CN100465643C

CN100465643C - Novel methods for high efficiency and rapid getting fine three dimensional structure of target protein composite body and target molecule target molecule

Info

Publication number: CN100465643C
Application number: CNB2007100874570A
Authority: CN
Inventors: 饶子和; 娄智勇; 孙玉娜; 马明
Original assignee: Tsinghua University; Institute of Biophysics of CAS; Nankai University
Current assignee: Tsinghua University; Institute of Biophysics of CAS; Nankai University
Priority date: 2007-03-19
Filing date: 2007-03-19
Publication date: 2009-03-04
Anticipated expiration: 2027-03-19
Also published as: CN101063682A

Abstract

This invention provides one effective and rapid method to extract target molecule and target compound find three dimensional structure from Chinese medicine product, which uses target protein transistor stricture as base and uses X ray transistor means to filter the product for active component and for study from one new respective.

Description

Therefrom obtain the new method of target molecule and target protein complex fine three dimensional structure in the medicines natural products mixed system efficiently, fast

Technical field

The present invention relates to obtain efficiently, fast in a kind of therefrom medicines natural products mixed system (natural products mixture alternate library) new method of target molecule and target protein complex fine three dimensional structure, belong to the drug screening method field.

Background technology

Natural products claims secondary metabolite (Secondary Metabolites) again, has diversity structure and bioactive diversity.Natural products and derivant thereof have been brought into play promising effect in disease treatment in the past, also be one of resource of tool potentiality in the current medicament research and development process (Newman DJ waits people Nat Prod Rep, 2000,17 (3): 215-234; Lee K-H.J NatProd.2004,67 (2): 273-283).But, in the past in 10 years, pharmaceuticals industry decreases to the research interest of natural products, this mainly be since traditional natural products crude extract storehouse and conventional high-throughout screening technique lack compatible (Frank E.and Guy T.Nature Rev.DrugDiscov.2005,4:206-220).

The appearance of the eighties of last century combinatorial chemistry nineties (Combichem) makes that the research of finding new drug from natural products has been treated coldly for many years.The compound that combinatorial chemistry can synthesize huge amount (has 10 in theory according to estimates ¹⁸To 10 ²⁰⁰Individual compound is commonly referred to be 10 ⁶⁰Individual), this just provides desirable screening compounds storehouse for the high-throughout screening technique of routine, therefore, the appearance of this technology of combinatorial chemistry, make most of drugmaker all turn on the high flux screening route based on combinatorial chemical library, they expect to adopt this technological means can accelerate the discovery speed of reactive compound greatly.Yet the new drug output capacity of pharmaceuticals industry does not in recent years improve as expection, but continues to fall.Cause this result's reason a lot, a most important reason may be exactly because after the combinatorial chemistry technique appearance, pharmaceuticals industry begins to adopt the synthetic compound library that contains huge amount of combinatorial chemistry to carry out high-throughout screening, therefore (the people .J.Nat.Prod.2003 such as David J. that decreases of the interest to the natural products research and development, 66,1022-1037).Combinational chemistry is from common stay in place form, selects to have the multiple establishment module of identical function, by the molecular diversity of key reaction realization of the same race.The structural framework lack of diversity of He Cheng compound like this.Therefore, combinatorial chemistry still has its limitation at present as the instrument of drug discovery, remains to be further developed and perfect.And the unique effect of natural products in medicament research and development causes people's attention in recent years again.

In decades, natural products is the important source of medicine and lead compound always, and natural products plays an important role in the discovery of new drug and lead compound.The nearest investigation of the U.S. state-run cancer research institute shows that in 877 kinds of micromolecule new chemical entities medicines of listing in 1981-2002 years, what derive from natural products accounts for 61%.Wherein, 6% is directed to natural products, and 27% is the derivant of natural products, 5% derives from the complex of natural products pharmacophore, the 23%th, according to the natural products structure carry out board design complex (people .J.Nat.Prod.2003 such as David J., 66,1022-1037).

At present, the natural products storehouse that is used for drug screening has three kinds of forms, and the one, natural products crude extract storehouse is compound library, includes 10-100 kinds of components; The 2nd, the natural product extraction component, half pure product storehouse includes 5-10 kinds of components; The 3rd, pure product storehouse, one-component (Frank E.and Guy T.NatureReviews Drug discovery.2005,4:206-220).

For the pure product of natural products storehouse, the detection method of hitting thing (hit) is identical with the pure product storehouse of artificial chemosynthesis, can be used for conventional high-throughout screening technique, therefore, the hardworking separation and purification work of carrying out natural products of current many chemists.Yet, for each natural products crude extract, separate the pure product compound that obtains its every kind composition, need long cycle and a large amount of work, and have tangible blindness, the accuracy that obtains active compound is very low.

For natural products crude extract storehouse, be simple and easy to, but with the poor reliability that conventional high-throughout screening technique screens, this is mainly reflected in following aspect: compound library composition concentration complicated and its each composition is also uncertain, might differ 10 times, 100 times even more.Therefore, for the low component of those concentration, may in the high-throughout screening active ingredients of routine, be left in the basket, and the more rich component of content might show nonspecific inhibition, such as the pH value of destroying detection architecture or other physical characteristics, cause false positive results (Frank E.and Guy T.Nature Reviews Drugdiscovery.2005,4:206-220).And of the present inventionly can overcome this shortcoming by the method for using biophysics such as X-ray crystallography, use this technology can detect compound than low-affinity is arranged with target proteins, therefore make natural products crude extract storehouse can be applied in the screening technique of X-ray crystallography, improve screening effeciency greatly, and the raising hit rate, be the revolutionary character progress of current medicament research and development.

Especially the research that derives from the natural products of Chinese medicine etc. for natural products is necessary, because Chinese medicine is the treasure-house of a greatness in the history that China used in existing thousands of years.But traditional Chinese medicine in use, and many methods that decoct with compound medicine, employing because the effective constituent relative content is lower, often need to take for a long time, just can obtain certain curative effect, and for a lot of acute diseases, the effect of Chinese medicine are relatively poor; And, because the traditional Chinese medicine composition is extremely complicated, with decoction for example after modes such as decoction are used, wherein chemical constitution may produce new variations such as chemical combination or decomposition again, be difficult to determine concrete which kind of composition in action, thereby uncontrollable medicinal material is planted, medicinal material processing, and drafts the quality standard behind the patent medicine, brings difficulty also for pharmacology, clinical trial.

But often content is humble for the complex chemical composition that Chinese medicine is contained, effective constituent.The separation of chemical composition of Chinese materia medica, purifying and evaluation then need to use repeatedly various chromatographic processes and multiple spectral method to go to finish, the chemical constitution study of Chinese medicine has been the work that the cycle is long, workload is big simply, and this is still only for the relatively large composition of those content.Seek effective constituent in the clearly various compositions of structure the inside and need pure product are carried out biological activity test in various external, the bodies again, this is a kind of filtering mode of blindness in fact, the pure product compound that often most of separation obtain is invalid, and the problem that this simple biological activity test model can illustrate also is limited.Even we can determine some effective constituents, these compositions are how some disease to be played therapeutic action so, they are how by with the interaction of body body being regulated, and, clear and definite elaboration is in detail arranged seldom almost at present about interaction of biomacromolecules details in these micromolecule compositions and the body.

Fast development along with genomics, bioinformatics etc. has produced the drug screening method based on molecular biology and structure biology.The genome plan of the mankind and microorganism provides quantity unprecedented potential drug target, only after the working draft of human genome is finished less than just having found 1778 disease genes in the time in 1 year.These work have promoted the research of protein expression (proteomics) and protein structure (structure biology), with the identification of study of human disease-related gene position, identify and the research of the structure of expressed protein and function is drug screening and find to have opened up limitless prospect, find to have brought opportunity to new drug.Along with further developing of science and technology, the crystal structure that the researcher begins with protein is the drug screening that lead compound is carried out on the basis, and this class screening technique mainly comprises:

(1) based on the virtual drug screening and the design (computer assisted drug design) of protein three-dimensional structure

The development of molecular biology and structure biology provides the three-dimensional structure of many target proteins matter, therefore, according to complementary principle, is widely used according to the drug design method of the three-dimensional structure of protein.It is different with traditional pharmaceutical chemistry method, is the three-dimensional structure according to protein, finds suitable with it small molecule structure at binding site.Generally can be divided into two kinds of approach: one is based on the virtual screening that is also referred to as of database search, promptly according to the shape and the architectural feature of receptor binding site, the compound of organic molecule library is docked to receptor binding site one by one with its three-dimensional structure, in the micromolecule database, finds out the molecule suitable with binding site.Another kind method is called brand-new drug design, promptly according to the shape and the architectural feature of receptor binding site, is spliced into complementary with it ligand molecular by atom, chemical group or segment.In fact, the former is equivalent to seek the key that can open lock in many existing keys, and the latter is the new key of structure manufacturing according to lock.

, the micromolecule database that is used for virtual screening at present mainly is micromolecule storehouse, Cambridge, and the noval chemical compound kind is less in this storehouse, renewal is slow, and the one-tenth property of medicine of chemical compound lot is relatively poor, has promptly allowed to activity, and further the potentiality of exploitation are little.The method of brand-new drug design can address the above problem, and the brand-new drug design of computing machine can be placed segment in the active pocket of target proteins matter, then by optimizing the molecule that static, Van der Waals and hydrogen bond action " growth " go out to be fit to active pocket.But this method often produces the molecule that is difficult to synthesize again.

(2) based on the screening of segment (fragment-based)

Refer to the micromolecule group of molecular weight based on the segment in the segment screening, normally form the basic group of compound, as naphthoquinones, phenyl ring or the like less than 200Da.Usually, this micromolecule segment has lower adhesion to target proteins, is not therefore often found by the high-throughput screening method of routine.

But this method also exists the limitation of itself: at first, the less group of molecular weight (is lower than 2.5 in low resolution

Resolution) electron density in be difficult for crystallization solution in the micromolecule (as DMSO etc.), ion (as acid ion etc.) that itself exist wait and differentiate, so the crystal mass of target proteins is had relatively high expectations, limited the scope of target protein greatly; Secondly, can stable existence, the micromolecule radical species of molecular weight on the 200Da level be limited, uses more being used for also only to have hundreds to thousands of kinds of compositions based on the alternative storehouse of segment screening at present, this has just limited the possibility of hitting greatly; Once more, this screening technique will much can not synthesize, can't be decomposed into the natural products of basic group and get rid of outside the scope of drug screening, limit the screening scope.

By up to the present, there is not to describe crystal structure in the prior art based on protein, therefrom obtain the method for target molecule and target protein fine three dimensional structure in the medicines natural products mixed system efficiently, fast.For research with structure diversity and the multifarious natural products of biologically active, still carry out in a conventional manner, promptly screen crude extract, if find to have activity with drug target protein, just this extract is divided into fractions, separates again and the identified activity compound.This process not only slowly, efficient is low, labour intensity is big, nor can guarantee that the lead compound that filters out chemically is being feasible.Therefore, remain to be developed a kind of brand-new natural products medicament research and development pattern, and press for and set up a kind of new, screening spare library of natural product mixture medicament that derives from Chinese medicine etc. that can satisfy the scientific technological advance needs, its can be used in from natural product mixture, carry out fast, efficient, comprehensive drug screening method.

Summary of the invention

The object of the present invention is to provide in a kind of therefrom medicines natural products mixed system efficient, obtain the new method of target molecule and target protein complex fine three dimensional structure fast, this method can efficiently be carried out drug screening, this method is based on protein structures, directly screen with the natural products mixture alternate library that derives from Chinese medicine etc. through the X-ray crystallography method that can be used in optimized, can be efficient, seek specific target molecule (the part micromolecule that comprises inhibitor that can combine) accurately, improved the efficient and the accuracy of screening with target proteins.Method of the present invention is simple relatively than the method for present already present drug screening, cost is lower, but screening effeciency height, quick, accurate, and can from the natural products mixed system, obtain the best target molecule of adhesion.

Particularly, the present invention uses the means of X-ray crystallography based on target proteins matter crystal structure, and natural productss such as China's traditional Chinese medicine are carried out the screening of active component, is to remove medicines natural products and biology the research from a kind of new angle.Its flesh and blood can comprise two aspects: the first, and the Collection and analysis of the expression of target protein, separation and purification, crystallization and even crystal diffraction data, this belongs to the research category of biomacromolecule; Second, break between the micromolecule of ten million kind of contained in this middle medicines natural products non-isolated existence original " life contact status " as organic life entity of a complexity, make these micromolecular compounds freely with the combining of being at war with property of target protein, this belongs to the especially category of natural product chemistry research of chemistry again.Each micromolecule separation and purification in the natural products crude extract is come out, carry out the screening active ingredients of target protein separately, can exclude the accidentalia in the experiment to greatest extent, can guarantee has the target molecule of effect (comprising inhibiting effect) to derive from certain micromolecule that structure is determined really to target protein, not only workload is huge but do like this, research cycle is long, and itself is blindly to screening.For this reason, the present invention has adopted following scheme: the crystal of target protein is placed on contains in a variety of micromolecular natural products crude extract blend sample, as fishing " angling out " target molecule to its ability of closing of signing an undertaking most.The advantage of doing like this is conspicuous, because it can carry out drug screening expeditiously at specific target proteins in the natural product mixture storehouse.In order will to remove (as compositions such as the contained biomacromolecule of crude extract itself, heavy metal, inorganic salts the factor that this process exerts an influence in the natural products crude extract sample, and because the too assorted intermolecular interaction that causes of sample itself), can keep again the target molecule that might combine with target protein provide maximum screened composition to guarantee this natural products crude extract sample, avoid the false negative that occurs regretting, need carry out certain optimization the natural product mixture sample that is used for the X-ray crystallography screening.

A kind of method of obtaining target molecule and target protein complex fine three dimensional structure from natural products mixture alternate library efficiently, fast provided by the invention, it comprises the steps: to obtain from natural products mixture alternate library based on protein structures the fine three dimensional structure of target molecule and target proteins complex.

Method provided by the invention can comprise the steps: the crystal of target proteins matter is contacted with the natural products mixture alternate sample, the crystal of analyzing target proteins matter and X-ray diffraction in crystals data after the natural products mixture alternate sample contacts, the electron density of the target molecule that acquisition combines with target proteins, and then the fine three dimensional structure of acquisition target molecule and target protein complex.

Method provided by the invention can comprise the steps:

(1) chooses target proteins matter;

(2) crystal of preparation target proteins matter;

(3) preparation natural products mixture alternate library;

(5) crystal with target proteins matter contacts with the natural products mixture alternate sample;

(6) collect and analytical procedure (5) in the crystal of target proteins matter and X-ray diffraction in crystals data after the natural products mixture alternate sample contacts, the electron density of the target molecule that acquisition combines with target proteins;

(7) identify target molecule, obtain the fine three dimensional structure of target molecule and target protein complex.

Method of the present invention also can comprise the steps:

(8) optimization of target molecule and further biological experiment.

In the method provided by the invention, the target proteins matter of choosing in step (1) is if report that it has the active determination in vitro method, then will carry out following steps (4) between step (3) and step (5): the natural products mixture alternate sample is to the active determination in vitro experiment of target proteins matter, choose target proteins matter is had the sample that suppresses active, these samples are called " have suppress active " sample, will " have suppress activity " sample and target proteins matter carry out step (5).

The preferred technical solution of the present invention is as follows:

1. target proteins matter chooses

The method of useful X-ray crystallography parse the protein of three-dimensional structure can be as the target proteins matter of the inventive method.At importance, expressing quantity size and the crystal growth complexity etc. of these protein, preferentially choose the protein relevant (for example human HIV related protein comprises (hiv protease, hiv integrase, HIV reverse transcriptase), HCV NS5B albumen, SARS main protease etc.) with human important diseases or important virus; Preferentially choose and in prokaryotic expression system (mainly referring to the E.coil expression system), to obtain great expression (comprising solubility expression or insoluble expression that exists with the inclusion body form), the protein that crystal can obtain in a large number.

After having determined target proteins, the method of buying, providing or clone from the cDNA library by the co-worker from company can be provided, with reference to the method in the clone that delivered, expression, the purifying document about this target proteins (can reference: Ferrari for HCV NS5B albumen, people .1999 such as Wright-Minogue; Lesburg, people .1999 such as Cable; Can reference for the SARS main protease: people .2003.The Crystal Structures of SARS Virus Main Protease Mpro and Its Complexwith an Inhibitor.PNAS such as Yang H, 100 (23): 13190-13195), obtain correct structure (construct), and with reference to delivered reported method in the document (can be for HCV NS5B albumen with reference to Ferrari, people .1999 such as Wright-Minogue; Can reference for the SARS main protease: people .2003.The Crystal Structures of SARS Virus Main Protease Mproand Its Complex with an Inhibitor.PNAS such as Yang H, 100 (23): 13190-13195) carry out the expression and the purifying of this target proteins matter, and can carry out suitable improvement (, and changing protein purification solution etc.) to the expression condition of this target proteins and the purification condition of this target proteins as change protein expression vector, change protein expression temperature.

The crystal of 2 preparation target proteinses

Use above-mentioned purified target proteins, (crystallization condition of optimizing target proteins comprises the crystallization concentration that changes target proteins to the crystallization condition of this target proteins that the crystallization condition of this target proteins that reference has been reported or process are optimized oneself, the composition of the crystallizing pond liquid of change target proteins etc.), the crystal that growth is a large amount of, diffracting power is strong, standby.

The preparation of 3 natural products mixture alternate libraries

The source of the primary raw materials of the sample in the natural products mixture alternate library is very extensive, and traditional Chinese medicine also is used for the treatment of various diseases so far in the application in existing several thousand of China, is the source that we screen the primary raw materials in alternative storehouse with standing in the breach.Prescriptions of traditional Chinese medicine extracts such as tradition classical prescription, proved recipe also can be used as the source of primary raw materials.In addition, we also are not limited to tradition Chinese herb medicine commonly used, also often contain novel, interesting micromolecular compound in the natural drug of some ethnic group's medicines, application among the people.Sea life provide the huge living resources of novel structure molecule, because ecologic environment and land are completely different, cause biosynthetic process difference in the body, make sea life compare with terrestrial life, have abundanter biomolecule diversity, so sea life also are the important sources of our alternative storehouse raw material.Other as metabolism product such as liver moss, fungi and microbial conversion product because its distinctive physiology course and growing environment also contain and abundant peculiar natural products micromolecule, also be the source of our primary raw materials.In a word, what we will prepare is the alternative sample of drug screening of natural products the most widely that comprises of maximum possible, and natural products mixture alternate library of the present invention is formed in the set of these samples.

Because the contained composition of natural products crude extract is quite complicated, therefore must the crude extract of natural products be optimized, purpose is to remove disturbing factor such as albumen, polysaccharide etc., is retained as property of medicine micromolecule and make it as far as possible to concentrate preferably, improves relative concentration.The present invention is for realizing this purpose, adopt the following technical scheme of this paper to prepare natural products mixture alternate library, its ultimate principle is: after primary raw materials is extracted, extract (crude extract) uses the solvent such as the ethyl acetate of opposed polarity to extract earlier, water after the extraction is handled with the method that macroreticular resin separates again, can guarantee to remove disturbing factor like this, can utilize the difference of two kinds of different separation mechanisms again, make micromolecule focus on the different section of polarity, obtain can be used in the natural products mixture alternate library of bio-physical methods such as X-ray crystallography thus.

The preparation method of natural products mixture alternate library comprises the steps:

(a) get a certain amount of primary raw materials, use the ethanolic solution refluxing extraction, merge extract, concentrate medicinal extract; Extract preparation for microbial metabolic products, then be that the nutrient solution that will contain an amount of microorganism filters, obtain solid microbe mycelium and nutrient solution filtrate, mycelium is used the acetone soln ultrasonication again, filter, filtrate concentrate medicinal extract, this medicinal extract and nutrient solution filtrate are all carried out next step processing respectively as the extract of microbial metabolic products;

(b) take by weighing the medicinal extract of gained in a certain amount of step (a), suspend, use organic solvent extraction, separate organic solvent phase and water with distilled water; Merge water, standby; Merge each organic solvent extraction thing, steam and remove organic solvent, obtain dried paste organic solvent phase constituent, standby;

Nutrient solution filtrate part for microorganism extracts is then directly used organic solvent extraction, merges each organic solvent extraction thing, steams and removes organic solvent, obtains dried paste organic solvent phase constituent, and is standby;

(c) aqueous portion after the extraction in the step (b) is steamed the middle a small amount of organic solvent that dissolves that dewaters, separate with macroporous resin column chromatography, water is the moving phase wash-out, discards the washing part; Use the ethanolic solution wash-out, the ethanolic solution wash-out is partly steamed desolventize, the shape sample that gets dry extract is standby;

(d) the dried paste sample that makes in the dried paste organic solvent phase part that makes in the above-mentioned steps (b), the step (c) is the natural products mixture alternate sample, constitute the natural products mixture alternate sample that a primary raw materials provides, the natural products mixture alternate sample that all primary raw materials provide has promptly been formed this natural products mixture alternate library.

Preparation method's preferred version of natural products mixture alternate library of the present invention is:

(a) preparation extract (crude extract) medicinal extract

Get a certain amount of primary raw materials, with alcohol reflux 2-4 time, each 1-3 hour, merge extract, concentrated medicinal extract with experiment.

Selected primary raw materials can be selected from above-mentioned raw material any material except that microorganism as natural products mixture alternate library of the present invention; Wherein said experiment concentration of alcohol is a 70%-95% ethanol, preferred 95% ethanol; Wherein the refluxing extraction number of times is preferably 3 times, each preferred 2 hours; Wherein used amount of alcohol and medicinal material amount mass ratio are 6:1-12:1.For the preparation of the extract medicinal extract of prescriptions of traditional Chinese medicine, then form primary raw materials according to medicinal material kind and the 1-6 times of consumption of regulation in the prescription, make medicinal extract one with 70%-95% ethanol by above-mentioned same method respectively; In addition, equally then form primary raw materials according to medicinal material kind and 1-6 times of consumption of regulation in the prescription, decoct 3 times with distilled water, each 1-3 hour, decoction liquor merged, concentrate medicinal extract two.Medicinal extract one and medicinal extract two all carry out next step processing as the extract medicinal extract of prescriptions of traditional Chinese medicine.Extract preparation for microbial metabolic products, then be that the nutrient solution that will contain an amount of microorganism filters, obtain solid microbe mycelium and nutrient solution filtrate, mycelium is used 80% acetone ultrasonication 20-60 minute again, filter, filtrate concentrate medicinal extract, this medicinal extract and nutrient solution filtrate are all carried out next step processing as the extract of microbial metabolic products.

(b) make the organic solvent extraction part

Take by weighing the medicinal extract in a certain amount of step (a), suspend, in the impouring separating funnel,, need shake well during extraction with the ethyl acetate extraction of Sq 4-6 time with distilled water, leave standstill layering in 5-8 hour after, separate organic solvent phase and water; Merge each organic solvent phase extract, steam with Rotary Evaporators and remove organic solvent, obtain dried paste organic solvent phase constituent, standby.

Nutrient solution filtrate part for microorganism extracts is then directly used Sq organic solvent extraction 4-6 time, merges each organic solvent extraction thing, steams with Rotary Evaporators and removes organic solvent, obtains dried paste organic solvent phase constituent, and is standby;

Wherein, with ethyl acetate or chloroform, medicinal extract is 1:5-1:15 with the mass ratio of suspension water before the extraction, preferably 1:10 with organic solvent in extraction; Separating funnel is selected suitable separating funnel for use, for example, if add 100ml water, then selects the separating funnel of 1000ml for use; Extraction is 6:1-10:1 with the volume ratio of volume of organic solvent and suspension water, preferred 8:1; The preferred EYELA N1001 of the Rotary Evaporators of selecting for use type Rotary Evaporators.

(c) make water macroreticular resin 50% ethanol and 95% ethanol elution part:

Aqueous portion after the extraction in the step (b) is steamed a small amount of organic solvent of the middle dissolving that dewaters, separate with macroporous resin column chromatography, water, 50% ethanol, 95% ethanol are the moving phase wash-out successively, water elution 6-10 time, preferred 7 times, each wash-out water is the water consumption that suspends in the step 2 of 2-4 times of volume, discards the washing part; 50% ethanol and each wash-out of 95% ethanol 4-6 time, preferred 5 times, water consumption suspends in the step 2 that each wash-out amount of alcohol is a 2-4 times of volume.Use the Rotary Evaporators steaming to desolventize respectively 50% ethanol and 95% ethanol elution part, the shape sample that gets dry extract is standby.

Wherein, the preferred EYELA N1001 of described Rotary Evaporators type Rotary Evaporators; Described wash-out is preferably with amount of alcohol with suspension water consumption and wash-out to suspend in the step 2 of 3 times of volumes uses distilled water; The preferred HP-20 type of described macroreticular resin, preferably, macroreticular resin is with 180 milliliters, glass column specification 30 * 300mm.

(4) make natural products mixture alternate library:

The organic solvent phase part that makes in the above-mentioned steps (b), the water macroreticular resin 50% ethanol elution part and water macroreticular resin 95% ethanol elution that make in the step (c) partly wait 3 part natural products mixture alternate samples, constitute 3 kinds of natural products mixture alternate samples that a primary raw materials provides (when primary raw materials is prescriptions of traditional Chinese medicine or microorganism, each part primary raw materials provides 6 kinds of natural products mixture alternate samples), the natural products mixture alternate sample that all primary raw materials provide (being made as the N* kind) has promptly been formed this natural products mixture alternate library.

The composition of above-mentioned natural products mixture alternate library is shown in Fig. 2

The alternative sample of 4 natural productss is to the mensuration of target proteins vitro inhibition activity

Selected target proteins in the step (1) has ripe active determination in vitro method if reported it, before the immersion test of the sample in carrying out target proteins crystal and natural products mixture alternate library (being called the natural products mixture alternate sample) (according to preparation in the step 3), preferably with the natural products mixture alternate sample to target proteins carry out external activity mensuration (the target proteins activity determination method with reference to pertinent literature (as HCV NS5B albumen, referring to: people .Journal of Virology such as Robert A., July2003:7575-7581; The sars coronavirus main protease, referring to: Yang H, people .2005PloS Biol 3 (10) such as Xie W: e324)), from different natural products mixture alternate samples, select target proteins is had the sample (sample that called after " has and suppresses active ") that suppresses active, carrying out next step crystal soaks or cocrystallization work, can select targetedly like this has the natural products mixture alternate sample that suppresses active to particular target albumen, the number of times of crystal immersion or cocrystallization in the following step 5 of minimizing, thereby effectively reduce workload, and improve hit rate.

The determination of activity condition of different target proteinses can be carried out with reference to the means known in the art of having reported, as the determination of activity condition of HCV NS5B albumen, referring to: people .Journal of Virology such as Robert A., July 2003:7575-7581; The determination of activity condition of sars coronavirus main protease, referring to: YangH, people .2005 PloS Biol 3 (10): e324 such as Xie W.

For other, attempt to carry out the target proteins (as SARS non-structural protein nsp10) that function is explored by method of the present invention, can not consider this step, and direct implementation step (5) to determine whether being combined with target molecule on target proteins, is used for studying the function of this target proteins.

5. the crystal of target proteins matter contacts with the natural products mixture alternate sample

For reporting target protein with active determination in vitro method, the sample that obtains " have and suppress active " in step 4 (may be a kind of, also may be multiple, be made as the N1 kind) afterwards, the sample through the target proteins behind the purifying and " have and suppress active " that the immersion test of the sample of the crystal of the target proteins of preparation and " have and suppress active " or use step 1 obtain in the use step 2 carries out cocrystallization and tests.Fundamental purpose be obtain target proteins and target molecule (ligand molecular of the target proteins that may exist in the sample of this target molecule for " have and suppress active ") complex can stable existence, and have the crystal that can supply the diffracting power of x-ray diffraction experiment.

The method of using comprises that mainly crystal soaks and cocrystallization, and the method that preferred crystal is soaked can adopt following three kinds of modes to carry out:

Mode (one): soak separately

(1) (crystallizing pond liquid is the solution of target protein crystal growth to the sample dissolution that the N1 kind that obtains in the step 4 " is had and suppresses active " in the crystallizing pond liquid of target proteins, usually by precipitation agent and buffering solution composition) (for example the crystallization damping fluid of SARS main protease consists of: 1.8～12% PEG 6000,3% dimethyl sulfoxide (DMSO) (DMSO), 1mM DTT, 100mM MES damping fluid (among the pH5.0～PH6.0), makes the crystal soak solution.

(2) utilize instrument such as nylon crystal rings, crystal is taken out from crystallizing pond liquid, be added to respectively to soak in the crystal soak solution described in above-mentioned (1) and do not wait to 48 hours in 2 hours.Adopt this mode to soak N1 time.

Mode (two) grouping is soaked

It is emphasized that especially, the sample that the N1 kind that obtains in the step 4 " has and suppresses active ", the not necessarily specific inhibiting effect of its inhibiting effect to target proteins, may there be false positive results, in order to reduce the immersion number of times, the sample of this N1 kind " have suppress active " can be suppressed active height divide into groups (supposing that 10 kinds is a group) according to it to target proteins, carry out according to mode () then, so only need to soak N1/10 time, reduce the immersion number of times, improved screening effeciency and hit rate.

Mode (three) is all mixed immersion

The sample that the N1 kind that obtains in the step 4 " is had and suppresses active " all mixes, prepare a kind of crystal soak solution according to (1) in step 5 mode (), and then carry out the crystal immersion test, adopt this mode only need soak once, farthest reduce the immersion number of times, farthest improved screening effeciency and hit rate.

For other, attempt to carry out the target proteins (as SARS non-structural protein nsp10) that function is explored by method of the present invention, with natural products mixture alternate sample and target proteins crystal directly the above-described mode of employing contact.

6. the Collection and analysis of crystal X ray diffracting data

On

step

4,5 basis, use to possess better diffracting power and (be commonly referred to as highest resolution and be not less than 3

) the crystal and crystal after the natural products mixture alternate sample contacts of target proteins matter, carry out the Collection and analysis of X ray diffracting data.

For the experiment that carry out in the laboratory, can select the X-ray machine that uses RigakuMM007 copper target rotary anode generator to produce X ray for use; Focus lamp by OSMIC company focuses on X ray, makes it can reach the highest intensity; Use the Mar345dtb imaging system of Rigaku RAXIS IV++ imaging plate detector or Marreseach company to collect diffraction data.

For the screening operation of high flux X-ray crystallography, can use the ACTOR automatic crystal installation manipulator system and the higher FR-E X-ray diffractometer of X ray intensity of Rigaku company, to accelerate speed of experiment.

When carrying out data aggregation, at first use the nylon crystal rings of Hampton Research company, (in order to reduce the infringement of soak solution to target proteins matter crystal, soak time is typically chosen between 2 hours to 48 hours to obtain the immersion certain hour from the crystal soak solution; For pick up speed, soak time was controlled between 2-6 hours) crystal; And use the cooling system of Rigaku company or the cooling system of OxfordCyrosystem company rapidly, crystal is refrigerated to-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 data processing according to following step:

At first, use the method for data 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 routine 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 data 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 amino acid of the avtive spot of this electron density and target proteins has rational chemical environments such as the hydrogen bond action of comprising, covalent effect, then can confirm in this natural products mixture alternate sample have ligand moleculars such as inhibitor micromolecule to combine with target proteins.

The target proteins (for example SARS non-structural protein nsp10) that uses the inventive method to carry out functional study for other hope, then need around whole protein molecule, examine, see if there is the unknown electron density existence 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, if there is unknown electron density to exist, then may be the avtive spot that a place has researching value herein.

7. evaluation target molecule obtains the fine three dimensional structure of target molecule and target protein complex

In case in step 6, find to be combined in the unknown electron density on the target proteins matter molecule, in general can carry out further work according to following two kinds of methods, grade in conjunction with micromolecular concrete one-tenth with definite:

(1), is often referred to resolution and surpasses 2.5 for the crystal structure of the higher complex of resolution

Can pass through Direct observation analytical electron density, analysis is in conjunction with the chemical action of micromolecule and target proteins matter molecule, and in conjunction with this natural products mixture alternate sample research, to reasonably infer (may be a kind of, also may be multiple) in conjunction with micromolecular composition and structure, obtain possible micromolecule by approach such as buying, synthetic then, and carry out control experiment, to determine the micromolecular ultimate constituent.

(2), be often referred to resolution and be lower than 2.5 for the crystal structure of the lower complex of resolution

At first, utilize mass spectral method, protein and natural products mixture alternate sample are analyzed, determine in conjunction with micromolecular molecular weight.Secondly, by consulting means such as pertinent literature, existing composition to this alternative sample is made a concrete analysis of, the statistics compound (may be a kind of, also may be multiple) that (being often referred in molecular weight ± 10) may exist about the above-mentioned molecular weight of determining with mass spectrometry method.Then, obtain this kind or these compounds by means such as synthetic, purchases, and carry out control experiment, to determine the micromolecular ultimate constituent.

8. the optimization of target molecule and further biological experiment

In case method according to step 1-step 7, from certain natural products mixture alternate sample, obtained the structure of the complex that target proteins matter molecule combines with target molecule, and after the concrete composition and structural formula of definite target molecule, can carry out structural design and optimization to target molecule according to the complex structure that obtains.Can design and synthesize out and the micromolecular compound that mate more in target proteins matter molecular activity site, binding ability is higher in the hope of obtaining, be called the target molecule of optimization.After the target molecule that is optimized, can carry out further biological experiment to the target molecule of target molecule, optimization, comprise biological experiments such as active determination in vitro, cytologic experiment, zoopery, further measure their biological parameter (as binding constant, enzyme kinetics parameter, cytotoxicity or the like), and to the curative effect of living animal, so that be that further clinical trial is ready.

" therefrom obtaining the new method of target molecule and target protein complex fine three dimensional structure in the medicines natural products mixed system efficiently, fast " provided by the invention, the method that X ray diffractive crystal is learned is applied in the natural products drug research, solved the bottleneck of the single component separation and purification that exists in the current natural products research, improved efficiency of research and development greatly, make the research of natural products enter a brand-new stage, for current pharmaceuticals industry has brought new opportunity.

Method provided by the invention is compared with existing method, and the advantage of utilizing the inventive method to carry out drug screening is:

(1) at first, this inventive method is used has structure diversity and the multifarious natural product mixture of the biologically active alternative storehouse as drug screening, solved with the protein structures be at present the basis carry out in the method for drug screening many shortcomings (particularly, difficult for the butt joint problem in the area of computer aided drug design, molecule novelty in the virtual library is relatively poor, classes of compounds is few, compound becomes the property of medicine poor, Theoretical Calculation is low to the indication degree of experiment results, for based on the less group of the molecular weight in the screening of segment the electron density of resolution in be difficult for crystallization solution in micromolecule difference itself that exist, molecular weight that can stable existence is limited etc. in the kind of the micromolecule group of 200Da level).

(2) secondly, the method of biophysicss such as this inventive method use X-ray crystallography, can detect the part micromolecule more weak with target proteins affinity, therefore make can not be used for conventional high-throughput screening method be simple and easy to blend sample such as natural products crude extract, can be used for high-throughout X-ray crystallography method for screening, so just save the purification procedures of loaded down with trivial details natural products, solved defectives such as present pharmaceuticals industry is low for the efficient that in the natural products research with the separation and purification is characteristics, labour intensity is big.

(3) this inventive method uses the blend sample of natural products crude extract as alternative storehouse, can effectively reduce active determination in vitro number of times in the screening, reduce the number of times of crystal immersion or cocrystallization, improve the hit rate that the target proteins crystal combines with the natural products micromolecule greatly, thereby effectively improve the efficient of screening.The inventive method and traditional contrast of carrying out the screening effeciency of method for screening for the basis based on protein structures are shown in following table 1 (shown in step 3, establish in the natural products mixture alternate sample that each part make and contain n kind natural products micromolecule)

(4) use the crystal of target proteins as " bait ", soak activated natural product mixture storehouse (the micromolecular pond of medicine is rich in likening to of image), can be target proteins there being specificity from potpourri, separate (process of " fishing ") in conjunction with the natural products micromolecule that comprises specificity inhibition combination, obtain the accurate three-dimensional model that target proteins matter combines with the natural products micromolecule, carry out further drug design and optimization according to the spatial configuration and the size of its atom, molecule.

(5) use the inventive method, soak the natural products mixture alternate sample by the crystal that uses target proteins, can from natural product mixture, obtain combining the best part micromolecule that comprises inhibitor (principle of competition combination) with target proteins, optimized choice comprise the micromolecule part of inhibitor.As everyone knows, for each natural products crude extract, the composition of a lot of similar is contained in the inside, with the Asian puccoon is example, the effective constituent of Asian puccoon mainly is alkannin and derivant thereof, and when we used the crystal immersion process Radix Arnebiae extract sample of target proteins, alkannin that contains in the Asian puccoon and the competition of various derivant thereof were in conjunction with the avtive spot of target proteins, can be attached to the target proteins avtive spot at last, must be the part micromolecule best with the target proteins adhesion.

Description of drawings

For the easier quilt of content of the present invention is clearly understood, below according to a particular embodiment of the invention and in conjunction with the accompanying drawings, the present invention is further detailed explanation, wherein

The experiment flow figure of Fig. 1 the inventive method

The composition synoptic diagram of Fig. 2 natural products mixture alternate library of the present invention

Fig. 3 curve 1-curve 11 is respectively the activity inhibition curve of the water 50% ethanol elution sample of honeysuckle, the capsule of weeping forsythia, mother chrysanthemum, reed rhizome, cape jasmine, Chinese holly, radix scrophulariae, Lasiosphaera fenzlii, blackberry lily, dayflower, the root bark of Chinese wolf-berry to the SARS main protease, sample concentration: 100 μ g/mL.Curve 12 is contrast (the activity curve figure that does not add the SARS main protease of other sample).

Fig. 4 curve 1 is contrast (the activity curve figure that does not add the SARS main protease of other sample).Curve 2-curve 7 is respectively the activity inhibition curve of the water 50% ethanol elution sample of subprostrate sophora, the wind-weed, Herba Andrographitis, cordate houttuynia, folium isatidis, the root of large-flowered skullcap to the SARS main protease, sample concentration: 100 μ g/mL.

Fig. 5 curve 1 is contrast (the activity curve figure that does not add the SARS main protease of other sample).Curve 2-curve 11 is respectively the activity inhibition curve of the ethyl acetate extraction sample of radix scrophulariae, honeysuckle, Herba Andrographitis, Chinese holly, dayflower, the root bark of Chinese wolf-berry, reed rhizome, folium isatidis, Lasiosphaera fenzlii, the root of large-flowered skullcap to the SARS main protease, sample concentration: 100 μ g/mL.

Fig. 6 curve 1 is contrast (the activity curve figure that does not add the SARS main protease of other sample).Curve 2-curve 8 is respectively the activity inhibition curve of the ethyl acetate extraction sample of the capsule of weeping forsythia, blackberry lily, mother chrysanthemum, cordate houttuynia, subprostrate sophora, the wind-weed, cape jasmine to the SARS main protease, sample concentration: 100 μ g/mL.

Fig. 7 curve 1 is contrast (the activity curve figure that does not add the SARS main protease of other sample).Curve 2-curve 6 is respectively the activity inhibition curve of the ethyl acetate extraction sample of cordate houttuynia, subprostrate sophora, the root of large-flowered skullcap, the wind-weed, cape jasmine to the SARS main protease, sample concentration: 10 μ g/mL

Fig. 8 curve 1 is contrast (the activity curve figure that does not add the SARS main protease of other sample).Curve 2 and curve 3 are respectively Rabdosia pseudo-irrorata C. Y. Wu water 50% ethanol elution sample and Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction sample the activity of SARS main protease are suppressed curve, sample concentration: 100 μ g/mL

Fig. 9 curve 1 is contrast (the activity curve figure that does not add the SARS main protease of other sample).Curve 2 and curve 3 are respectively Rabdosia pseudo-irrorata C. Y. Wu water 50% ethanol elution sample and Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction sample the activity of SARS main protease are suppressed curve, sample concentration: 10 μ g/mL

Near electron density comparison diagram Figure 10 SARS main protease of the present invention avtive spot.A is that the electron density of SARS main protease parent avtive spot shows; B is the electron density demonstration that SARS main protease and Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction sample soak back crystal avtive spot; C is that SARS main protease and the Rabdosia pseudo-irrorata C. Y. Wu third plain electron density of soaking back crystal avtive spot show.Electron density shown in the figure, dark-coloured (white circle is outer) be the 2fofc electron density, be 1.0sigma; Light tone (white circle in) be 1fofc difference electron density, be 2.5sigma.

The micromolecular electron density of the existence in Figure 11 Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction of the present invention sample and SARS main protease avtive spot Cys145 residue electron density form the displayed map of covalent bond.Protein molecule represents that with ribbon figure the Cys145 residue of avtive spot is represented with ball-and-stick model; Electron density is represented the electron density of Cys145 in the circle 2, and electron density is represented unknown micromolecular electron density in the circle 1.

Mass spectrogram before and after Figure 12 SARS main protease of the present invention and the Rabdosia pseudo-irrorata C. Y. Wu extract ethyl acetate extraction sample effect.The mass spectra peak of SARS main protease itself is represented at the peak on the left side; The mass spectra peak behind SARS main protease and the Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction sample effect is represented at the peak on the right.

Figure 13 curve 1 is contrast (the activity curve figure that does not add the SARS main protease of other sample).Curve 2 is the activity inhibition curve of the Rabdosia pseudo-irrorata C. Y. Wu third plain sample to the SARS main protease, sample concentration 20 μ M.

Figure 14 curve 1 is contrast (the activity curve figure that does not add the SARS main protease of other sample).Curve 2 is the activity inhibition curve of the Rabdosia pseudo-irrorata C. Y. Wu third plain sample to the SARS main protease, sample concentration 2 μ M.

Figure 15 is the result after the improving for HCV NS5B crystal described in the present invention.Wherein a is the crystal that carries out HCV NS5B after the crystal growth according to the crystallization solution of describing in the document (Ago, people .1999 such as Adachi), and b is the crystal that carries out HCV NS5B after the crystal growth after the crystallization solution in the document improves

The ethyl acetate sample that Figure 16 A1-A12 is respectively Asian puccoon, Radix Isatidis, rhizoma cyperi, semen litchi, Chinese yam, the root bark of Chinese wolf-berry, pyrrosia lingua, root of Chinese trichosanthes, the tuber of stemona, the flower bud of lily magnolia, ramulus mori, brave account is contrast (the active figure of HCV NS5B that does not add other sample) to activity inhibition figure, the A13 of HCV NS5B albumen.T5: sampling, T10 when the time is 5 minutes: sampling, T15 when the time is 10 minutes: sampling, T20 when the time is 15 minutes: the sampling when time is 20 minutes.G/mL:12 Chinese medicine ethyl acetate of a:20 μ sample concentration is 20 μ g/mL.G/mL:12 Chinese medicine ethyl acetate of b:10 μ sample concentration is 10 μ g/mL.

Near electron density comparison diagram Figure 17 HCV NS5B albumen His502 avtive spot.A is the electron density of HCV NS5B albumen parent avtive spot; B is that the electron density that the crystal of HCV NS5B albumen soaks the HCV NS5B protein active site behind the Asian puccoon ethyl acetate extraction sample shows; C is the demonstration behind the b half-twist.Electron density shown in the figure, dark-coloured (white circle is outer) be the 2fofc electron density, be 1.0sigma; Light tone (white circle in) be 1fofc difference electron density, be 2.5sigma.Figure 18. coincideing of Acetylshikonin micromolecule and unknown electron density.1 mallet is the amino acid residue of protein, the unknown electron density of 2 expressions, and 3 mallets are the structural formula of Acetylshikonin.

Embodiment

Embodiment 1

The preparation of the sample in the natural products mixture alternate library

The preparation of the alternative sample of Rabdosia pseudo-irrorata C. Y. Wu (I.pharicus (Prain) Murata)

1 preparation extract medicinal extract:

Get the Rabdosia pseudo-irrorata C. Y. Wu medicinal material of 500 gram drying and crushing,, extracts 3 times with 4500 milliliter of 95% alcohol reflux, each 2 hours, merging extract, concentrated medicinal extract;

2 make the ethyl acetate part:

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 in 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 sample that gets dry extract is standby;

3 make macroreticular resin 50% ethanol and 95% ethanol elution part:

Aqueous portion after the extraction steams the amount of ethyl acetate of the middle dissolving that dewaters with EYELA N1001 type Rotary Evaporators, (macroreticular resin is with 170 milliliters with the separation of HP-20 type macroporous resin column chromatography, glass column specification 30 * 300mm), water, 50% ethanol, 95% ethanol are the moving phase wash-out successively.Water elution 8 times, 250 milliliters of each wash-outs discard the washing part.Each wash-out of 50% ethanol and 95% ethanol 5 times, 300 milliliters of each wash-outs.Every part is all used EYELA N1001 type Rotary Evaporators to steam and is desolventized, and the shape sample that gets dry extract is standby;

4 obtain the alternative sample of Rabdosia pseudo-irrorata C. Y. Wu:

The ethyl acetate phase part that above-mentioned steps (2) makes, 50% ethanol elution part and 95% ethanol elution that step (3) makes partly wait 3 parts promptly to form 3 parts of mixture alternate samples that the primary raw materials Rabdosia pseudo-irrorata C. Y. Wu provides.

Other 17 kinds of (cordate houttuynia, mother chrysanthemum, folium isatidis, reed rhizome, honeysuckle, capsules of weeping forsythia, the root of large-flowered skullcap, the wind-weed, Chinese holly, Herba Andrographitis, dayflower, cape jasmine, blackberry lily, subprostrate sophora, Lasiosphaera fenzlii, radix scrophulariae, the root bark of Chinese wolf-berry) preparation of the alternative sample that provides of primary raw materials and the preparation of the alternative sample of Rabdosia pseudo-irrorata C. Y. Wu be basic identical.

Embodiment 2

Based on the crystal structure of sars coronavirus main protease, from the natural products mixture alternate sample of embodiment 1, obtain the method for Rabdosia pseudo-irrorata C. Y. Wu third element and SARS main protease complex fine three dimensional structure fast.Comprise the steps:

1. choosing of target proteins matter: sars coronavirus main protease

2003, the SARS epidemic situation was broken out in the whole world on a large scale, and causes that the pathogen of SARS finally is confirmed to be a kind of coronavirus-sars coronavirus of the unknown (Drosten, C., Gunther, S., Preiser, people .N Engl J Med.2003 such as W., 348:1967-76).Further discover, the genome encoding of sars coronavirus two big replicase polyprotein (replicasepolyproteins) ppla (486kDa) and pplab (790kDa), these two albumen are by the genome encoding that accounts for coronavirus 2/3 to 3/4.These two albumen are hydrolyzed a lot of function subunits that the back produces the virus replication complex.In this hydrolytic process, the major protein hydrolytic enzyme (main of sars coronavirus

Protease abbreviates main protease as, is abbreviated as M ^Pro, molecular weight 33.8kDa is also referred to as 3C-LikeProtein sometimes) and played very crucial effect.Main protease also is a section among ppla and the pplab, and its release is finished by the self-catalysis hydrolysis, and the self-catalysis hydrolysis occurs in the Gln of this proteinase side, and (finish by trans-splicing (bimolecular reaction) in Ser, Ala) site.Under the effect of main protease, replicase polyprotein ppla and pplab are hydrolyzed into more than ten functional peptide fragment, thereby further play a role.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 proteins of anti-SARS drug screening.Sars coronavirus main protease (amino acid sequence is seen sequence table part 1) is expressed the further separation and purification of laggard row (referring to Yang Het al.2003.The Crystal Structures of SARS Virus Main Protease Mproand Its Complex with an Inhibitor.PNAS, 100 (23): 13190-13195) in coli strain BL21 (DE3).

The crystal growth of 2 sars coronavirus main proteases

The crystallization of the main protease of SARS virus is still referring to Yang H et al.2003.The CrystalStructures of SARS Virus Main Protease Mpro andIts Complex with anInhibitor.PNAS, 100 (23): 13190-13195.Particularly: adopt hanging drop gas phase diffusion method, under 289K-297K, carry out with tissue culturing plate.Crystal growth kit (Crystal Screening Kit I ﹠amp with Hampton Research company; II, PEG/ION Kit, PEG 6000 GridKit and (NH4) 2SO4 Kit) the primary crystallization condition is screened.In the drop under the C3 of PEG 6000 Grid Kit (20% PEG 6000,0.1M MES (the pH6.0)) condition, red nodular precipitation is arranged, and crystallite occurs at the edge of precipitation.Under this crystallization condition, we have attempted the damping fluid (for example the pH value from 5.5 to 6.5) of different pH values, the PEG of different polymerization degree (as carrying out), different protein concentration (for example 2mg/ml is to 30mg/ml) and various adjuvant (using the Additive Screen Kit (adjuvant screening reagent box) of Hampton Research to carry out) and have waited and optimize crystal from PEG 3350 to PEG 10000.At last, we are at 1.8-12% PEG 6000,3% dimethyl sulfoxide (DMSO)s (DMSO), and 1mM DTT, 100mM MES damping fluid (pH6.0) and protein concentration are the crystal that has obtained suitable diffraction under the situation of 9-12mg/ml.Typical crystalline size is 0.03 * 0.3 * 0.5mm.

The preparation of 3 natural products mixture alternate samples

With reference to embodiment 1

4. the natural products mixture alternate sample suppresses active mensuration to the SARS main protease

The determination of activity of SARS 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 amino acid sequence of this fluorogenic substrate derives from the N end of SARS 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 dried paste natural products mixture alternate sample that obtains among the embodiment 1 is dissolved in to make its final concentration in the dimethyl sulfoxide (DMSO) (DMSO) be 50mg/mL

At buffer solution (50mM Tris-HCl (pH 7.3), 1mM EDTA (containing or do not contain DTT)) adds SARS main protease (final concentration 0.5 μ M) in, the DMSO dissolved matter of natural products mixture alternate sample (as the DMSO dissolved matter of Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample) makes its final concentration be: 100 μ g/mL, concentration of substrate is 20 μ M, 298K placed after 10 minutes, add fluorescence labeling substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH2, final concentration 20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 330nM and 395nm, 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. 3-Fig. 9.

Fig. 3-Fig. 6 and Fig. 8 are that the natural products mixture alternate sample that 18 kinds of Chinese medicines make suppresses active measurement result to the SARS main protease, and wherein the concentration of every kind of alternative sample is 100 μ g/mL.

By above Fig. 3-Fig. 9 as can be seen, wherein most of alternative sample all has the activity of inhibition to the SARS main protease, wherein suppressing activity has 6 kinds preferably, is respectively the acetic acid ethyl ester extract sample of the root of large-flowered skullcap, cordate houttuynia, subprostrate sophora, cape jasmine, the wind-weed, Rabdosia pseudo-irrorata C. Y. Wu.

Fig. 7 and Fig. 9 are above 6 kinds and suppress activity preferably: during concentration dilution to the 10 μ g/mL of the acetic acid ethyl ester extract sample of the root of large-flowered skullcap, cordate houttuynia, subprostrate sophora, cape jasmine, the wind-weed, Rabdosia pseudo-irrorata C. Y. Wu the SARS 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, cordate houttuynia, subprostrate sophora, cape jasmine, the wind-weed, Rabdosia pseudo-irrorata C. Y. Wu is 10 μ g/mL, the SARS main protease is still had stronger inhibition activity.

This just illustrates may contain the activity that certain composition can effectively suppress the SARS main protease in the ethyl acetate extraction sample of these 5 kinds of Chinese medicines, illustrate that also these 5 kinds of Chinese medicines might become the active drug of treatment sars coronavirus.In order to understand them to the definite molecule mechanism of SARS main protease inhibiting effect, the crystal that we can select the ethyl acetate extraction sample of these 5 kinds of Chinese medicines to carry out the SARS main protease soaks or the cocrystallization experiment.

5.SARS the main protease crystal soaks Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction sample

With Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction sample is example, owing to contain not only a kind of compound in the Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction sample, if adopt the method for cocrystallization, the influence of target protein crystallization is bigger, can't obtain that the diffraction quality is good, the target proteins matter crystal of abundant amount, even possibly can't grown crystal, the therefore main method that adopts crystal to soak is used for obtaining the SARS main protease and contacts crystal afterwards with Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample.

In order to guarantee on the basis of crystal mass as much as possible, improve when soaking the concentration of micromolecular compound in the alternative sample, adopt two kinds of methods to prepare the crystal soak solution.

Method one, at first the dissolved matter (according to step 4 preparation) with Rabdosia pseudo-irrorata C. Y. Wu extract acetic acid ethyl ester extract DMSO is diluted to 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)), 13000-15000rpm high speed centrifugation is got supernatant afterwards, get soak solution 1, standby.

Method two, dried paste Rabdosia pseudo-irrorata C. Y. Wu extract acetic acid ethyl ester extract (according to preparation among the embodiment 1) directly is dissolved in (1.8～12% PEG 6000 in the pond liquid of crystal growth, 3% dimethyl sulfoxide (DMSO) (DMSO), 1mM DTT, 100mM MES damping fluid (pH 5.0～PH6.0)) is after treating fully to dissolve, 13000-15000rpm high speed centrifugation, get supernatant afterwards, get soak solution 2, standby.

When guaranteeing that crystal soaks, be unlikely to make the diffraction quality of too high micromolecule concentration affects crystal, therefore soak

solution

1 and 2 is diluted to separately 10 times of original liquid concentration, standby.

During immersion, utilize instruments such as nylon crystal rings, the protein crystal of the good SARS main protease of growing is taken out from crystallizing pond liquid, add respectively soak

solution

1 and 2 and the soak solution of corresponding 10 times of dilutions in, soak after 4-6 hours the crystal after making the SARS main protease and the Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract contacting.

6. the Collection and analysis of crystal diffraction data

On the basis of step 5, 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.

The screening experiment that carries out in the laboratory can be selected the X-ray machine that uses Rigaku MM007 copper target rotary anode generator to produce X ray for use; Focus lamp by OSMIC company focuses on X ray, makes it can reach the highest intensity; Use the Mar345dtb imaging system of Rigaku RAXIS IV++ imaging plate detector or Marreseach company to collect diffraction data.

For high-throughout drug screening work, can use the ACTOR automatic crystal installation manipulator system and the FR-E X-ray diffractometer of Rigaku company.

When carrying out data aggregation, at first use the nylon crystal rings of Hampton Research company, from soak solution, obtain the crystal that soaks certain hour (being generally 2-48 hours); And use the cooling system of Rigaku company or the cooling system of Oxford Cyrosystem company rapidly, crystal is refrigerated to-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.

(resolution is more preferably greater than 2.6 to select the measured crystal of diffraction matter

) carry out data aggregation, processing, whether detect has micromolecular combination.The statistics of data aggregation such as following table 2:

Complex data aggregation statistics after table 2:SARS main protease parent, SARS main protease and Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample soak

By observing the SARS main protease parent and the complex avtive spot electron-density map (Figure 10) (dead color is the electron density of protein itself) of equal angular, can find, can observe the electron density of nonprotein itself (light tone is represented in the white circle) clearly at the avtive spot of complex, prompting has micromolecule and is combined on the protein molecule.What show among Figure 10 a is near the electron density of avtive spot of SARS main protease precursor structure, and Figure 10 b is that SARS main protease and Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract 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 Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample and the electron density of the SARS 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 (as shown in figure 11) of SARS main protease.

7. in conjunction with micromolecular analysis and evaluation

We mix with Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample SARS main protease (concentration is the SARS main protease aqueous solution of 12mg/ml) according to the ratio of mass concentration ratio 1:1-1:4, and act on 6-18 hours down at 4 ℃-16 ℃.With the sample after the effect, 13,000-15, the 000rpm high speed centrifugation discards post precipitation and is ready for use on mass spectrophotometry.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 Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample, tangible skew has appearred in molecular weight, and this mainly is because the reason that protein molecule combines with micromolecule in the Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample.Can calculate (deducting lower mass spectra peak) by the mass spectrum result and find with higher mass spectrum peak value, in conjunction with the micromolecule molecular weight be about 390Da.

By analyzing existing Rabdosia pseudo-irrorata C. Y. Wu composition, we find, on this molecular weight level, mainly have compound as follows

We can find in the principal ingredient of Rabdosia pseudo-irrorata C. Y. Wu, in this molecular weight ranges, principal ingredient 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 micromolecule at SARS main protease active pocket place and the γ sulphur atom of Cys145 has formed covalent bond, knowing this micromolecule 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 micromolecule of combination is Rabdosia pseudo-irrorata C. Y. Wu third element or Isodomedin.

For further checking, we have obtained the pure compound of Rabdosia pseudo-irrorata C. Y. Wu third element and Isodomedin from Kunming, Chinese Academy of Sciences Yunnan plant, prepare the crystal of Rabdosia pseudo-irrorata C. Y. Wu third element and Isodomedin and SARS main protease complex according to above-described method, and carried out data aggregation (data statistics the results are shown in table 3).By observing electron density, we have only found similar electron density at the crystal of the Rabdosia pseudo-irrorata C. Y. Wu third plain SARS main protease that soaked, shown in Figure 10 .c.Therefore can determine that the inhibitor micromolecule of finding that combines with the SARS main protease should be Rabdosia pseudo-irrorata C. Y. Wu third element from Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample.

Crystal avtive spot at Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample and the Rabdosia pseudo-irrorata C. Y. Wu third plain SARS 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 Rabdosia pseudo-irrorata C. Y. Wu third element is sure lower than pure product in Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract sample, therefore may occupation rate also descend to some extent.

Therefore, the micromolecule that can determine combination is Rabdosia pseudo-irrorata C. Y. Wu third element.

The complex data aggregation statistics of table 3:SARS main protease parent, SARS main protease and Rabdosia pseudo-irrorata C. Y. Wu third element

Thus, we have obtained the three-dimensional structure of sars coronavirus main protease and the Rabdosia pseudo-irrorata C. Y. Wu third plain complex.

Here, we can see the huge advantage that new method of the present invention is compared with traditional drug screening method.According to our method, (1) just only need to screen once can be from the Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract discovery micromolecule that can combine fast with the SARS main protease, (2) this micromolecule is a micromolecule best with the binding ability of SARS in the Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract, and (3) need not to prepare numerous pure product compounds when drug screening; And if utilize classic 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.

8 further biological experiments: Rabdosia pseudo-irrorata C. Y. Wu third element suppresses active mensuration to the SARS main protease and suppresses kinetic parameter and measure

We use pure product compound micromolecule (Rabdosia pseudo-irrorata C. Y. Wu third element), have measured its inhibition activity to the SARS main protease.Rabdosia pseudo-irrorata C. Y. Wu third element suppresses active mensuration to the SARS main protease to carry out according to following steps: at buffer solution (50mM Tris-HCl (pH 7.3), 1mM EDTA (containing or do not contain DTT)) adds SARS main protease (final concentration 0.5 μ M) in, (final concentration is Rabdosia pseudo-irrorata C. Y. Wu third element: 20 μ M), concentration of substrate is 20 μ M, 298K placed after 10 minutes, add fluorescence labeling substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH2, final concentration 20 μ M) rapidly.Excitation wavelength and emission wavelength are respectively 330nM and 395nm, and temperature keeps 298K, writes down first order fluorescence reading (the results are shown in Figure 13) per 2 seconds.Change the concentration of Rabdosia pseudo-irrorata C. Y. Wu third element then, measure its inhibition activity (the results are shown in Figure 14) the SARS 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 Rabdosia pseudo-irrorata C. Y. Wu third element of 20 μ M has stronger inhibition activity to the SARS main protease, when its concentration is 2 μ M, the SARS main protease still be had the activity of inhibition.This Rabdosia pseudo-irrorata C. Y. Wu third element that just illustrates that Chinese medicine Rabdosia pseudo-irrorata C. Y. Wu the inside is contained can effectively suppress the activity of SARS main protease, and the active drug that Rabdosia pseudo-irrorata C. Y. Wu third have may become the treatment sars coronavirus also is described.

Simultaneously, we have measured the accurate inhibition kinetic parameter of Rabdosia pseudo-irrorata C. Y. Wu third element to the SARS main protease.Measuring principle is as follows: by with the course of reaction curve of time-dependent inhibitor according to the match of single order exponential form, promptly can obtain apparent one-level and suppress rate constant (k _Obs).Wherein P is the fluorescence intensity of product, v ₀Be initial reaction rate, t is the time, and D is in order to solve the zero non-vanishing and displacement parameter introduced of fluorescence intensity constantly.Can try to achieve k by formula (1) _ObsIn formula (2), utilize 1/k _ObsTo 1/[I] carry out linear fit, can try to achieve K _iAnd k ₃Wherein [I] is the concentration of inhibitor, and [S] is the concentration of substrate, K _mBe Michaelis constant, k ₃Be the reaction rate constant that inhibitor makes enzyme deactivation, K _iBe inhibitor with enzyme between the non-covalent equilibrium constant that combines.

P＝(v ₀/k _obs)(1-exp(-k _obst))+D (1)

\frac{1}{k_{obs}} = \frac{1}{k_{3}} + \frac{K_{i}}{k_{3}} (1 + [S] / Km) \cdot \frac{1}{[I]} - - - (2)

In the Rabdosia pseudo-irrorata C. Y. Wu third plain kinetic parameter determination experiment, enzyme activity determination method and embodiment 2 steps 4 are basic identical, the concentration of SARS main protease ([E] expression): [E]=0.2uM, the concentration of Rabdosia pseudo-irrorata C. Y. Wu third element (with [I] expression) is at [I]=25～100[E] in the scope, choose 8 different values and measure 8 groups of fluorescence intensities, the fluorescence intensity result is carried out nonlinear fitting to the response curve of time with the Origin program, calculate the K of Rabdosia pseudo-irrorata C. Y. Wu third element thus _iAnd k ₃Value.By aforesaid method, the kinetic parameter that we record Rabdosia pseudo-irrorata C. Y. Wu third element is:

k ₃：7.5±0.710 ^-3s ^-1

K _i：10.3±1.7μM

Embodiment 3

The preparation of Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction sample is basic identical among the preparation of the ethyl acetate extraction sample of Asian puccoon, Radix Isatidis, rhizoma cyperi, semen litchi, Chinese yam, the root bark of Chinese wolf-berry, pyrrosia lingua, root of Chinese trichosanthes, the tuber of stemona, the flower bud of lily magnolia, lichee, 12 kinds of medicinal materials of brave account and the embodiment 1.

Embodiment 4 obtains the new method of the fine three dimensional structure of Acetylshikonin and HCV NS5B protein complexes fast based on the crystal structure of HCV NS5B albumen from the alternative sample of embodiment 3, comprise the steps:

1. choosing of target proteins matter: HCV NS5B albumen

HCV (Hepatitis C Virus) belongs to flaviviridae (flaviviridae), is the pathogen of hepatitis C.Because lack vaccine and effective medicine, HCV infects has become a kind of global epidemic disease.Owing to lack suitable animal model and tissue culture system, can't effectively carry out the amplification and the corresponding virological investigation of virus at present, therefore exploitation becomes a global difficult problem at the specific medicament of HCV.Parsing along with the accurate three-dimensional structure of the important albumen of some HCV reproduction processes and these albumen and associated ligands or inhibitor, based on the three-dimensional structure design of these albumen, the medicine that screening treatment HCV infects, become the important means of present exploitation treatment HCV medicine.NS5B is that (RNA-dependent RNA polymerase RdRp), plays a part crucial (Behrens, people EMBO J 1996:15 (1) 12-22 such as S.E) to the RNA polymerase that relies on of the RNA of HCV in the HCV reproduction process.But the HCV NS5B protein solubility of total length is relatively poor, Ferrari (Ferrari, Deng people 1999) etc. for improving the dissolubility of NS5B, when making up the NS5B expression vector, excise 21 amino acid (being called HCVNS5B Δ 21) of C end, in E.Coil, obtained to keep the soluble albumen of height of enzymatic activity.Though in HCV different genotype and hypotype, the protein sequence of NS5B is not quite similar, the related activity site is but very conservative, this just for we from the NS5B of analysis result, screening provides important basis at the medicine of different genotype.

Up to the present, on NCBIPubMed, can retrieve existing 53 pieces of the article relevant with HCV NS5B inhibitor, but at protein structure database (Protein Database Bank, the three-dimensional structure of the compound that the NS5B that can retrieve PDB) combines with inhibitor has only 15, the exploitation that is to say quite a few inhibitor only proceeds to external enzyme activity determination, and do not obtain the complex three-dimensional structure of corresponding inhibitor and protein bound, therefore do not provide this inhibitor and suppress the definite molecular mechanism of HCV NS5B albumen.

The present invention is a target proteins with HCV NS5B albumen, introduces therefrom and obtains target molecule and target protein fine three dimensional structure fast in the medicines natural products mixture alternate library, carries out the new method of anti-HCV drug screening efficiently.

Be structured in the HCV NS5B Δ 21:genotypelb on the pET-21b carrier; isolate BK (NS5B of 21 residues of hepatitis c virus genotype 1b separated strain BK disappearance C end: amino acid sequence is seen sequence table part 2) plasmid is received in the Gao Guangxia of biophysics institute of Chinese Academy of Sciences professor laboratory; this plasmid is expressed the further separation and purification of laggard row referring to (Ago waits people 1999) in coli strain BL21 (DE3).

2.HCV the crystal growth of NS5B albumen

Crystal growth list of references (the Ago of HCV NS5B, Deng people 1999), and carried out preliminary experiment, found that (21-28% (w/v) polyethylene (PEG) 4000 under the albumen crystallization condition of reporting in the literature, 0.2-0.35M ammonium acetate, 0.1M sodium acetate, and0.02M TES (pH 6.0-7.5) .) though can grow the crystal of bibliographical information, but this crystal not only size is less, twinning is serious, and be difficult to obtain the measured crystal of diffraction matter (Figure 15 .a), be unfavorable for that we carry out further crystal immersion and cocrystallization test, thereby carry out corresponding drug screening work.Therefore we have carried out corresponding improvement to crystallization condition, and crystallization condition is as follows: 20-25% (w/v) PEG4000,10% (v/v) glycerine, 5mM DTT, 50mM MES pH 5.0～6.0.Under the protein concentration of this crystallization condition and 25 ~ 30mg/ml, can access size big (can reach 3 * 1.5 * 1mm usually, Figure 15 .b), crystal that diffracting power is very strong, for further drug screening work provides good crystallography basis.

3. the preparation of natural products mixture alternate sample

With reference to embodiment 3

4. the natural products mixture alternate sample suppresses active mensuration to HCV NS5B

The active determination in vitro method of HCV NS5B is referring to (people .Journal ofVirology such as Robert A., July 2003:7575-7581) the natural products mixture alternate sample is to the active following mensuration of the inhibition of HCV NS5B: at buffer solution 20mM Tris-HCl (pH7.5), 5mM MgCl ₂, 0.5mM MnCl ₂, 1mM DTT, 1mg/ml polyA, 1mg/ml OligodT (18T), 1mg/ml UTP, 1mg/ml P ³²Add HCV NS5B albumen (final concentration 0.8 μ g/ml) among-UTP, the DMSO dissolved matter of natural products mixture alternate sample (final concentration 20ug/ml, 10ug/ml), sampling in per 5 minutes is once dripped on the DEAE filter paper of handling well the 2-4 μ l reacted solution that arrives sample time with cessation reaction.After reaction is finished, successively wash DEAE filter paper 4 times with 2 * SSC solution and 95% ethanol, to remove remaining P ³²-UTP after drying, uses the exposure of PhosphorImage or egative film, to measure the inhibition activity of Chinese medicine mixture alternate sample to HCV NS5B, the results are shown in Figure 16.The ethyl acetate extraction sample of Asian puccoon, Chinese yam, brave account has better inhibited activity to HCV NS5B albumen as seen from Figure 16.Selected these samples carry out next step crystal immersion work.

5.HCV the crystal of NS5B albumen soaks Asian puccoon ethyl acetate extraction sample

The method that adopts crystal to soak makes the crystal of HCV NS5B albumen contact with Asian puccoon ethyl acetate extraction sample.

The preparation method of the crystal soak solution of 5 SARS main proteases is basic identical among the preparation of the crystal soak solution of HCV NS5B albumen and the embodiment 2.The method of the method for the crystal immersion Asian puccoon ethyl acetate extraction sample of HCV NS5B albumen and the crystal immersion Rabdosia pseudo-irrorata C. Y. Wu ethyl acetate extraction sample of SARS main protease is basic identical.

6, the Collection and analysis of crystal diffraction data

Basic identical in the data analysis of the crystal of HCV NS5B albumen and crystal (abbreviation complex) after Asian puccoon ethyl acetate extraction sample contacts and embodiment 2 steps 6.

The crystal and the complex avtive spot electron-density map (Figure 17) of the HCV NS5B albumen parent by observing equal angular, can find, can observe the electron density of nonprotein itself (light tone part in the white circle) clearly at the avtive spot of complex, prompting has micromolecule and is combined on the protein molecule.

7 in conjunction with micromolecular analysis and evaluation

Another analysis that the step 7 of employing the inventive method embodiment proposes is analyzed the micromolecule of combination in conjunction with micromolecular method and is identified.

Asian puccoon (Sinkiang) belongs to the medicinal per nnial herb of Boraginaceae, and it is sweet, cold in nature to distinguish the flavor of, thoughts of returning home lung channel.Remarkable efficacy because of its cooling and activating blood, detoxifcation promoting eruption all is documented in each traditional Chinese medicine ancient books and records.In Compendium of Material Medica, be listed in top grade (being used as medicine) with root with the cool blood that induces sweat, clearing heat and detoxicating, wide spectrum curative effect such as activate blood circulation and disperse blood clots.In modern medicine study, because of its have anti-infective, promote numerous effects such as wound healing, antitumor, antimicrobial and antithrombotic, be widely used in medical research and disease treatment (people such as Papageorgiou, " 1999. The Chemi stry and Biology of Alkannin; Shikonin, and Related Naphthazarin Natural Products. " Angew.Chem.Int.Ed.39:270-300).

Up to the present, identified the multiple principal ingredient in the Asian puccoon, these principal ingredients play critical effect to Asian puccoon in the curative effect aspect the treatment disease.And correlative study (people such as Papageorgiou, 1999. " The Chemistry and Biology of Alkannin; Shikonin; and RelatedNaphthazarin Natural Products. " Angew.Chem.Int.Ed.39:270-300) these compositions of table name mainly all are the derivants of alkannin (skikonin/alkannin) parent nucleus, the structural formula of this parent nucleus is:

The structural formula of the parent nucleus of principal ingredient in the Asian puccoon.Wherein R group difference is represented different compounds.Known R group comprises as shown below 36 kinds at present.We are at first according to the demonstration of unknown electron density, and it is excessive and can not put into the compound of this place's electron density to remove some groups.Prepare candidate's micromolecular PDB data then, read in COOT after, utilize the function of the Realspace Refinement of COOT, different micromolecule is revised in electron density.The result shows that coincideing of Acetylshikonin and unknown electron density is better, and the result as shown in figure 18.Obtained the fine three dimensional structure of Acetylshikonin and HCV NS5B protein complexes thus.

In the present invention, the proposition of our initiative based on the crystal structure of protein, therefrom carry out drug screening in the medicines natural products mixture alternate library, obtain the new method of part micromolecule complex fine three dimensional structures such as target proteins matter molecule and inhibitor.Its main innovate point and significant values are: at first, this method is that this ancient medical science essence of China of Chinese medicine has been opened up a new development course, illustrates the scientific basis of Chinese medicine curative effect with the method for modern science; Secondly, the preparation and the optimization method of the Chinese medicine screening spare library of natural product mixture medicament such as that proposes in this method, not that the effective constituent in the Chinese crude drug (or composition) separation and purification is one by one come out, but with of the set of a kind of Chinese crude drug as a natural products compound, again it is carried out certain optimization, guaranteeing the rational while of small numerator medicament sifting motion, as much as possible the chemical constitution in every kind of medicinal material is kept, significantly reduce workload, and improved screening effeciency and hit rate greatly; Once more, because it is potpourri (the alternative sample of each part is the set of a variety of micromolecular compounds) that the inventive method is used for the alternative sample in the alternative storehouse of drug screening, make when utilizing method of the present invention to carry out drug screening, natural just carried out different ligands molecule and the emulative combination of target protein in the alternative sample, thereby can therefrom screen and obtain the best micromolecular compound of binding ability.

In embodiment 2 and embodiment 4, we have showed the inhibitor that utilizes method of the present invention to screen the SARS main protease that obtains from the Rabdosia pseudo-irrorata C. Y. Wu acetic acid ethyl ester extract, and obtained the fine three dimensional structure of the plain and SARS main protease complex of Rabdosia pseudo-irrorata C. Y. Wu third, accurately measured the kinetic constant of Rabdosia pseudo-irrorata C. Y. Wu third element to the inhibition of SARS main protease, the result hints that this Rabdosia pseudo-irrorata C. Y. Wu third have the active drug that prestige becomes treatment SARS.Also screen the inhibitor that has obtained HCV NS5B simultaneously from the Asian puccoon acetic acid ethyl ester extract, obtained the fine three dimensional structure of Acetylshikonin and HCV NS5B protein complexes, the hint Acetylshikonin is expected to become the active drug of treatment HCV.

Based on the crystal structure of protein, therefrom carry out the drug screening further deep development and perfect that awaits in the alternative storehouse of medicines natural products mixture drug screening.In work from now on, we also will develop corresponding software and be used for to analyzing in conjunction with micromolecular electron density, set up the virtual data base of the micromolecular compound that has identified in the various middle medicines natural products, and use software from this storehouse, the electron density that observes to be compared, so that micromolecular evaluation is provided prompting, even directly identifies.Secondly, obtain in screening after the precursor micromolecule such as possible part, inhibitor, can use the method for chemistry, these precursors are carried out corresponding transformation and optimization, to improve its binding ability.Once more, comprise the biological experiment of cell experiment, virology experiment or the like, also can further going deep into as work of the present invention.

Obviously, the foregoing description only is for example clearly is described, and is not the qualification to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here need not also can't give exhaustive to all embodiments.And conspicuous variation of being extended out thus or change still are among the protection domain of the invention.

Sequence table

＜110〉Tsing-Hua University, Nankai University, Institute of Biophysics, Academia Sinica

＜120〉therefrom obtain the new method of target molecule and target protein complex fine three dimensional structure in the medicines natural products mixed system efficiently, fast

<160>2

<210>1

<211>306

<212>prt

＜213〉sars coronavirus (SARS Coronavirus)

<400>1

<210>2

<211>566

<212>PRT

＜213〉hepatitis c virus genotype 1b separated strain BK (Hepatitis C Virus HCV, genotype 1b, isolate BK)

<400>2

Claims

1, a kind of method of obtaining target molecule and target protein complex fine three dimensional structure from natural products mixture alternate library efficiently, fast is based on protein structures obtains target molecule and target proteins complex from natural products mixture alternate library fine three dimensional structure; It is characterized in that comprising the steps: that the crystal with target proteins matter contacts with the natural products mixture alternate sample, the crystal of analyzing target proteins matter and X-ray diffraction in crystals data after the natural products mixture alternate sample contacts, the electron density of the target molecule that acquisition combines with target proteins, and then the fine three dimensional structure of acquisition target molecule and target protein complex.

2, method according to claim 1 is characterized in that comprising the steps:

(1) chooses target proteins matter;

(2) crystal of preparation target proteins matter;

(3) preparation natural products mixture alternate library;

3, method according to claim 2 is characterized in that comprising the steps:

(8) optimization of target molecule and further biological experiment.

4, method according to claim 2, it is characterized in that the target proteins matter of choosing in the step (1) is if report that it has the active determination in vitro method, then will carry out following steps (4) between step (3) and step (5): the natural products mixture alternate sample is to the active determination in vitro experiment of target proteins matter, choose target proteins matter is had the sample that suppresses active, these samples are called " have suppress active " sample, will " have suppress activity " sample and target proteins matter carry out step (5).

5, method according to claim 2 is characterized in that in the step (1), and the target proteins matter of choosing parses the protein of its three-dimensional structure for the method for learning by X ray diffractive crystal.

6, method according to claim 2 is characterized in that the preparation of described natural products mixture alternate library comprises the steps:

7, method according to claim 6 is characterized in that in the step (a), when elementary raw material is prescriptions of traditional Chinese medicine, removes and use the ethanolic solution refluxing extraction, outside the medicinal extract, also get primary raw materials in addition, water decocts, decoction liquor merges, concentrated medicinal extract; Extract two parts of medicinal extract that obtain with ethanolic solution and water, all carry out next step processing respectively as the extract medicinal extract of prescriptions of traditional Chinese medicine.

8, method according to claim 7 is characterized in that step (a) is: get a certain amount of primary raw materials, use the 70%-95% alcohol reflux, merge extract, concentrate medicinal extract; For the preparation of the extract medicinal extract of prescriptions of traditional Chinese medicine, then form primary raw materials according to medicinal material kind and the 1-6 times of consumption of regulation in the prescription, make medicinal extract one with 70%-95% ethanol by above-mentioned same method respectively; In addition, equally then form primary raw materials according to medicinal material kind and 1-6 times of consumption of regulation in the prescription, decoct with distilled water, decoction liquor merges, concentrate medicinal extract two; Medicinal extract one and medicinal extract two all carry out next step processing respectively as the extract medicinal extract of prescriptions of traditional Chinese medicine; Extract preparation for microbial metabolic products, then be that the nutrient solution that will contain an amount of microorganism filters, obtain solid microbe mycelium and nutrient solution filtrate, mycelium is used the ultrasonication of 60%-90% acetone again, filter, filtrate concentrate medicinal extract, this medicinal extract and nutrient solution filtrate are all carried out next step processing respectively as the extract of microbial metabolic products.

9, method according to claim 8 is characterized in that used amount of alcohol and primary raw materials mass ratio are 6:1-12:1 in the step (a); Acetone and mycelium mass ratio are 8:1-30:1; The mass ratio of medicinal extract and suspension water is 1:5-1:15 in the step (b), extraction is ethyl acetate or chloroform with organic solvent, the volume ratio of its volume and suspension water is 6:1-10:1, and extraction is 1:8-1:1 with the volume ratio of volume of organic solvent and nutrient solution filtrate.

10, method according to claim 11 is characterized in that step (c) is:

Aqueous portion after the extraction in the step (b) is steamed a small amount of organic solvent of the middle dissolving that dewaters, separate with macroporous resin column chromatography, water, 50% ethanol, 95% ethanol are the moving phase wash-out successively, discard the washing part; 50% ethanol and 95% ethanol elution part steamed respectively desolventize, the shape sample that gets dry extract is standby.

11, according to the described method of each claim of claim 1-10, the primary raw materials of wherein said natural products mixture alternate library is selected from any in following: mixing medicinal material, microorganism, sea life that medicinal animals and plants, the medicinal material kind of pressing the prescriptions of traditional Chinese medicine regulation and consumption are formed.

12, method according to claim 11 is characterized in that described sea life are marine alga or sponge.

13, method according to claim 11 is characterized in that the target molecule in the step (5) is the ligand molecular that comprises inhibitor that can combine with target proteins matter that exists in the natural products mixture alternate sample.

14, method according to claim 11 is characterized in that making the target proteins crystal to contact with the natural products mixture alternate sample, and the method for using is soaked or cocrystallization as crystal.

15, method according to claim 14 is characterized in that crystal soaks the mode that can take " soaking separately ", " grouping is soaked " or " all mix and soak ".

16, method according to claim 11 is characterized in that described target proteins is the sars coronavirus main protease.

17, method according to claim 11 is characterized in that described target proteins is a HCVNS5B albumen.