CN103184264B - HIV type I protease inhibitor screened out from crude extract of Berberis nummularia Bge, and application thereof - Google Patents
HIV type I protease inhibitor screened out from crude extract of Berberis nummularia Bge, and application thereof Download PDFInfo
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Abstract
The invention provides a method for screening an HIV protease inhibitor based on combination of a fluorogenic substrate and crystal soak. The method comprises the steps of measuring inhibitory activity of a series of compounds that are separated and purified from natural products to the HIV-1 protease; and finally separating and purifying the series of compounds into a single component, wherein the single component is a small molecular inhibitor of the natural products. With the above method, the extract of the Berberis nummularia Bge is found to have effective inhibitory activity for the HIV-1 protease; small molecules with relatively strong combination capacity for the HIV-1 protease are found via the crystal soak method; then the inhibitory activity of the small molecules to the HIV-1 protease is determined via enzyme activity assay; and finally a small molecular inhibitor capable of effectively inhibiting the activity of the HIV-1 protease is provided. Besides, a method for screening anti-HIV materials by the combination of the small molecular substance and an HIV protease compound is provided, novel sites that the HIV protease can interact to the inhibitor are provided, and a method for screening the anti-HIV active materials by using the sites is provided.
Description
Technical field
The present invention relates to a kind of screening technique of anti-AIDS virus substance and its application, especially by fluorogenic substrate and crystal
Immersion joint screening HIV (human immunodeficiency virus) protease inhibitor, is isolated and purified out in natural product using biological activity tracking and testing
A series of inhibitory activity of compound components to HIV-1 protease, until be finally recovered being purified into the single natural product of composition
Thing micromolecular inhibitor.
Background technology
Acquired immune deficiency syndrome (AIDS), complete entitled " acquired immune deficiency syndrome (AIDS) " (the Acquired Immune Deficiency of medical science
Syndrome--AIDS), present scientific research is widely considered to be human infection HIV (human immunodeficiency virus) (Human
Immunodeficiency virus, abbreviation HIV) the caused infectious disease of institute.HIV (human immunodeficiency virus) belongs to Retroviridae
(Retroviridae), slow viruss (lentivirus) category, primatess immunodeficiency subgenus.HIV point is confirmed for amphitypy：
HIV-1 types and HIV-2 types, wherein IV-2 is distributed mainly on African western part, also tested in some the infecteds in Europe and America
Measure, but virulence and transmissibility are below HIV-1, and the acquired immune deficiency syndrome (AIDS) course of disease for causing is relatively slow and relatively relaxes.HIV-1 is distributed widely in generation
Boundary various places, are the cause of diseases for causing whole world AIDS popular, and the research of current HIV is also to be carried out based on HIV-1.Acquired immune deficiency syndrome (AIDS) disease
Oral cavity, genitals, anus of the permeable directly contact mucous membrane tissue of poison etc. or with virulent blood, seminal fluid, vaginal secretion,
Milk and infect.HIV (human immunodeficiency virus) is after since 20th century, the eighties is spread by the African Territories on the south the Sahara, so far into
For one of global pandemic disease, at present, scientist not yet has found the vaccine that can completely resist HIV (human immunodeficiency virus), does not also have
Finding can effect a radical cure the medicine of acquired immune deficiency syndrome (AIDS), therefore, exploitation is always a global difficult problem for the specific medicament of HIV.
The auxiliary type T cell being widely present in HIV (human immunodeficiency virus) main infection human immune system, macrophage and tree
Prominent shape cell.After cell entry is intracellular, will start by Reverse Transcriptease (reverse transcriptase) leading transcription
Process, is double-stranded DNA by the viral single stranded RNA reverse transcription of itself, then in the presence of integrase (intergrase), by disease
In the DNA of malicious DNA insertions human body itself, follow the duplication of host DNA itself and replicate.And using the copy machine table of host
Up to the albumen that itself needs.After viral protein expression is completed, by HIV (human immunodeficiency virus) protease by immaturity Protein cleavage shape
Into ripe albumen, assembling obtains continuing to infect next cell after ripe virion.
The genome main code of HIV structural protein, modulin and auxilin.Wherein structural protein include
Gag, Pol and Env.Gag gene expresses the albumen to form one section of 55kD, in HIV-1Protease (HIV (human immunodeficiency virus) albumen
Enzyme) in the presence of, cutting forms four sections of little albumens, is respectively matrix protein A (p17), capsid protein CA (p24), nucleocapsid egg
White NC (p9), and p6 albumen；Gag-Pol fusion protein is in the presence of HIV-1Protease (HIV (human immunodeficiency virus) protease)
Four Ge little albumens are cut to, are respectively protease (p10), reverse transcriptase (p50), RNase H (p15) and intergrase (p31).Env
It is initially to synthesize in endoplasmic reticulum, is the protein that a segment molecule amount is 88kD, it is sugared in the transport process to Golgi body
Base and become the envelope glycoprotein precursor of molecular weight 160kD (gp 160).Gp160 is segmented into gp by the proteolytic cleavage in host's body
120 and gp41 two parts, gp120 is located at the surface of infection cell and virion, claims outer membrane protein, gp41 to be embedded in the lipid of virus
In bilayer, claim transmembrane protein, the fusion of mediate retroviral adipose membrane and cell membrane is capable of in virus infection.In addition, HIV
The regulation and control such as tat, nef, rev, vpu, vpr, vif and auxilin are also encoded in genome.
From the biocycle of HIV (human immunodeficiency virus) and genomic constitution HIV (human immunodeficiency virus) protease is can be seen that for acquired immune deficiency syndrome (AIDS) disease
The existence of poison has vital effect with breeding, if it is possible to effectively suppress the activity of HIV (human immunodeficiency virus) protease, that
HIV (human immunodeficiency virus) will be unable to form new ripe virion, so as to prevent sending out for acquired immunodeficiency disease (AIDS)
It is raw.
HIV-1 protease belongs to aspartic protease family, is made up of with symmetric same two same subunits
Source dimer, each subunit contains 99 aminoacid.According to the research of protease structure and substrate, people are constantly looking for albumen
The inhibitor of enzyme specific bond, totally 9 kinds of the PIs of current Jing FDA approvals：Saquinavir, ritonavir, indinavir,
Nelfinavir, amprenavir, lopinavir, atazanavir, tipranavir, darunavir.Except
Outside tipranavir, in addition 8 kinds of inhibitor are all polypeptide analog (bioavailability are low), and competitive inhibitor all contains one
Individual hydroxy vinyl division center, tipranavir is then a dihydro pyrone ring structure.Due to " HAART " HAART
The application of (Highly Active Antiretroviral Therapy), these inhibitor really can during previous tretament
Effectively suppress the propagation of HIV, but because patient will undertake high medical expenses, higher injection dosage, fever, abdomen
Rush down, the toxic and side effects such as nausea, and the high mutation rate of inhibition of HIV develops immunity to drugs, acquired immune deficiency syndrome (AIDS) endangers always the health of the mankind, although
The numerous medical researchers in the whole world have paid huge effort, and the specific medicament of radical cure acquired immune deficiency syndrome (AIDS) is not yet developed so far,
Not can be used for the effective vaccine for preventing, this also promotes people constantly looking for new anti-HIV-1 medicines.
With accurate the three of the important albumen and these albumen in some HIV reproduction processes and associated ligands or inhibitor
The parsing of dimension structure, based on the three-dimensional structure modeling of these albumen, the medicine of screening treatment HIV, becomes exploitation at present and controls
Treat the important means of HIV medicines.Although in HIV different genotype, hypotype and mutant, the protein sequence of protease
Row are not quite similar, but its main assembly and related activity site are but very conservative, and this is just us from analytic structure
Protease set out, screen the protease medicines that are directed to of specificity and provide important foundation.
Natural product has structured multiformity and life also known as secondary metabolite (Secondary Metabolites)
The multiformity of thing activity.Natural product and its derivant have played promising effect in conventional disease treatment, are also
One of most potential resource in current drug development process.Now to the conventional medicaments such as Chinese medicine, marine organisms and microorganism
Natural products research in metabolic process is in the ascendant, and the compound that can all have a large amount of structures novel every year is found to provide
Come, these compounds are the important sources of the medicine that synthetic method to be realized and lead compound, in new drug
With play an important role in the discovery of lead compound.
Medicine or the main source of its lead are always using external drug model screening natural product.In-vitro screening one
As carry out enzymatic reaction by substrate of small peptide, then carried out respectively according to the characteristics of the presence or absence of substrate labelling and label HPLC,
Radioactivity, fluorescence analysiss or ELISA detection.According to these methods, people obtain some from separate sources suppression hiv protease
The material of activity.Illustrate prior art as a example by our fluorescence analysiss below：
List of references Matayoshi ED, Wang GT, Krafft GA, Erickson J.Novel fluorogenic
substrates for assaying retroviral proteases by resonance energy
transfer.Science.1990Feb 23；247(4945)：954-8.
FRET (fluorescence resonance energy transfer) (fluorescence resonance energy transfer, FRET) technical measurement
It is a kind of detection method the most frequently used at present for the activity of the inhibitor of HIV-1 protease, Main Basiss HIV-1 protease
Recognition site designs and synthesizes artificial polypeptide substrate：
DABCYL-g-Abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS, when inhibitor is to HIV-1 eggs
When white enzyme does not have inhibitory action, HIV-1 protease can cut the peptide bond between substrate Tyr-Pro, quenching group (DABCYL)
Away from fluorophor (EDANS), fluorophor absorbs 336nm wavelength, inspires the wavelength of 472nm, detects glimmering by detector
Light intensity, if inhibitor has inhibitory action to HIV-1 protease, peptide bond is not cut open, between quenching group and fluorophor
It is closer to the distance, promote the wavelength rear portion energy that fluorophor absorbs 336nm to be transferred to quenching group, 336nm's swashs
Sending out intensity of wave will weaken, and inspection does not measure the change of fluorescence intensity, judge that suppression of the inhibitor to HIV-1 protease is imitated with this
Really.
According to testing compound presence or absence of when fluorescence intensity change, compound can be calculated to proteinase activity
Inhibition level or residual activity, so as to complete the preliminary screening to protease inhibitor.Can be in the short time using the method
The screening operation to a large amount of samples is inside completed, the model can be used for the known material for having an Anti-HIV-1 Active, to check it to make
Whether it is protease with position target spot.
Fluorogenic substrate HIV-1 protease inhibitor screening models are to commonly use screening model at present, the method operation letter
It is single, it is easier to realize the high flux screening of compound, but this system there is also certain defect, such as compound candidate not
Too high fluorescent value can be contained, false positive easily occurs in the selection result.With the development of bioinformatics and structure biology, medicine
Materialization scholars increasingly focus on the compound for having an inhibitory activity found during enzyme kineticss are determined, with egg on atomic level
Interaction sites and model of action between white matter.This is also that the enzyme activity determination being currently based on molecular level is unable to reach
's.
The content of the invention
The present invention is provided a kind of immersion based on fluorogenic substrate and crystal and combines screening HIV (human immunodeficiency virus) protease inhibitor
Method.At present, a series of compound groups for generally being isolated and purified out step by step in natural product using biological activity tracking and testing
The inhibitory activity (including complex components and one-component) to HIV-1 protease, final separation is divided to be purified into single group of composition
Point, this one-component is exactly natural product micromolecular inhibitor, and the present invention has found Berberis nummularia Bge. using fluorogenic substrate screening technique
Complex components be capable of the activity of effective Inhibit HIV-1 Protease, the method soaked by crystal is found and HIV-1 protease
The stronger small molecule of binding ability, then passes through again inhibitory activity of the enzyme activity determination small molecule to HIV-1 protease.Finally, carry
For a kind of micromolecular inhibitor that can effectively suppress HIV (human immunodeficiency virus) proteinase activity.
The present invention provides a kind of Traditional Chinese Herb inhibitor that can effectively suppress HIV (human immunodeficiency virus) proteinase activity.
The present invention provides a kind of noncompetitive inhibitor that can effectively suppress HIV (human immunodeficiency virus) proteinase activity.
The present invention is also provided a kind of combination with HIV (human immunodeficiency virus) proteinase complex by small-molecule substance and screens anti-AIDS
The method of viral material.
The present invention also provides the crystal structure of a kind of micromolecular inhibitor and HIV (human immunodeficiency virus) proteinase complex and is somebody's turn to do
The preparation method of crystal structure.
Present invention also offers a kind of HIV (human immunodeficiency virus) protease can be with the novel site of inhibitor interaction and utilization
The method that anti-HIV activity material is screened in the site.
The invention provides a kind of Traditional Chinese Herb inhibitor is being prepared for virus infection for the treatment of or prevent AIDS
Medicine in purposes.
The invention provides one kind comes from screen to obtain in middle medicines natural products mixture alternate sample targeting and HIV-
1protease protein binding, and the best micromolecular inhibitor of binding ability --- berberine hydrochloride.
Due to composition contained in Berberis nummularia Bge. it is considerably complicated, it is therefore necessary to its crude extract is optimized, it is therefore an objective to
Remove to the interference factor in HIV (human immunodeficiency virus) protease active determination in vitro and crystal immersion test such as albumen, polysaccharide etc., protect
The preferable small molecule of the retained property of medicine simultaneously is allowed to concentrate as far as possible, improves its relative concentration, is applied to HIV (human immunodeficiency virus) egg with preparing
The alternative sample of inhibitor screening is carried out based on white enzyme crystal structure.The present invention provide not only can realize this purpose system
The technical scheme of standby alternative sample, and have the sample of inhibitory activity to screening, separated, identified with various chromatographs, spectral method
The wherein structure of micromolecular inhibitor-berberine hydrochloride.
Present invention separation section also each to Berberis nummularia Bge. is (i.e. with each isolated portions of method such as extraction, macroporous resins
Point sample) carried out the vitro inhibition determination of activity of HIV (human immunodeficiency virus) protease.In the Berberis nummularia Bge. ethyl acetate for filtering out
Berberine hydrochloride still stronger inhibitory activity is shown to HIV (human immunodeficiency virus) protease in 100 μM of concentration, be preferable
Medicine or potential prodrug.
The present invention explains the inhibitory action mode between compound and albumen also from structure, there is provided an acquired immune deficiency syndrome (AIDS) disease
The novel site that toxalbumin enzyme can interact with inhibitor, there is provided one can effectively suppress HIV (human immunodeficiency virus) proteinase activity
Noncompetitive inhibitor, this provides foundation and the reference of preciousness for further drug design and screening.
Abbreviation used in this application and Key Term are defined as：
HIV-1PR：1 type hiv protease
AIDS：Acquired immune deficiency syndrome (AIDS)
LB：Luria-Bertani culture medium
SOC：Containing nutrition than a kind of culture medium of LB culture medium more horn of plenty, can be used for the competent cell after electricity conversion
Recovery
IPTG：Isopropylthio-β-D galactosides, a kind of lactose analog.
PCR：Polymerase chain reaction
FRET：FRET (fluorescence resonance energy transfer), when a fluorescence molecule (being also called donor molecule) fluorescence spectrum with it is another
When the excitation spectrum of individual fluorescence molecule (being also called acceptor molecule) overlaps, the excitation energy induction acceptor molecule of donor fluorescent molecule
Fluorescence is sent, while the donor fluorescent molecule fluorescence-intensity decay of itself.
The screening technique of the AIDS virus resisting material that the present invention is provided specifically includes following steps：
(1). HIV (human immunodeficiency virus) protease is prepared, and crystallization obtains the crystal of HIV (human immunodeficiency virus) protease；
(2). natural extracts are prepared, the extract is included by extraction, solvent extraction or macroporous resin, silica gel
Deng each position that column chromatographic isolation and purification is obtained；
(3). the natural extracts that step (2) is obtained are dissolved inOrganic solvent；UseBuffer solutionTo in step (1)
The dissolving of HIV (human immunodeficiency virus) protease, dilution, then two kinds of solution are mixed to get into mixed solution, prepare fluorescent substrate solution,
Fluorescent substrate solution is added in above-mentioned mixed solution again；
(4). the fluorescence intensity change of detection sample, and compare with the negative controls for being added without natural extracts,
Obtain that there is the bioactive natural product extract for suppressing HIV (human immunodeficiency virus) protease.
In said method, the HIV (human immunodeficiency virus) protease is 1 type hiv protease, is prepared used
The aminoacid sequence of HIV-1protease plasmids is PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKM
IGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF。
In said method, further include using the method for tracking activity, pair active natural product for determining is carried
Take thing further to isolate and purify, to the component repeat step (3) that isolates and purifies and (4), such multiple stage separation, purification and step
(3) determination of activity of-(4), finally gives active monomeric compound.
In said method, described organic solvent is preferred：Pentane, hexane, octane, hexamethylene, Ketohexamethylene, toluene hexamethylene
Ketone, chloroform, dichloromethane, methanol, ethanol, isopropanol, ether, methyl acetate, ethyl acetate, propyl acetate, acetone,
Espeleton, methylisobutylketone, glycol monoethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, acetonitrile, pyridine, dimethyl are sub-
Sulfone, dichloro Methanamide.
The fluorogenic substrate is preferably：
MCA-γ-Abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(Dnp)-Lys-NH2.The fluorescence bottom
Specific cleavage sequence of the aminoacid sequence of thing from HIV (human immunodeficiency virus) protease.Instrument for fluorescent strength determining is
The ripple of Fluoraskan Ascent luminoscopes (ThermoLabsystems, Helsinki, Finland), exciting light and launching light
It is long to be respectively 320nm and 405nm.
According to the middle medicines natural products for carrying out the inhibitor screening of HIV-1protease albumen prepared by the present invention
Sample in mixture alternate library is referred to as natural products mixture alternate sample.It is preferred that natural products mixture alternate sample is molten
Its final concentration of 50mg/mL is made in dimethyl sulfoxide (DMSO), the DMSO dissolvings of natural products mixture alternate sample are obtained
Thing.
Buffer solution (50mM NaAc, pH5.2,1mMEDTA, 2.5mM DTT, 1.0M are preferably used in the inventive method
NaCl, 2.5% (v/v) glycerol) 10mg/ml HIV (human immunodeficiency virus) protease is carried out into 100 times of dilutions.Dissolved using DMSO
Fluorogenic substrate, makes final concentration of 10mM, then with buffer solution (50mM sodium acetate, pH5.2,1mMEDTA, 2.5mM
DTT, 1.0M NaCl, 2.5% (v/v) glycerol) ten times of dilution fluorogenic substrates.
The inventive method preferably uses 96 hole elisa Plates, and the protein solution of the above-mentioned dilutions of 97 μ L is added in every hole, adds 1
The reaction buffer solute of the above-mentioned natural products mixture alternate samples of μ L is (as Berberis nummularia Bge. ethyl acetate extracts the anti-of sample
Answer buffer solution thing), after 30 degree are placed 10 minutes, it is rapidly added the fluorescence labeling substrate of the above-mentioned dilutions of 2 μ L.Excitation wavelength and
Launch wavelength is respectively 320nm and 405nm, and temperature is kept for 30 degree, and first order fluorescence reading was recorded per 3 seconds, reads 60 times altogether.
In said method, negative controls are preferably added without natural extracts, are replaced with 1 μ L100%DMSO, remaining
Preparation condition is identical with natural extracts.
Natural extracts are preferably Berberis nummularia Bge. through ethanol extraction, ethyl acetate extraction in the method for the invention
Then separated with HP-20 types macroporous resin column chromatography, obtain the alcohol elution of 30%-80%.The monomeric compound is excellent
Elect berberine or its pharmaceutically-acceptable salts as.
The present invention also provides a kind of bioactive natural product extract, activated monomer chemical combination obtained by above-mentioned screening technique
The application of thing and berberine or its pharmaceutically-acceptable salts in Anti-AIDS Drugs are prepared.
The present invention also provides a kind of method that anti-hiv drug is screened in site by hiv protease, and its feature exists
In：The site is the binding site of berberine or its pharmaceutically-acceptable salts and HIV-1 protease.
The present invention also provides the binding site on a kind of HIV-1 protease, it is characterised in that：The binding site is Radix Berberidis Amurensis
The binding site of alkali or its pharmaceutically-acceptable salts and HIV-1 protease.
Description of the drawings
Fig. 1 is the activity suppression curve chart of the screening sample to HIV (human immunodeficiency virus) protease of Berberis nummularia Bge. of the present invention, sample
Concentration：50mg/mL.1：Control (being added without the activity curve of the HIV (human immunodeficiency virus) protease of any sample) 2：Berberis nummularia Bge. is always carried
Take activity suppression curve 3 of the thing sample to HIV-1 protease：Berberis nummularia Bge. ethyl acetate extracts sample to HIV-1 protease
Activity suppression curve 4：Activity suppression curve of the ethanol elution sample of Berberis nummularia Bge. macroporous resin 50% to HIV-1 protease.
Fig. 2 is half suppression curve figure of the berberine hydrochloride of the present invention to HIV (human immunodeficiency virus) protease.HIV-1 protease is dense
Degree：500nM, 20 μM of concentration of substrate, the IC of berberine hydrochloride50For 114.8 ± 17.6 μM.
Fig. 3 is the Michaelis-Menten equation curve of HIV (human immunodeficiency virus) protease of the present invention and substrate reactions.HIV-1 protease concentrations：
500nM, concentration of substrate is respectively 200.000 μM, 180.000 μM, 150.000 μM, 120.000 μM, 100.000 μM, 50.000 μ
M, 25.000 μM, 12.500 μM, 6.250 μM, 3.125/ μM.Michaelis constant Km24.29 ± 3.7 μM, 96.24 μm of oL/L/ of Vmax
s。
Fig. 4 is that HIV (human immunodeficiency virus) protease is first with substrate reactions when berberine hydrochloride of the present invention is present in reaction system
Speed and concentration of substrate curve.Reaction system berberine hydrochloride final concentration is respectively 0 μM, 80 μM, 120 μM, and protein concentration is
500nM, concentration of substrate is respectively 200.000 μM, 180.000 μM, 150.000 μM, 120.000 μM, 100.000 μM, 50.000 μ
M, 25.000 μM, 12.500 μM, 6.250 μM, 3.125/ μM, the Ki values of berberine hydrochloride are 180.2 ± 16.8 μM, hydrochloric acid Radix Berberidis Amurensis
It is noncompetitive inhibitor that alkali suppresses type.
Fig. 5 is the electron density map of HIV-1 protease and berberine hydrochloride compound crystal.What dotted line inframe was represented is salt
The cloud density of sour berberine, what is represented outside dotted line frame is the cloud density of HIV-1 protease.By the AIDS of 10mg/ml
Sick protease mixes with Berberis nummularia Bge. ethyl acetate extraction sample with 4: 1, and crystal growth is carried out after high speed centrifugation, finally gives salt
The crystal of sour berberine and HIV-1 proteinase complex.
Specific embodiment
The following examples can make those skilled in the art that the present invention is more fully understood, but limit never in any form
The present invention.
Embodiment 1：The expression of HIV (human immunodeficiency virus) protease, purification and crystal growth
Professor's laboratory chivalrous in biophysicss institute of Chinese Academy of Sciences bloom is received in HIV-1cDNA libraries (pNL4-3), using polymerization
Polymerase chain reaction obtain HIV-1Protease genetic fragment, is built and pET-11a carriers on.
PCR upstream and downstream primers are respectively：
5’protease-11a-s ATCATATGGCCGATAGACAAGGAAC
5’protease-11a-as GCGGATCCCTAAAAATTTAAAGTGC
The gene order of HIV-1protease：
GCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAGCGACCCCTCGTCACA
ATAA
AGATAGGGGGGCAATTAAAGGAAGCTCTATTAGATACAGGAGCAGA
TGATACAGTATTAGAAGAAATGAATTTGCCAGGAAGATGGAAACCAA
AAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGAT
CAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATT
AGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTC
AGATTGGTTGCACTTTAAATTTTTAG
Aminoacid sequence：
PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGH
KAIGTVLVGPTPVNIIGRNLLTQIGCTLNF
(1) the HIV-1protease plasmids by above-mentioned structure on pET-11a carriers are converted to E.coli BL21 (DE3)
Bacterial strain, and with LB flat boards (ampicillin containing 100mg/L) screening positive clone.
(2) the LB culture medium of 1L is proceeded on the flat board described in (1) after picking positive colony overnight incubation (containing 100mg/
L ampicillin), when OD600 reaches 0.6-0.8,200 μM of IPTG is added, cultivate 6 hours or so at 37 DEG C.
(3) 5000-8000rpm/min centrifugations 10-15min collects cell, then ice-bath ultrasonic broken bacterium 20-30 minutes；It is broken
Bacterium solution 13000-15000rpm is centrifuged 20-40min.
(4) abandoning supernatant, by precipitation 40mL 20mM Tris-HCl, pH8.0,500mMNaCl, 2mM EDTA, 2%
Triton-X100 is fully washed, 13000-15000rpm centrifugation 20-40min.
(5) it is repeated two more times with (4) method.
(6) supernatant discarded, will precipitate and uses 40mL 20mM Tris-HCl, pH8.0,500mM NaCl, 2mM EDTA, and 2%
Tween is fully washed, 13000-15000rpm centrifugation 20-40min.
(7) supernatant discarded, by precipitation 80ml 20mM Tris, pH8.0,10mM NaCl, 8mM Urea dissolve,
13000-15000rpm is centrifuged 20-40min.Collect supernatant
(8) in supernatant being added molecular cut off for the Amicon concentration tubes of 30kD, upper strata residue 1ml or so is concentrated into,
Collect part under centrifuge tube.
(9) in the Amicon concentration tubes for partly adding molecular cut off to be 5kD under the pipe obtained in (8), it is concentrated into
Layer residue 1mL or so, collects part on centrifuge tube.
(10) the upper part protein concentration of pipe is determined, uses 20mM Tris, pH8.0,10mM NaCl, 8mM Urea to be diluted to
The concentration of 0.1mg/mL.
(11) in albumen being added into molecular cut off for the bag filter of 3500Dalton, it is put into the dialysis buffer of 4 degree of pre-coolings
In liquid A, its composition is as follows：20mM NaH2PO4-Na2HPO4, pH7.2,25mM NaCl, 10% glycerol, 0.2%beta- sulfydryls
Ethanol, dialysed overnight.
(12) bag filter is transferred in elution buffer B of 4 degree of pre-coolings, its composition is as follows：20mMNaAc-HAc,
PH5.2,0.2%beta- mercaptoethanol, dialyses 3 hours.
(13) solution in bag filter, 13000-15000rpm centrifugation 20-40min are taken out.Collect supernatant.
(14) in the supernatant obtained in (13) being added molecular cut off for the Amicon concentration tubes of 5kD, in 20mM
10mg/mL is concentrated in the buffer of NaAc-HAc, pH5.2.
(15) crystal growth of HIV-1protease：Crystallization condition is as follows：10-15% (w/v) PEG3350,5mM DTT,
0.2M MgCl2.Under the crystallization condition, the protein concentration of 5mg/mL obtains that size is larger, and diffraction resolution is higher (to be higher than) crystal.
Embodiment 2：The preparation of the alternative sample of Berberis nummularia Bge.
Berberis nummularia Bge. herb is removed into deadwood dead leaf and other alloys, is cleaned and is shredded, in being placed in 50 DEG C of vacuum drying ovens
It is dried 12 hours.The Berberis nummularia Bge. medical material of 500 grams of drying and crushing is accurately weighed, 95% ethanol is added in 2500mL round-bottomed flasks
1000mL, extracts 4 hours, sucking filtration while hot in 80 DEG C of water-baths.Then in residue the ethanol of 1000mL 95% is added to carry out the again
Second extraction, 3 hours time, equally sucking filtration while hot, filtrate is merged.It is left followed by the filtrate after merging is concentrated into into 20mL
The right side, culture dish is proceeded to respectively carries out at normal temperatures natural evaporation drying, drying 24 in 50 DEG C of vacuum drying ovens is then proceeded to again little
When, obtain extractum.
20.5 grams of this extractum is weighed, is suspended with 150 milliliters of distilled waters, in being poured into 1000 milliliters of separatory funnels, use acetic acid second
Ester is extracted 3 times, every time 650 milliliters of ethyl acetate of extraction, shake well, stands the little layereds of 6-10.Acetic acid is obtained after extraction
Ethyl ester phase and water phase.Acetic acid ethyl ester extract is evaporated off solvent with EYELA N1001 type Rotary Evaporators, and get dry extract shape Berberis nummularia Bge.
Ethyl acetate extracts sample, standby.
Aqueous portion after extraction EYELA N1001 type Rotary Evaporators are evaporated off a small amount of ethyl acetate dissolved in water,
Separate (macroporous resin with 170 milliliters, glass column 30 × 300mm of specification) with HP-20 types macroporous resin column chromatography, successively with water,
50% ethanol is mobile phase eluting.First wash with water 8 times, 250 milliliters of each eluting discards water-wash section.Then 50% second is used
Alcohol eluting 5 times, 300 milliliters of each eluting, the eluent that each 50% ethanol elution of merging is obtained.By 50% ethanol elution part
Solvent, the ethanol elution of the shape Berberis nummularia Bge. water phase that gets dry extract macroporous resin 50% are removed using EYELA N1001 types Rotary Evaporators
Sample, it is standby.
Above-mentioned ethyl acetate extraction sample and the ethanol elution sample of macroporous resin 50% are Berberis nummularia Bge. to acquired immune deficiency syndrome (AIDS) disease
The screening sample of toxalbumin enzyme vitro inhibition activity.
Embodiment 3：The alternative sample of Berberis nummularia Bge. is determined to HIV (human immunodeficiency virus) protease inhibiting activity
The active determination in vitro method of HIV-1protease is referring to (Dieter Hoffmann et al., Journal of
Clinical Virology 32 (2005) 294299), and be suitably modified.
The determination of activity of acquired immune deficiency syndrome (AIDS) protease is to use fluorogenic substrate MCA- γ
- Abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln--Lys (Dnp)-Lys-NH2 (purity is more than 95%,
Shanghai gill biochemistry company limited) come what is completed.The aminoacid sequence of the fluorogenic substrate is from HIV (human immunodeficiency virus) protease
Specific cleavage sequence.Instrument for fluorescent strength determining is Fluoraskan Ascent luminoscopes
The wavelength of (ThermoLabsystems, Helsinki, Finland), exciting light and launching light is respectively 320nm and 405nm.
According to the middle medicines natural products for carrying out the inhibitor screening of HIV-1protease albumen prepared by the present invention
Sample in mixture alternate library is referred to as natural products mixture alternate sample.Natural products mixture alternate sample is dissolved in into two
Its final concentration of 50mg/mL is made in methyl sulfoxide (DMSO), the DMSO solutees of natural products mixture alternate sample are obtained.
Using buffer solution (50mM NaAc, pH5.2,1mMEDTA, 2.5mM DTT, 1.0M NaCl, 2.5% (v/v)
Glycerol) 10mg/ml HIV (human immunodeficiency virus) protease is carried out into 100 times of dilutions.Fluorogenic substrate is dissolved using DMSO, is made dense eventually
Spend for 10mM, then with buffer solution (50mM sodium acetate, pH5.2,1mMEDTA, 2.5mM DTT, 1.0M NaCl,
2.5% (v/v) glycerol) ten times of dilution fluorogenic substrates.
Using 96 hole elisa Plates, the protein solution of the above-mentioned dilutions of 97 μ L is added in every hole, add the above-mentioned natural products of 1 μ L
The reaction buffer solute of mixture alternate sample is (as Berberis nummularia Bge. ethyl acetate extracts the reaction buffer dissolving of sample
Thing), after 30 degree are placed 10 minutes, it is rapidly added the fluorescence labeling substrate of the above-mentioned dilutions of 2 μ L.Excitation wavelength and launch wavelength are distinguished
For 320nm and 405nm, 30 degree of temperature holding, first order fluorescence reading was recorded per 3 seconds, read 60 times altogether.
According to testing compound presence or absence of when fluorescence intensity change, compound can be calculated to proteinase activity
Inhibition level or residual activity, so as to complete the preliminary screening to protease inhibitor.Circular is, enzymatic reaction
In, according to course of reaction curve (Fig. 1), the growing amount (i.e. fluorescence intensity) of product and the curve in response time, slope of a curve
The change of product formation in the unit interval is represented, so the slope of any point is exactly the reaction speed of the corresponding time on curve
Rate, in the range of base consumption is less than 10% (reaction started in the very short time), the slope of curve is the first rate of enzyme reaction
(V0) in the presence of, having different inhibitor, the reaction first rate (Vi) of enzyme has different degrees of decline, the residual activity (Vi/ of enzyme
V0 10%) is less than, is positive findingses.
Control：Negative control is added without alternative sample, is replaced with 1 μ L100%DMSO, and remaining condition is identical.As a result it is shown in
In Fig. 1.As shown in Figure 1 Berberis nummularia Bge. acetic acid ethyl ester extract has stronger inhibitory activity, illustrates little point in Berberis nummularia Bge.
Sub- inhibitor is primarily present in ethyl acetate extraction sample.The sample of inhibitory activity will be screened, with various chromatographs, wave spectrum
Method separates, identifies the structure of wherein micromolecular inhibitor-berberine hydrochloride.
Embodiment 4：IC of the berberine hydrochloride to HIV (human immunodeficiency virus) albumen enzyme level50Determine and suppress the judgement of type
IC50The measure of (half-inhibition concentration)：Concentration of substrate is respectively 20 μM, and protein concentration is 500nM, by hydrochloric acid Radix Berberidis Amurensis
Alkali carries out 1/2 Concentraton gradient dilution, typically determines 15 points, then calculates the corresponding residual activity of each compound concentration, profit
With the software analysis of software GraphPad Prism 5, the curve of residual activity value and the logarithm value of compound concentration is made, calculated
Go out IC50Value.The IC of berberine hydrochloride50For 114.8 ± 17.6 μM (Fig. 2).
The measure of Km and Ki and the judgement of suppression type, protein concentration is 500nM, and concentration of substrate is respectively 200.000 μ
M, 180.000 μM, 150.000 μM, 120.000 μM, 100.000 μM, 50.000 μM, 25.000 μM, 12.500 μM, 6.250 μM,
3.125/ μM, the corresponding initial velocity of reaction of each concentration of substrate is calculated, using the software analysis of software GraphPad Prism 5, made
Go out the curve of initial velocity of reaction and concentration of substrate, calculate Km values (Fig. 3), Km is 24.29 ± 3.7 μM.
Berberine hydrochloride is added in reaction system, makes berberine hydrochloride final concentration be respectively 80 μM, 120 μM, protein concentration
For 500nM, concentration of substrate is respectively 200.000 μM, 180.000 μM, 150.000 μM, 120.000 μM, 100.000 μM,
50.000 μM, 25.000 μM, 12.500 μM, 6.250 μM, 3.125 μM, the corresponding initial velocity of reaction of each concentration of substrate is calculated,
Using the software analysis of software GraphPad Prism 5, react initial velocity and the curve of concentration of substrate calculate hydrochloric acid little
Bark of a cork tree alkali Ki values (Fig. 4), Ki is 180.2 ± 16.8 μM, and it is noncompetitive inhibitor that berberine hydrochloride suppresses type.
Embodiment 5：HIV (human immunodeficiency virus) protease and the extraction sample immersion of Berberis nummularia Bge. ethyl acetate and cocrystallization
According to document report, due to the active pocket of HIV (human immunodeficiency virus) protease it is larger, the suppression of protease and macromolecule
Preparation will to a great extent change the configuration of protease after combining, therefore the method soaked by crystal is more difficult to get macromole
The inhibitor of amount.But on the other hand, because the middle medicines natural products for the screening of HIV (human immunodeficiency virus) protease inhibitor are mixed
Each sample in the alternative storehouse of compound containing many kinds of natural product small molecules (therefore, be referred to as natural product mixing
The alternative sample of thing), according to the method for cocrystallization, the impact to HIV (human immunodeficiency virus) protease crystallizing system is again larger.Therefore, I
Employ two methods simultaneously, on the one hand the method by soaking, expects the inhibitor for obtaining molecular weight, on the other hand
Then optimize albumen and the cocrystallization system in natural product mixing storehouse, expect to obtain the larger inhibitor of molecular weight.
The crystal infusion method of method 1
It is little in Berberis nummularia Bge. ethyl acetate sample when improving immersion in order to as far as possible on the basis of crystal mass is ensured
The concentration of molecular compound, can adopt the following two kinds method to prepare the crystal soak of HIV (human immunodeficiency virus) protease：
Berberis nummularia Bge. ethyl acetate is extracted into 10 times of dilutions of DMSO solutees (preparing according to the method in step 3) of sample
Into HIV (human immunodeficiency virus) protease crystals growth pool liquid (10-15% (w/v) PEG3350,5mM DTT, 0.2M MgCl2), with
13000-15000rpm high speed centrifugations, take afterwards supernatant, obtain soak, standby；
During in order to ensure that crystal soaks, it is unlikely to make the micromolecular compound in Berberis nummularia Bge. ethyl acetate extraction sample dense
Spend height and affect the diffraction quality of crystal, soak is diluted to respectively 5 times of original liquid concentration, 10 times.
During immersion, using instruments such as nylon crystal rings, by the crystal of the HIV (human immunodeficiency virus) protease for having grown from crystallization
In the liquid of pond take out, be separately added in soak and corresponding 5 times, 10 times of diluents in, soak 2-48 hours.
Method 2：Cocrystallization method
Berberis nummularia Bge. ethyl acetate is extracted into 10 times of dilutions of DMSO solutees (preparing according to the method in step 3) of sample
Into HIV (human immunodeficiency virus) protease crystals growth pool liquid (10-15% (w/v) PEG3350,5mM DTT, 0.2M MgCl2), with
13000-15000rpm high speed centrifugations, take afterwards supernatant, standby.
By the acquired immune deficiency syndrome (AIDS) protease of 10mg/ml with above-mentioned supernatant respectively with 4: 1,2: 1,1: 1,1: 2,1: 4 four gradient
Mixing, after 1-5 hour of effect, with 13000-15000rpm high speed centrifugations, takes afterwards supernatant, standby.
The crystal growth of HIV-1protease- inhibitor complexes and single HIV-1protease crystal growth conditions
Identical, crystallization condition is as follows：10-15% (w/v) PEG3350,5mM DTT, 0.2MMgCl2.Obtain size larger, diffraction is differentiated
Rate is higher (to be higher than) crystal.
Embodiment 6：The collection and analysis of crystal diffraction data
Using possessing preferable diffracting power, (resolution is preferably greater than) crystal, carry out the receipts of X ray diffracting data
Collection and analysis.
When carrying out data collection, first by the nylon crystal rings of Hampton Research companies, from soak 1 and leaching
The crystal of immersion certain hour (usually 2-48 hours) is obtained in bubble liquid 2；And rapidly using the cooling system of Rigaku companies
Or the cooling system of Oxford Cyrosystem companies, crystal is cold in the cryogenic nitrogen air-flow that above-mentioned refrigeration system is produced
Freeze to subzero 150 DEG C -180 DEG C；Make X-ray by crystal, using precession method X ray diffracting data is collected.
(resolution is preferably greater than to select the good crystal of diffraction quality) carry out data collection, process, detect whether
The combination of small molecule.
In the X-ray for collecting the crystal after the crystal of target proteins matter is contacted with natural products mixture alternate sample
After diffraction data, need to carry out corresponding data processing as steps described below：
First, it is known in the art using HKL2000 etc. (including Mosflm, D*trek etc.) diffraction data processing routine bag etc.
Conventional data processing method, process collecting the diffraction data for obtaining in previous step, obtain complete data
File；
Secondly, using the various molecular replacement (Molecular such as Phaser, Molrep in CNS or CCP4 program bags
Replacement) the conventional data processing method known in the art such as program, using molecular replacement (Molecular
Replacement method), the parent crystal structure with target proteinses obtains the target proteinses and target of candidate as initial model
The fine three dimensional structure of the complex of molecule；
Again, using the conventional data processing known in the art such as the visualization procedure bag such as program COOT, XtalView
Method, observe possible target proteinses with inhibitor or the electron density of part small molecule complex.If in this case, it was found that
In the corresponding avtive spot of target proteinses, have be not belonging to protein molecule itself, be not belonging to solvent molecule, be not belonging to crystallization
The unknown electron density of molecule present in solution is present, and the aminoacid of the avtive spot of the electron density and target proteinses
There is generation including rational chemical environment including hydrogen bond action, covalent effect etc., then can confirm that this natural product mixture is standby
In sampling product, there are the ligand moleculars such as inhibitor small molecule to be combined with target proteinses.
The statistics of data collection such as table 1 below：
Table 1：HIV (human immunodeficiency virus) protease parent, HIV (human immunodeficiency virus) protease and Berberis nummularia Bge. acetic acid ethyl ester extract sample
Complex data collection statistics after immersion
By observing the HIV (human immunodeficiency virus) protease parent of equal angular and the avtive spot electron density map (figure of complex
5) (black is the electron density of protein itself), it is found that it is clear to be able to observe that above the avtive spot of complex
Nonprotein itself electron density (redness represent), show may have micromolecular compound to combine on protein molecule.
It is shown that the electron density near the avtive spot top of HIV (human immunodeficiency virus) protease precursor structure, dotted line outside the dotted line frame of figure five
Crystal avtive spot top is attached after the HIV (human immunodeficiency virus) protease that inframe is represented soaks with Berberis nummularia Bge. ethyl acetate extraction sample
Near electron density.
The electron density of the vicinity of the avtive spot of complex is examined, we can be found that and are present in Berberis nummularia Bge. second
The electron density of the berberine hydrochloride in acetoacetic ester extract sample is close with HIV (human immunodeficiency virus) proteinase activity pocket top electronics
Degree is closely linked, and points out us to be that some interactions are defined between berberine hydrochloride and protein atoms,
So as to inhibit the activity of HIV (human immunodeficiency virus) protease.Found by the aminoacid sequence of HIV-1 protease, the ammonia around small molecule
Base acid is mainly the G of 48-52 positions48G49I50G51G52, all it is some hydrophobic amino acids, they form hydrophobic phase with berberine hydrochloride
Interaction, is the porch of peptide substrate in the top loop ring regions of active pocket, hinders substrate to enter avtive spot, so as to hinder
Break cutting of the HIV-1 protease to polypeptide.The HIV-1 protease inhibitor having now been found that, it is in the majority with competitive type inhibitor, it is main
Will be with reference in avtive spot, the enzyme activity determination result and eutectic of berberine hydrochloride and HIV-1 protease in the majority with polypeptide analog
Data display, the berberine hydrochloride extracted from natural product is not only a kind of good HIV-1 protease inhibitor, and presses down
Preparation is also incorporated on a new binding site of HIV-1 protease, and the research and development for HIV-1 protease inhibitor are provided newly
Thinking.
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding can carry out various changes, modification, replacement to these embodiments without departing from the principles and spirit of the present invention
And modification, the scope of the present invention be defined by the appended.
List of references (such as patent/paper/standard)
1.Zhiqiang Yu a, Tsutomu Kabashima a, Chenhong Tang a, Takayuki Shibata
A, Kaio Kitazato a, Nobuyuki Kobayashi a, b, Myung Koo Lee c, Masaaki Kai a, b*
.Selective and facile assay of human immunodeficiency virus protease activity
by a novel fluorogenic reaction.Analytical Biochemistry.2010(397)：197-201.
2.ULRIKA NILLROTH, 1LOTTA VRANG, 2PER-OLOF MARKGREN, 1JOHAN HULTE ' N,
3ANDERS HALLBERG, 3AND U.HELENA DANIELSON 1*, Human Immunodeficiency Virus TyPe
1Proteinase Resistance to Symmetric Cyclic Urea Inhibitor
Analogs.ANTIMICROBIAL AGENTS AND CHEMOTHERAPY.1997Nov：41(11)：2383-2388.
3.EDMUND D.MATAYOSHI, * GARY T.WANG, GRANT A.KRAFFT, JOHN ERICKSON.Novel
Fluorogenic Substrates for Assaying Retroviral Proteases by Resonance Energy
Transfer.SCIENCE.1990September：247：954-958.
4.Riham Salah El Dine, Ali M.El Halawany, Chao-Mei Ma, and Masao
Hattori*.Anti-HIV-1Protease Activity of Lanostane Triterpenes from the
Vietnamese Mushroom Ganoderma colossum.J.Nat.Prod.2008,71,1022-1026.
5.John M.Louis§, Ewald M.WondraAlan R.KimmelPaul T.Wingfieldi, and
Nashaat T.Nashed**.Proteolytic Processing of HIV-1Protease Precursor, Kinetics
And Mechanism*.THE JOURNAL OF BIOLOGICAL CHEMISTRY.1999,274,23437-23442.
Claims (4)
1. a kind of screening technique of AIDS virus resisting material, it is characterised in that：Comprise the steps：
(1). HIV (human immunodeficiency virus) protease is prepared, and crystallization obtains the crystal of HIV (human immunodeficiency virus) protease；
(2). prepare natural extracts, the post such as the extract is included by extracting, solvent extraction or macroporous resin, silica gel
Each position that chromatography purification is obtained；
(3). the natural extracts that step (2) is obtained are dissolved in into organic solvent；Using buffer solution to the Chinese mugwort in step (1)
Sick virus protease dissolving, dilution are grown, then two kinds of solution mixed solution is mixed to get into, fluorescent substrate solution is prepared, then will
Fluorescent substrate solution is added in above-mentioned mixed solution；
(4). the fluorescence intensity change of detection sample, and compare with the negative controls for being added without natural extracts, obtain
With the bioactive natural product extract for suppressing HIV (human immunodeficiency virus) protease；
Wherein, the HIV (human immunodeficiency virus) protease is 1 type hiv protease, prepares HIV- used
The aminoacid sequence of 1protease plasmids is PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGI
GGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF；
Using the method for tracking activity, pair active natural extracts for determining further are isolated and purified, to separating
The component repeat step (3) of purification and (4), the determination of activity of such multiple stage separation, purification and step (3)-(4), finally give
Active monomeric compound, the monomeric compound is berberine or its pharmaceutically-acceptable salts；
Natural extracts described in step (2) are Berberis nummularia Bge. through ethanol extraction, ethyl acetate extraction and then use HP-20
Type macroporous resin column chromatography is separated, and obtains the alcohol elution of 30%-80%.
2. screening technique according to claim 1, it is characterised in that：Organic solvent described in step (3) is selected from：Pentane, hexane,
Octane, hexamethylene, Ketohexamethylene, toluene Ketohexamethylene, chloroform, dichloromethane, methanol, ethanol, isopropanol, ether, tumer
Ester, ethyl acetate, propyl acetate, acetone, espeleton, methylisobutylketone, glycol monoethyl ether, ethylene glycol monoethyl ether, second two
Alcohol monobutyl ether, acetonitrile, pyridine, dimethyl sulfoxide, dichloro Methanamide.
3. screening technique according to claim 1, it is characterised in that：Fluorogenic substrate described in step (3) is MCA- γ-Abu-
Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(Dnp)-Lys-NH2。
4. screening technique according to claim 1, it is characterised in that：Negative controls described in step (4) are natural to be added without
Product extract, is replaced with 1 μ L100%DMSO, and remaining preparation condition is identical with natural extracts.
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| CN101063682A (en) * | 2007-03-19 | 2007-10-31 | 南开大学 | Novel methods for high efficiency and rapid getting fine three dimensional structure of target protein composite body and target molecule target molecule |
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| WO2001088113A3 (en) * | 2000-05-03 | 2003-01-30 | Upjohn Co | Hepatitis c virus helicase crystals, crystallographic structure and methods |
| CN101063682A (en) * | 2007-03-19 | 2007-10-31 | 南开大学 | Novel methods for high efficiency and rapid getting fine three dimensional structure of target protein composite body and target molecule target molecule |
| CN101063234A (en) * | 2007-04-04 | 2007-10-31 | 南开大学 | Method for screening SARS coronaviruses main Protease inhibitors from natural product such as Chinese medicinal materials mixture prepared storeroom |
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