit the scope of the invention by any way.
The specific embodiment
1. the expression of sars coronavirus main protease, purification and crystal growth
The sars coronavirus main protease is carried out further separation and purification and crystallization (referring to Yang H et al. 2003. The Crystal Structures of SARS Virus Main Protease Mpro and Its Complex with an Inhibitor. in coli strain BL21 (DE3)
PNAS, 100 (23): 13190-13195).
The preparation of the alternative sample of henry rabdosia leaf
Get the henry rabdosia leaf medical material of 500 gram drying and crushing,, extract 3 times with 4500 milliliter of 95% alcohol reflux, each 2 hours, merge extractive liquid,, concentrated extractum.
Take by weighing this extractum 20.5 grams, suspend, in 1000 milliliters of separatory funnels of impouring, use ethyl acetate extraction 3 times with 150 milliliters of distilled waters, the each extraction with 650 milliliters of ethyl acetate, shake well leaves standstill layering in 6-10 hour.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 henry rabdosia leaf ethyl acetate extraction sample that gets dry extract is standby.
Dissolved amount of ethyl acetate during aqueous portion after the extraction dewaters with the steaming of 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 * 300 mm), water, 50% ethanol are the mobile phase eluting successively.Elder generation's water eluting 8 times, 250 milliliters of each eluting discard the washing part.Use 50% ethanol elution 5 times then, 300 milliliters of each eluting merge the eluent that each time 50% ethanol elution obtains.Partly use EYELA N1001 type Rotary Evaporators to steam 50% ethanol elution and desolventize, the shape henry rabdosia leaf water macroporous resin 50% ethanol elution sample that gets dry extract, standby.
Above-mentioned ethyl acetate extraction sample and water macroporous resin 50% ethanol elution sample are henry rabdosia leaf to the active screening sample of sars coronavirus main protease vitro inhibition.
The alternative sample of henry rabdosia leaf 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 320 nm and 405 nm to Fluoraskan Ascent luminoscope for ThermoLabsystems, Helsinki.
The natural products mixture alternate sample of 2 dried pastes of preparation in the step 2 is dissolved in the dimethyl sulfoxide (DMSO), and making its final concentration is 50 mg/mL, makes the DMSO solute of 2 natural products mixture alternate samples respectively.
At buffer solution (50mM Tris-HCl(pH 7.3), add sars coronavirus main protease (final concentration 0.5 μ M) among the 1mM EDTA, the DMSO solute (as the DMSO solute of henry rabdosia leaf ethyl acetate extraction sample) that adds above-mentioned natural products mixture alternate sample makes its final concentration be: 100 μ g/mL, above the fluorogenic substrate concentration of sars coronavirus main protease is 20 mM, 298 K placed after 10 minutes, add fluorescent labeling substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH2, final concentration 20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 320 nm and 405 nm, and temperature keeps 298 K, write 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.The henry rabdosia leaf acetic acid ethyl ester extract has stronger inhibition activity as shown in Figure 1, illustrates that the micromolecular inhibitor in the henry rabdosia leaf mainly is present in the ethyl acetate extraction sample.
Sars coronavirus main protease crystal soaks henry rabdosia leaf ethyl acetate extraction sample
Owing to (therefore 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 micromolecule, be referred to as 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 grown crystal, therefore, the method that mainly adopts crystal to soak obtains the crystal of sars coronavirus main protease and micromolecular inhibitor complex.
In order to guarantee on the basis of crystal mass as much as possible, improve when soaking the concentration of micromolecular compound in the henry rabdosia leaf ethyl acetate sample, can adopt following two kinds of methods to prepare the crystal soak of sars coronavirus main protease:
Method one, the DMSO solute of henry rabdosia leaf ethyl acetate extraction sample (according to the preparation of the method in the step 3) is diluted to sars coronavirus main protease crystal growth pond liquid (1.8～12% PEG 6000 for 10 times, 3% dimethyl sulfoxide (DMSO), 1mM DTT, in the 100 mM MES buffer (pH 5.0～PH6.0)), with the 13000-15000rpm high speed centrifugation, get supernatant afterwards, get soak 1, standby;
Method two, the henry rabdosia leaf ethyl acetate extraction sample of dried paste directly is dissolved in sars coronavirus main protease crystal growth pond liquid (1.8～12% PEG 6000,3% dimethyl sulfoxide (DMSO), 1mM DTT, in the 100 mM MES buffer (pH 5.0～PH6.0)), with the 13000-15000rpm high speed centrifugation, get supernatant afterwards, get soak 2, standby;
When guaranteeing that crystal soaks, be unlikely to make the micromolecular compound excessive concentration in the henry rabdosia leaf ethyl acetate extraction sample and influence crystalline diffraction quality, soak 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 1 and 2 respectively and in corresponding 10 times of diluents, soaked 2-48 hour.
The Collection and analysis of crystal diffraction data
On the basis of step 4, use possesses better diffracting power, and (resolution is preferably greater than 2.6
) crystal, carry out the Collection and analysis of X ray diffracting data.
When carrying out data collection, at first use the nylon crystal rings of Hampton Research company, from soak 1 and soak 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, crystal is refrigerated to subzero 150 ℃-180 ℃ in the cryogenic nitrogen air-flow that above-mentioned refrigeration system produces; Make X ray pass through crystal, use the precession method to collect X ray diffracting data.
(resolution is preferably greater than 2.6 to select the measured crystal of diffraction matter
) carry out data collection, processing, whether detect has micromolecular combination.The statistics of data collection such as following table 1:
Complex data collection statistics after table 1:SARS coronavirus proteolytic parent, sars coronavirus main protease and henry rabdosia leaf acetic acid ethyl ester extract sample soak
By the sars coronavirus main protease parent of observation equal angular and the avtive spot electron density map (Fig. 2) (black is the electron density of protein itself) of complex, can find, can observe the electron density of nonprotein itself (red expression) clearly at the avtive spot of complex, show that having micromolecular compound is combined on the protein molecule.What left side figure showed is near the electron density of avtive spot of sars coronavirus main protease precursor structure, and the right figure is that sars coronavirus main protease and henry rabdosia leaf ethyl acetate extraction sample soak near the electron density of back crystal avtive spot.
Examine the electron density of the avtive spot of complex, we can find to be present in unknown micromolecular electron density in the henry rabdosia leaf 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 micromolecule and Cys145 has formed covalent bond, thereby suppressed the activity of sars coronavirus main protease.
In conjunction with micromolecular analysis and evaluation
In order to determine to be present in the chemical constitution of the unknown micromolecular inhibitor in the henry rabdosia leaf acetic acid ethyl ester extract sample, we have carried out analyzing and processing to henry rabdosia leaf acetic acid ethyl ester extract sample again.Concrete steps are as follows:
Henry rabdosia leaf acetic acid ethyl ester extract (9.2 gram) is used dissolve with methanol, admix 25 gram silica gel (column chromatography 160-200 order), separate (180 gram column chromatography silica gel 160-200 order) with silica gel column chromatography, with chloroform-acetone system is mobile phase, carry out eluting with chloroform, chloroform-acetone (30:1), chloroform-acetone (20:1), chloroform-acetone (15:1), chloroform-acetone (10:1), chloroform-acetone (7:1), acetone respectively, each gradient elution 5-10 retention volume.Detect with thin layer chromatography, merge and form similar receiving unit.Wherein chloroform-acetone (30:1) eluting part detects the speckle that contains more 10% ethanol solution of sulfuric acid heating colour developing through thin layer chromatography, and has removed most of pigment.This chloroform-acetone (30:1) eluting is partly continued to separate with Sephadex LH-20 gel filtration chromatography, is the mobile phase eluting with chloroform-methanol (1:1), receives altogether to have merged 7 part: a1-a7.
Each part of a1-a7 is contacted with sars coronavirus main protease crystal respectively, and carry out X-ray diffraction, data are carried out collection and treatment, obtain near 7 parts of sars coronavirus main protease crystal avtive spots electron density map (referring to Fig. 3-Fig. 9).By Fig. 3-Fig. 9 as can be known: a3 and a4 part mainly contain tool and suppress active micromolecule, so a3 and a4 two parts are merged, analyze (chromatographic column: YMC ODS-A with HPLC, 150 * 4.6 mm, 10 μ m, analysis condition: 0-10 minute, methanol-water (50:50) eluting; 10-25 minute, the methanol-water (gradient elution of 50:50 → 75:25); 25-40 minute, methanol-water (75:25 → 100:0) gradient elution), analysis result is seen Figure 10.
As shown in Figure 10, the pairing chemical compound of the chromatographic peak that retention time is the longest (compile and be chemical compound 1), it is main contained chemical compound in a3 and the a4 assembling section, screening sample just shows stronger inhibition activity under very low concentration because enzyme is lived, and chemical compound 1 is exactly the sars coronavirus main proteinase inhibitor probably.A3 and a4 assembling section are separated with half preparation HPLC (chromatographic column: YMC ODS-A, 150 * 10 mm, 10 μ m), and (44:56) is the mobile phase eluting with methanol-water, obtains pure product chemical compound 1(3 milligram).The chemical constitution of chemical compound 1 through the various Wave Spectrum data of integrated use (comprise ORD, ESIMS,
1H NMR,
13C NMR, HMQC, HMBC sees Figure 11-Figure 17) be accredited as 3 β, 7 α, 14 β-trihydroxy-mapping-kaur-16-ene-15-ketone, i.e. Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element (wangzaozin A).The structure and the spectral data of chemical compound 1 are summarized as follows:
Chemical compound 1: white unformed solid, [α]
D 20-61.6 °
(c0.10, MeOH);
ESIMS?
m/z?357?[M?+?Na]
+,?691?[2M?+?Na]
+,?1025?[3M?+?Na]
+；
1H NMR (MeOH-
d 4 , 500 MHz)
δ6.03 and 5.35 (each 1H, br s, H
2-17), 4.83 (1H, H-14), 4.14 (1H, dd,
J=11.5,4.0 Hz, H-7), 3.31 (1H, br s, H-3), 1.06 (3H, s, Me-20), 0.92 (3H, s, Me-18), 0.84 (3H, s, Me-19);
13C?NMR?(MeOH-
d 4 ,?125?MHz)?
δ?33.9?(C-1),?26.3?(C-2),?76.5?(C-3),?40.8?(C-4),?47.6?(C-5),?29.5?(C-6),?75.4?(C-7),?62.4?(C-8),?56.1?(C-9),?38.4?(C-10),?18.3?(C-11),?32.3?(C-12),?47.2?(C-13),?76.1?(C-14),?208.8?(C-15),?149.7?(C-16),?117.7?(C-17),?29.1?(C-18),?22.6?(C-19),?18.4?(C-20)；
Its
1H NMR,
13C NMR data and Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first prime number are according to (Wang Xianrong, Wang Zhaoquan, stone distance of travel of roc etc.The new diterpene of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen-Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element.Anhui medical science, 1982,2:50-53) in full accord.
, Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element suppresses active mensuration to the main protease of sars coronavirus
We use and separate the pure product chemical compound micromolecule (Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element) (its source, such as certain producer commodity when please be indicated if special) that obtains, and have measured its inhibition activity to the sars coronavirus main protease.Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element suppresses active mensuration to the sars coronavirus main protease to carry out according to following steps: at buffer solution (50mM Tris-HCl (pH 7.3), 1mM EDTA(contains or does not contain DTT)) the middle sars coronavirus main protease (final concentration 0.5 μ M) that adds, (final concentration is Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element: 200 μ M), the fluorogenic substrate concentration of sars coronavirus main protease is 20mM, 298 K placed after 10 minutes, add fluorescent labeling substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH2, final concentration 20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 320 nm and 405 nm, and temperature keeps 298 K, write down the first order fluorescence reading per 2 seconds.Measure its inhibition activity at 20 μ M again then to the sars coronavirus main protease.Contrast: do not add alternative sample, all the other conditions are identical.Determination of activity the results are shown in Figure 18.Can find that from Figure 18 the Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element of 200 μ M has stronger inhibition activity to the sars coronavirus main protease, when its concentration is 20 μ M, the sars coronavirus main protease still be had the activity of inhibition.This Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element that just illustrates that Chinese medicine henry rabdosia leaf the inside is contained can effectively suppress the plain active drug for the treatment sars coronavirus of activity, Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first of sars coronavirus main protease.
Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element and sars coronavirus main protease compound crystal are through X-ray diffraction, data are carried out finding after the collection and treatment, the same with the henry rabdosia leaf acetic acid ethyl ester extract with sars coronavirus main protease compound crystal, can observe the electron density of nonprotein itself (Figure 19-20) clearly at the avtive spot of complex, and the electron density of micromolecular electron density and the sars coronavirus main protease avtive spot Cys145 of place is closely linked, show that this should be that the plain γ sulphur atom with Cys145 of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first has formed covalent bond, thereby suppressed the activity of sars coronavirus main protease.
, Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element suppresses active mensuration to the main protease of transmissible gastro-enteritis virus (TGEV)
At buffer (20 mM Tris-HCl pH 7.0,1 mM DTT) in, the main protease (0.5 μ M) that adds transmissible gastro-enteritis virus, Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element (20 μ M), 298 K placed after 10 minutes, add fluorescent labeling substrate (MCA-AVLQSGFRL (DNP) L-NH2,20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 320 nm and 405nm, and temperature keeps 298 K, write down the first order fluorescence reading per 2 seconds.Change inhibitor concentration then, under 200 μ M, measure and suppress active.Contrast: do not add inhibitor, all the other conditions are identical.The results are shown among Figure 21.
The expression and purification list of references of TGEV: Conservation of substrate specificities among coronavirus main proteases. J. Gen. Virol. 2002; 83 (Pt 3): 595-9.
. Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element suppresses active mensuration to the main protease of avian infectious bronchitis virus (AIBV)
At buffer (20 mM Tris-HCl pH 7.0,1 mM DTT) in, the main protease (1 μ M) that adds avian infectious bronchitis virus, Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element (100 μ M), 298 K placed after 10 minutes, add fluorescent labeling substrate (MCA-AVLQSGFRL (DNP) L-NH2,20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 320 nm and 405 nm, and temperature keeps 298 K, write 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 22.
The expression and purification list of references of AIBV: Preliminary crystallographic analysis of avian infectious bronchitis virus main protease.Acta.Cryst.F.2007; 63 (Pt1): 24-6.
By Figure 18, Figure 21 and Figure 22 as can be seen, Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element all has the main protease of SARS, TGEV, AIBV and suppresses active, 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 Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element all has the main protease of whole coronavirus family and suppress active.
, based on the Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first plain with sars coronavirus main protease complex fine three dimensional structure, carry out the drug design of similar diterpene-kind compound
By anatomizing the plain and sars coronavirus main protease complex fine three dimensional structure of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first, we have obtained the binding site figure (Figure 23) of sars coronavirus main protease and Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element.Wherein the carbon atom of plain 17 of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first forms covalent bonds with 145 cysteine of sars coronavirus main protease, 141 leucines of hydrogen on plain 12 carbon atoms of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first and sars coronavirus main protease and 144 serine formation hydrogen bond.Because one of catalysis disome that 145 cysteine are sars coronavirus main protease functionatings, therefore both this kind effects have not only caused the inactivation of 145 cysteine catalytic action, and the hydrogen on 12 carbon atoms and the hydrogen bond action of other residues of protein make Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element more closely be combined in the active pocket of sars coronavirus main protease, finally caused the active inhibition of sars coronavirus main protease.Can reasonably infer to have this type of mapping-kaurane diterpene chemical compound with the plain similar structures of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first, also have similar even stronger inhibitory action.Therefore, active according to Figure 23 to mapping-kaurane diterpene chemical compound inhibition of similar Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first element, can draw following results: owing to will form covalent bonds, α with 145 cysteine, the structure of alpha, beta-unsaturated ketone (i.e. two keys of 15 carbonyl and 16,17) is essential; If 12 have the oxygen of containing to replace,, may form hydrogen bond by easier hydroxyl with 141 leucic carbonyls or 144 serines as hydroxyl and carbonyl; Because the other parts in the plain structure of Isodon amethystoides (Benth.) C.Y.Wuet Hsuen first are especially failed and protein residues generation effect from protein residues space length part far away, therefore 3,7 and 14 s' substituent group can have tiny variation not influence to suppress active.According to above-mentioned conclusion, can write out following general structure (I)
R=H wherein, OH, OAc, OCH
3, OCH
2CH
3, OCH (CH
3)
2, OXO (wherein OAc represents acetoxyl group, and on behalf of this position, OXO form carbonyl).Consult document, the natural product of having found that meets above-listed general formula has following array structure:
Among the related herein various experimental articles (including but not limited to: chemical reagent, biological product, cell, organism, instrument etc.), special or be difficult for obtaining for those, Wen Zhongjun has indicated manufacturer, list of references 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.
Should be noted that the above-listed natural product of having found combines and produce inhibitory action with the chemical compound that meets above-mentioned general formula with the sars coronavirus main protease, therefore it should be understood that they all should fall within protection scope of the present invention.
Claims (2)
1. the purposes of henry rabdosia leaf in the medicine of preparation treatment coronavirus infection.
2. purposes according to claim 1, wherein said coronavirus are sars coronavirus, transmissible gastro-enteritis virus or avian infectious bronchitis virus.
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