CN101916330A - Virtual screening method for novel cancer-preventing or anti-cancer medicament by taking Keap1 as target point - Google Patents
Virtual screening method for novel cancer-preventing or anti-cancer medicament by taking Keap1 as target point Download PDFInfo
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Abstract
The invention relates to a virtual screening method for a novel cancer-preventing or anti-cancer medicament by taking Keap1 as a target point. The method comprises the following steps of: 1) determining a PDB structural data of a Keap1 protein structure; 2) determining an active center according to an active cysteine residue site of the Keap1 by adopting molecular docking software, and setting active bags; 3) screening small molecule ligand; 4) establishing a small molecule ligand database for docking; 5) docking the small molecule ligand in the small molecule ligand database for docking and the active bags one to one according to the set active bags by using the molecular docking software; and 6) primarily determining a pilot medicament with chemical cancer-preventing or anti-cancer effect according to the sequencing of the docking results. The method can obtain a clue of an active compound in short time, and then screens the cancer-preventing or anti-cancer medicament by using animal horizontal screening or high-pass molecule platform screening so as to greatly improve the speed and the efficiency and shorten the research period of a new medicament.
Description
Technical field
The present invention relates to drug screening method, to relate in particular to keap1 be target spot, utilize the screening technique of the novel anticancer medicine that the medicine virtual screening method of molecular docking carries out.
Background technology
Cancer is the arch enemy of man, and especially morbidity such as lung cancer, liver cancer, cancer of the stomach, cancer of the esophagus, colorectal cancer, breast cancer, nasopharyngeal carcinoma and mortality ratio are still high in recent years.For beat cancer, almost each country has all spent substantial contribution and manpower.According to World Health Organization's report, global New Development cancer patient 1,010 ten thousand people in 2000, dead 6,200,000 people, existing cancer stricken case 2,240 ten thousand.Whole world cancer is still in rising trend, and according to The World Health Organization's scholarly forecast, the year two thousand twenty whole world total population will reach 8,000,000,000, and the cancer new cases will reach 2,000 ten thousand, and 1,200 ten thousand people die from cancer, and cancer will become the new century mankind's first killer.Generally admit through medical circle, cancer is not the god of death.From the nineties in 20th century, western developed country common cancers such as the U.S., Canada, Australia begin to descend, and Japanese cancer mortality has become and steadily no longer raise.This variation, exciting and inspiration.And developing country increases quickening, and 60% occurs in developing country in the cancer patient.China's pathogenesis of cancer number in 2000 is 2,000,000, dead 1,500,000.According to statistics, cancer mortality has risen 18.1% among 1991-2000 China city dwellers, and cancer mortality has risen 11.03% among the urban residents, and average every dead 5 people just have a people to die from cancer.China is in the period to developed country's cancer spectrum transition by developing country.
Under various carcinogen effects, it is a long-term and hidden process that normal cell develops into tumour cell.Continuous damage causes to DNA or/and the inactivation of tumor suppressor gene is owing to carcinogen to have abundant proof to show the continuous activation of oncogene.The canceration process is to be caused by multifactor, experiences a plurality of stages and relates to the unusual of many barss transmission path.Studies show that the canceration process can be slowed down, stop and reverse in nearly several 10 years.Therefore, stoping the canceration process is the available strategy of prophylaxis of cancer.
Although ultraviolet ray, virus etc. can cause canceration, chemical carcinogens is the primary factor of canceration.In general, chemical carcinogen at first can be by the detoxification system metabolism of human body after entering body.The detoxification machine of liver is shaped on two road programs in the human body: be called Phase I and Phase II detoxification system.Phase I is first road detoxifcation program, this system mainly is made up of Cytochrome P450 enzyme, playing the part of an oxidation and the role who divides toxolysin in detoxification processes, but the toxin after Phase I activation might have more toxicity than original molecule, therefore needs to remove toxicity by the Phase II enzyme system in second road; The function of Phase II enzyme system promptly is the performance detoxication, these molecules is lost activity, the injury of neutralize a toxin molecule pair cell, DNA.The protection system of biosome is receiving that the quantity of stimulation back by the II phase enzyme that increases sharply plays a protective role.This kind of enzyme is the ability of counteract toxin damage dna and cause cancer effectively.Phase II enzyme system comprises Glutathione transferase, and Quinone reductase etc. have the function of superpower cancer-resisting and prevention cell mutation.For example Quinone reductase wherein (QR, quinone reductase) be study one of Phase II enzyme the most widely.Think that at present the increase of phase II enzyme level can provide a suitable valid approach resisting cancer.And can be regarded as a very effective method of cancer chemoprotective new drug development by the compound that screening has a phase II enzyme induction.
No matter traditional drug screening is screening of animal level or the screening of high-flux cell platform, all exist natural drug and composition of food complexity, from confirming that extract has potential anti-cancer effect to definite effective constituent, need spend great amount of manpower to finally isolating monomer component, time and efforts, the speed of screening compounds and efficient are extremely low, and the cycle is long, positive rate low (0.01%～0.1%).Therefore, development of virtual screening method in recent years and the application in new drug research have been subjected to paying attention to widely.So setting up efficient, effective, the quick-acting Chemistry for Chinese Traditional Medicine of cover anti-cancer medicine screen body is tied to form and is the task of top priority.
Summary of the invention
The objective of the invention is to set up a kind of novelty, fast, is the medicine virtual screening method of target spot with keap1 efficiently, to remedy the deficiency of existing method.
Mechanism of the present invention is:
Studies show that, with the adjusting of Phase II enzyme relation the closest be the Keap1/Nrf2/ARE path, this be one with oxygenant and the closely-related signal conditioning system of electrophilic reagent carcinogenesis.Under the physiological status, Nrf2 combines and is anchored in the endochylema with the Keap1 coupling and with actin.Keap1 (Kelch-like ECH-associated protein 1) is a kind of ubiquitin ligase, and the PROTEIN C UL3 that is connected ubiquitin with another kind forms complex and links to each other with target protein such as transcription factor Nrf2.Research finds that also some has α, the natural drug of β-unsaturated structural unit (michael reaction acceptor molecule, contain α, the chemical micromolecule part of β-unsaturated structural unit, the electron withdraw group that its conjugated structure produces can with nucleopilic reagent generation Michael addition reaction), by with the sulfydryl generation Michael addition reaction of Keap1 cysteine residues, modify its cysteine residues with covalent bond, promote Keap1 ubiquitinization and degraded, thereby the Nrf2 that makes combination with it dissociates out and enters nucleus, after other transcription factors combine, be combined in the anti-oxidant response element ARE of adjusting district (the antioxidantresponse element of II phase enzyme gene 5 ' upstream again, ARE), start II and separate transcribing of toxenzyme and anti-oxidation stress protein gene mutually, thus the ability prophylaxis of cancer (see figure 1) of raising cell anti-oxidative damage.Therefore, activation Keap1/Nrf2/ARE path is the most promising strategy of prophylaxis of tumours.
People Keap1 albumen contains 27 cysteine residues sites, and being which cysteine residues on earth brings into play active function in michael acceptor molecule performance II phase enzyme induction active process, is the focus in cancer chemoprotective Mechanism Study field in recent ten years always.The detection method of probe that Keap1 researcher's employing combines with inducer and zymetology, radio-labeled, UV, chromatogram has determined that Keap1 inserts four high activity cysteine sulfydryl site (Cys257 in district, Cys273, Cys288 and Cys297) be the identification II enzyme induction thing and the important cells " sensor " of reaction with it mutually.And it is wherein important with Cys273 and Cys288.Inside and outside research all proves the variation in Cys273 and two sites of Cys288 or Sulforaphane or the GSSG addition to wild type Keap1, all can directly cause dissociating and the increase of II phase enzyme and anti-oxidation stress expression of target gene of Nrf2.In addition, the Cys151 site also can be by being modified by the addition of michael acceptor molecular compound, adds in the dissociating of the Keap1 that induces and Nrf2 playing a significant role at oxidative stress derivant and Sulforaphane.Therefore, can become the potential drug of cancer chemoprotective with the compound of the active cysteine residues generation Michael addition reaction of the Cys273 of keap1 and Cys288 and Cys151.
The known α that contains, micromolecule part of β-unsaturated structural unit (being called the Michael addition reaction acceptor molecule) and Keap1 go up the sulfydryl generation Michael addition reaction of Cys273 and cysteine residues such as Cys288 and Cys151 can effectively raise the expression of quinone reductase, thereby plays protective effect on cancer risk.But in fact be not all michael reaction acceptor micromolecule part can both with this activity residue generation addition reaction, should have the pharmacophoric group (α that michael reaction takes place except micromolecule part itself, β-unsaturated structural unit) outside, between near itself and the Keap1 protein macromolecule active pocket other amino acid residues interaction is arranged also, and this interaction can impel the easier generation addition reaction of sulfydryl of ligand molecular and active cysteine residues.
Design of the present invention is based on above-mentioned mechanism: at first, utilize the homology mould to build the PDB data that software prediction goes out keap1 avtive spot place functional domain (IVR); Then on this basis, be that the macromolecular avtive spot of acceptor makes up active pocket with active cysteine residues site Cys151, the Cys273 of keap1 albumen and Cys288 etc. respectively, utilize molecular docking software, to the micromolecule part database that makes up in advance, especially from the natural products database, carry out virtual screening, determine the michael reaction acceptor micromolecule part that can be complementary with this active pocket, as novel lead drug, further verify with the high flux screening of cell platform again with cancer chemoprotective and antitumaous effect.
Provided by the invention a kind of be the virtual screening method of the novel anticancer medicine of target spot with Keap1, may further comprise the steps:
1) determines the PDB structured data of Keap1 protein structure;
2) adopt molecular docking software, determine the activated centre, set active pocket according to the active cysteine residues site of keap1;
3) the micromolecule part is screened, the screening project is the screening that contains that there is something special of screening of toxicity screening, quasi-medicated property and pharmacophore;
4) set up butt joint micromolecule part database;
5) according to the active pocket of setting, utilize molecular docking software, butt joint is made with active pocket one by one of the micromolecule part in the micromolecule part database docked;
6), tentatively determine to have the lead drug of cancer chemoprotective or anticancer effect according to butt joint result's ordering.
Above-mentioned virtual screening method: step 3) can place step 1) to any one step of step 6).That is: can to do not carry out molecular docking as yet or docked after the micromolecule database in the micromolecule part carry out toxicity, quasi-medicated property and pharmacophore and contain that there is something special screens, and be not subjected to the ordering restriction.
In order further to improve speed, efficient and the accuracy of screening, above-mentioned virtual screening method: step 6) can for: according to the butt joint result ordering, micromolecule part pharmacophore contains that there is something special and the interaction mode in micromolecule part and activated centre, carry out comprehensive evaluation, tentatively determine to have the lead drug of cancer chemoprotective or anticancer effect.
The invention has the beneficial effects as follows: the present invention is by the virtual screening of medicine, can obtain the clue of reactive compound at short notice, goal in research is focused on a hundreds of compound from millions of compounds, and the positive rate of virtual screening (5%～30%) is higher than high flux experiment screening (0.01%～0.1%) far away.And then the lead compound behind virtual screening, utilizing screening of animal level or high-flux cell platform, screening has anticancer medicine, has therefore improved the speed and the efficient of screening compounds greatly, shortens the cycle of new drug research.
Description of drawings
The molecular mechanism of Fig. 1 II phase enzyme inducer protective effect on cancer risk.
The prlmary structure of protein sequence of Fig. 2 hKeap1.
The tertiary protein structure mode chart in the active function territory of Fig. 3 hkeap1.
The assessment of Fig. 4 hkeap1 prediction tertiary structure.
The SHELL script that uses in Fig. 5 micromolecule part docking procedure.
The interaction diagram in the Cys151 activated centre of Fig. 6 Compound D EM and keap1.
The cluster analysis in the Cys151 activated centre of Fig. 7 A positive control molecule DEM and keap1.The interaction diagram in the Cys151 activated centre of Fig. 7 B positive control molecule DEM and keap1.
The cluster analysis in the Cys151 activated centre of Fig. 8 A positive control molecule isoliquiritigenin and keap1.The interaction diagram in the Cys151 activated centre of Fig. 8 B positive control molecule isoliquiritigenin and keap1.
The cluster analysis in the Cys151 activated centre of Fig. 9 A positive control molecule xanthohumol and keap1.The interaction diagram in the Cys151 activated centre of Fig. 9 B positive control molecule xanthohumol and keap1.
The interaction diagram in the Cys151 activated centre of Figure 10 A isoflavones and keap1.The cluster analysis in the Cys151 activated centre of Figure 10 B isoflavones and keap1.
The process flow diagram of Figure 11 high-flux cell platform screening cancer chemoprotective cancer therapy drug method.
The interpretation of result that Figure 12 isoflavones induces quinone reductase to express at cellular level.
The interaction diagram in the Cys273 activated centre of Figure 13 A positive control molecule 1 5D-PGJ2 and Keap1.The interaction diagram in the Cys288 activated centre of Figure 13 B positive control molecule 1 5D-PGJ2 and Keap1.
The interaction diagram in the Cys273 activated centre of Figure 14 A Ligustilide and Keap1.The interaction diagram in the Cys288 activated centre of Figure 14 B Ligustilide and Keap1.
Figure 15 is the interpretation of result that Ligustilide induces quinone reductase to express at cellular level.
Embodiment
Following examples will help those of ordinary skill in the art further to understand the present invention, but not limit the present invention in any form.
1 one kinds of cysteine residues Cys151 with keap1 albumen of embodiment are avtive spot, carry out the virtual screening of cancer chemoprotective cancer therapy drug.
(1) the virtual screening step is as follows:
1, utilize the homology mould to build the structured data that software is determined the PDB in Keap1 protein structure active function territory
Utilize Swiss Model online service, extract amino acid sequence (the FASTA form of human Keap1, Fig. 2), submit the template function of search of Swiss Model to, with the tertiary protein structure (3i3nb) with the KLHL11 of the BTB functional domain high conservative of keap1 is template, the homology modeling, determine keap1 the active function territory tertiary protein structure (1-314) as shown in Figure 3.
The model that obtains by the homology modeling method often has some irrational mixes, therefore, adopts online model analysis instrument Molprobity (http://molprobity.biochem.duke.edu/index.php) to assess.Evaluation criteria is: the adosculation (atom clashes) excessively that red expression is comparatively serious; The irrational dihedral angle of green expression; Purple is represented irrational Cb; Crocus is represented irrational rotamer.Assessment result by Fig. 4 can clearly find out, is that the pocket scope in activated centre exists unreasonable structure hardly or crosses adosculation with the active cysteine residues site Cys151 of keap1.Summary statistics shows that this model is reasonable substantially.Therefore, the model of the tertiary protein structure in the active function territory of keap1 shown in Figure 3 can not taked further optimization and be directly used in subsequent operation.
2, adopt Autodock molecular docking software, determine the activated centre, set active pocket according to the active cysteine residues site Cys151 of keap1;
In this step, at first utilize the known molecule that addition reaction can take place with the active cysteine residues site Cys151 of keap1 to do the positive control molecule, according to its in the active pocket that with avtive spot Cys151 is center construction with the interactional binding energy of pocket and whether to help taking place Michael addition reaction be standard, determine in the keap1 albumen with Cys151 to be the size of the active pocket at center.
2.1 utilize Autodock molecular docking software to carry out the setting of molecular docking Program for Calculation.
Key step is as follows:
1), with in the step 1, the tertiary protein structure in the keap1 active function zone that the homology modeling is determined is handled in MGLTools.Mainly comprise the removal hydrone, add nonpolar hydrogen, add the Gasteiger electric charge.Save as hKeaplforMD.pdbqt at last.
2), calculate lattice point energy (AutoGrid), in Grid＞Set Map Types＞Directly, import into carrying out the atomic type that the lattice point energy calculates.Operation AutoGrid can generate a series of files after finishing, and is mainly the map file of each atom.
3), be that each micromolecule part is prepared the required file of butt joint, this process will use the SHELL script to realize (Fig. 5).Make earlier the listing file ligands.list of a micromolecule part, then according to the order of SHELL script shown in Figure 5 input command Run Script a.csh successively; Will be file of each micromolecule part generation after finishing top operation, this document clip pack contains the required All Files of butt joint: the map file that part, acceptor, AutoGrid produce, dpf file etc.Next step promptly can dock.
4), circular flow AutoDock: circular flow AutoDock, dock each micromolecule part, and extract butt joint result such as binding energy and the butt joint sort result.This script can move a plurality of simultaneously, does not disturb mutually, therefore can utilize computer resource fully.
5), extract the butt joint result: obtain summary.sort at last: virtual screening is so far finished.
2.2 the active cysteine residues site Cys151 with keap1 is the activated centre, sets active pocket;
(network address: http://www.modelling.leeds.ac.uk/pocketfinder/), found that near the Cys151 does not have tangible binding pocket to utilize Pocket-Finder to search near Cys151 possible active binding pocket.Consider that Cys151 is a hydrophilic amino-acid residue, may have hydrophilic binding pocket near the supposition Cys151.Autodock4.2 program and corresponding Graphical Analysis Software MGLTools-1.5.4 are adopted in molecular docking.In MGLTools, adjust display mode, show the keap1 surface characteristics, the results are shown in Figure 6.Yl moiety is represented the position of Cys151 among Fig. 6, can see near the Cys151 that by Fig. 6 the groove that obviously impales is arranged, and the right is that the known positive that can combine with the Cys151 position of keap1 is with reference to molecule DEM6 among Fig. 6.Clearly, that groove solvent accessible surface among Fig. 6 is bigger, is the active pocket setting area.Because it is bigger to consider that pocket is influenced by water, therefore the grade of fit of micromolecule part in this pocket investigated with Autodock4.2, further optimize the setting of pocket.
The setting of active pocket during butt joint: the coordinate file of at first opening keap1, find the coordinate of each atom of Cys151, average in x, y, three directions of z respectively, draw the centre coordinate of Cys151, and write down numerical value (be that x is-10.094, y for-6.533, z is-32.398).When active pocket was set, x, the y of active pocket center correspondence, z filled out earlier above-mentioned numerical value respectively.The default value of sizing grid is 40 * 40 * 40, and step-length (spacing) is 0.375nm, then by adjusting the numerical value of sizing grid repeatedly, further determines the position and the size of active pocket, makes it can just encase groove.With the active cysteine residues site Cys151 of keap1 is that the active pocket in activated centre is: centre coordinate is that x is-10.094, y for-6.533, z is-32.398, the size of active pocket decides according to the molecular docking result and the interaction mode of the positive control molecule in following 2.3 steps.
2.3 adjust and the checking active pocket
The Lamarckian genetic algorithm (LGA) of computing machine widespread use is adopted in butt joint, and each part produces 50 butt joint conformations, and ga_pop_size is made as 300ga_num_evals and is made as 250000, and all the other parameters adopt the program default value.The micromolecule part can freely rotate during butt joint, and protein molecule then is fixed.Calculate and finish the back with the foundation of RMSD value as cluster analysis.
Utilize known molecule (1) xanthohumol that addition reaction can take place with the active cysteine residues site Cys151 of keap1; (2) isoliquiritigenin; (3) DEM (structural formula is as follows) does the positive control molecule, according to they in active pocket with the interactional binding energy of pocket and whether to help taking place Michael addition reaction be standard, be the active pocket in activated centre with Cys151 in the checking keap1 albumen.
According to checking repeatedly, we have finally determined the condition that is provided with of active pocket.Specific as follows: for the active cysteine residues site Cys151 with keap1 is the active pocket in activated centre: centre coordinate is that x is-10.094, y for-6.533, z is-32.398, the sizing grid value is 30 * 34 * 34, and step-length (spacing) is 0.375nm.
Be below under this active pocket of determining, three positive control molecules reach the interactional analysis of group on every side with the activated centre in active pocket.
(1) xanthohumol structural formula (2) isoliquiritigenin structural formula (3) DEM structural formula
Big and also higher the analyzing of binding energy of conformation number in the action diagram intercepting bunch, the result is respectively as Fig. 7 to Fig. 9.Can draw as drawing a conclusion by observing these three the butt joint results with reference to molecule: binding energy is all not high, is between-3 to-6.The micromolecule part with active pocket around amino acid residue do the time spent, this nonbonding effect (binding energy) can not too greatly can not be too little: too little, poor specificity on the one hand, part also has little time to be stabilized in and just may be subjected to the influence of other disturbing factor near the Cys 151 and leave pocket on the other hand; Too big words have limited the spatial orientation of part again, can not make unsaturated unit in the part take the sulfydryl generation addition reaction of best spatial orientation and Cys151.The formation that examines hydrogen bond finds that Val132, Arg135, these four residues of Lys131, Lys150 are promoting part and acceptor generation Michael addition reaction to play key effect.At can seeing on of DEM and Keap1: in the most conformation in Lys131 and the Val132 residue N-H in the peptide key unit form hydrogen bond with ether oxygen and the ketonic oxygen of DEM respectively, hydrogen in the epsilon-amino and another one ketonic oxygen form hydrogen bond on the Lys150 residue simultaneously, this space has promoted DEM generation Michael addition reaction in conjunction with making the sulfydryl of Cys151 relative with carbon-carbon double bond on the DEM just.At can seeing on of isoliquiritigenin and Keap1: the N-H in the most conformation in the Arg135 residue guanidine radicals and the phenolic hydroxyl group oxygen of a side form hydrogen bond, in the part conformation in Lys131 and the Val132 residue N-H of peptide key unit form hydrogen bond with ketonic oxygen, the phenolic hydroxyl group oxygen formation hydrogen bond of the hydrogen in the epsilon-amino and a side on the Lys150 residue in a part of conformation.This space is in conjunction with the sulfydryl generation addition reaction that promotes part and Cys151 residue too.The structure of xanthohumol is more similar to isoliquiritigenin, and hydrogen bond generation type and xanthohumol are almost roughly the same.
By xanthohumol, isoliquiritigenin, DEM does positive control, and having determined above-mentioned is the activated centre in activated centre and the rationality that active pocket is provided with Cys151 in the keap1 albumen, can carry out next step micromolecule ligand screening.
3, the micromolecule part is screened, the screening project is the screening that contains that there is something special of screening of toxicity screening, quasi-medicated property and pharmacophore
Before formal butt joint, the micromolecule part in the micromolecule part database is screened.The screening project is the screening (three kinds of screening and sequencing in no particular order) that contains that there is something special of screening of toxicity screening, quasi-medicated property and pharmacophore.
Micromolecule part database is selected free international natural products database (http://bioinformatics.charite.de/supernatural/), contain in this database 45917 kinds that reported in the world at present and be the structured data of buying the natural organic-compound that obtains easily from company.Certainly, in addition, can also be according to the newest research results of herbal pharmacology, according to scientific research and bibliographical information, collect a collection of α that contains, the micromolecular compound and the structural information thereof of β-unsaturated structural unit (pharmacophoric group) merge structure micromolecule part database with international natural products database.
3.1 toxicity screening: utilization toxicity of compound forecasting software Osiris carries out toxicity prediction to the micromolecule part in the micromolecule part database, eliminates the toxicity prediction result and shows positive molecule.
3.2 quasi-medicated property screening: the ADME forecast function of utilization ADME (pharmacokinetics) forecasting software or related software calculates the quasi-medicated property index of each micromolecule part in the micromolecule part database, index comprises multinomial, such as: molecular weight, hydrogen bond receptor, hydrogen-bond donor, rotatable bond number, lipid, water-soluble index, polar molecular surface area, molecular volume, based on the medicine similarity index of experience, chemical enthalpy change index (stability of molecule index).Molecule quasi-medicated property quality according to the reflection of each index gives each index again and marks with quality, and the superior of index is 1, and the inferior of index is 0.10 index superiority-inferiorities scoring of each molecule added and, draw total quasi-medicated property scoring.
3.3 screening that pharmacophore contains that there is something special: determine that according to this screening purpose pharmacophoric group is α, β-unsaturated structural unit, so our mode of adopting structural formula to search for will contain α in the database, and β-unsaturated structural unit micromolecule part is searched for out.
4, set up butt joint micromolecule part database
Toxicity screening result in the comprehensive step 3, quasi-medicated property assessment result and the Search Results that contains the micromolecule part of pharmacophore are set up and are applicable to this screening purpose butt joint micromolecule part database.
5, according to the active pocket of setting, utilize molecular docking software, butt joint is made with active pocket one by one of the micromolecule part in the micromolecule part database docked;
According to active pocket and the butt joint micromolecule part database that above-mentioned steps is determined, the molecular docking program that is provided with in the operation 2.1.The micromolecule part done one by one with active pocket dock, determine lead drug.Be divided into screening in enormous quantities and accurately screen two steps.
Screening in enormous quantities: parameter ga_num_evals=2500000 and ga_run=10 among the Autodock are set, and the molecular docking program that is provided with in the operation 2.1 is docked one by one.
Certainly also can carry out the setting of ga_num_evals and ga_run according to the size of other that make up or selected micromolecule part database.
Accurately screening: the ga_num_evals in the Autodock software and ga_run parameter are set to the highest, i.e. ga_num_evals=250000, ga_run=50, the molecular docking program that is provided with in the operation 2.1 is accurately screened.
6,, tentatively determine to have the lead drug of cancer chemoprotective or anticancer effect according to butt joint result's ordering.
Through the accurate screening in the 5th step, obtain butt joint back micromolecule part database, according to butt joint result's ordering, tentatively determine to have the lead drug of cancer chemoprotective or anticancer effect.
Certainly, for the number of further dwindling lead drug can also adopt: comprehensive evaluation butt joint result's ordering, micromolecule part pharmacophore contains that there is something special and the interaction mode in micromolecule part and activated centre, determines to have the lead drug of cancer chemoprotective or anticancer effect.
(2) utilize the screening of cell high flux screening platform to have anticancer medicine
By above-mentioned virtual screening, from free natural products data, filter out a collection of and the active pocket that with Cys151 the is center construction candidate compound preferably that interacts.According to process flow diagram shown in Figure 11, utilize cell high flux screening platform in vitro QR activity assay system then, further screen candidate compound, determine II phase enzyme inducer medicine, thereby determine to have anticancer medicine.
1, our candidate compound behind virtual screening, choose at random a compound: isoflavones, Genistein, molecular structure is as follows,
According to process flow diagram shown in Figure 11, carry out the experiment of cell high flux.
Quinone reductase (NAD (P) H:quinone reductase, QR), it is a kind of typical drug metabolism Phase II enzyme, two electron reductions that can the catalysis quinones substance generate quinhydrones, blocked the generation of semiquinone free radical, prevented the formation of a large amount of oxygen radicals, therefore brought into play important tumor prevention effect in the starting stage that tumour generates.Owing to utilize mouse hepatoma cell strain Hepa 1c1c7 cell can measure inducing of QR, have stability and high-throughout advantage easy and conveniently.Cell is through Genistein (1,5,10,50,100M) and after control group (group of solvents) handles, add QR reaction substrate (menadione) and corresponding reactant liquor, 595nm place absorbance is measured in order to measuring the QR activity in the reaction back, and the QR induced activity of medicine is judged in the variation of the absorbance by comparing administration group and control group unit's protein concentration.
The statistical result of isoflavones as shown in figure 12, various dose group (1,10,50, isoflavones 100M) induces QR to express, reach about 1.82,3.05,3.85 and 4.42 times of control group, and the increase that these QR express is compared with control group or significant difference (*, p＜0.05 all variant; *, p＜0.01).Conclusion: determine that isoflavones is an II phase enzyme inducer medicine, thereby determine that it is medicine with cancer-resisting.
Analyze: isoflavones, Genistein contains typical α in the molecule, β-unsaturated structural unit (pharmacophoric group), its molecular docking result is shown in Figure 10 A and Figure 10 B, and LYS150 has participated in the interaction of nearly all conformation and keap1 active pocket, and the mode of action and DEM are similar.Therefore isoflavones is by the sulfydryl site with the Cys151 of Keap1 Michael addition reaction to take place, thereby has changed the configuration of keap1, causes dissociating and having induced the expression of II phase enzyme of Nrf2, thereby brings into play its protective effect on cancer risk.
2, by other candidate compound behind the virtual screening, can proceed the experiment of cell high flux, thereby determine whether it is II phase enzyme inducer medicine according to the method for isoflavones, finally determine whether it is medicine with cancer-resisting.
21 kinds of cysteine residues Cys273 and Cys288 with keap1 albumen of embodiment are avtive spot, carry out the virtual screening of cancer chemoprotective cancer therapy drug.
When the Cys273 of Keap1 albumen and Cys288 simultaneously Michael addition reaction take place, can change the configuration of Keap1, and the Nrf2 transcription factor of correspondingly dissociating, impel it to enter the effect of performance increase II phase expression of enzymes in the nuclear.According to this basic theories, determine with Cys273 and two cysteine residues of Cys288 simultaneously as the activated centre.
1, utilize the homology mould to build the structured data that software is determined the PDB in Keap1 protein structure active function territory
With embodiment 1
2, adopt Autodock molecular docking software, determine the activated centre, set active pocket according to the active cysteine residues site CYS151 of keap 1;
With embodiment 1, be not both:
2.2 active cysteine residues site Cys273 and Cys288 with keap1 are the activated centre, set active pocket;
The setting of active pocket during butt joint: the coordinate file of at first opening keap1, find the coordinate of Cys273 and each atom of Cys288, average in x, y, three directions of z respectively, draw the centre coordinate of Cys273 and Cys288, and write down numerical value (be that x is-17.717, y for-4.149, z is-78.05).When active pocket was set, x, the y of active pocket center correspondence, z filled out earlier above-mentioned numerical value respectively.The default value of sizing grid is 40 * 40 * 40, and step-length (spacing) is 0.375nm, then by adjusting the numerical value of sizing grid repeatedly, further determines the position and the size of active pocket, makes it can just encase groove.Active cysteine residues site Cys273 and Cys288 with keap1 are the active pocket in activated centre: the activated centre coordinate is: x for-17.717, y for-4.149, z is-78.05, the size of active pocket decides according to the molecular docking result and the interaction mode of the positive control molecule in following 2.3 steps.
2.3 adjust and the checking active pocket
Utilize the known 15-deoxy prostaglandin J2 (15-Deoxy-12 that Michael addition reaction can all take place with Cys273 and Cys288,14-Prostaglandin J2,15D-PGJ2, structural formula is as follows) do the positive control molecule, according to they in active pocket with the interactional binding energy of pocket and whether to help taking place Michael addition reaction be standard, checking and to establish in the keap1 albumen with Cys273 and Cys288 be the active pocket in activated centre, and carry out next step butt joint.
According to checking repeatedly, we have finally determined the condition that is provided with of active pocket.Specific as follows: for active cysteine residues site Cys273 and Cys288 with keap1 are the active pocket in activated centre: centre coordinate is that x is-17.717, y for-4.149, z is-78.050, the sizing grid value is 60 * 62 * 62, and step-length (spacing) is 0.375nm.
Be below under this active pocket of determining, the positive control molecule reaches the interactional analysis of group on every side with the activated centre in active pocket.
15D-PGJ2 and Cys73 and Cys288 activated centre results of interaction are shown in Figure 13 A and Figure 13 B.Has good interaction between positive control molecule 1 5D-PGJ2 and the Cys273 activated centre, binding energy reaches-4.6, and present the pharmacophoric group (α of 15D-PGJ2, β-unsaturated group) face the position of Cys273 cysteine residues (yl moiety) from spatial orientation, this kind mode of action makes that Michael addition reaction is easier to be carried out.The mode of action of 15D-PGJ2 and Cys288 also is the generation that more helps Michael addition reaction from the space similarly.
By positive control, further determine and verified that obtained above-mentioned is the rationality of the active pocket in activated centre with Cys273 and Cys288 in the keap1 albumen, can carry out next step micromolecule ligand screening.
3, the micromolecule part is screened, the screening project is the screening that contains that there is something special of screening of toxicity screening, quasi-medicated property and pharmacophore
With embodiment 1.
4, set up butt joint micromolecule part database
With embodiment 1.
5, according to the active pocket of setting, utilize molecular docking software, butt joint is made with active pocket one by one of the micromolecule part in the micromolecule part database docked;
With embodiment 1.
6,, tentatively determine to have the lead drug of cancer chemoprotective or anticancer effect according to butt joint result's ordering.
With embodiment 1.
(2) utilize the screening of cell high flux screening platform to have anticancer medicine
By above-mentioned virtual screening, from free natural products data, filter out a collection of with Cys273 and Cys288 be the active pocket of the center construction candidate compound preferably that interacts.According to process flow diagram shown in Figure 11, utilize cell high flux screening platform in vitro QRactivity assay system then, further screen candidate compound, determine II phase enzyme inducer medicine, thereby determine to have anticancer medicine.
1, our candidate compound behind virtual screening, choose at random a compound: Ligustilide ligustilide, Ligustilide are the effective constituent in the famous Chinese traditional medicine angelica, account for about 45% of Chinese angelica volatile oil composition.Its molecular structure is as follows,
According to process flow diagram shown in Figure 11, carry out the experiment of cell high flux.
Because mouse hepatoma cell strain Hepa 1c1c7 cell is through Ligustilide (1,5,10,50,100M) and after control group (group of solvents) handles, add QR reaction substrate (menadione) and corresponding reactant liquor, 595nm place absorbance is measured in order to measuring the QR activity in the reaction back, and the QR induced activity of medicine is judged in the variation of the absorbance by comparing administration group and control group unit's protein concentration.Its statistical result as shown in figure 15, various dose group (1,10,50, Ligustilide 100M) induces QR to express about 2.6,3.4,4.7 and 42.6 times that reach control group respectively, and the increase that these QR express is compared all variant or significant difference (*, p＜0.05 with control group; *, p＜0.01).Conclusion: determine that Ligustilide is an II phase enzyme inducer medicine, thereby determine that it is medicine with cancer-resisting.
Analyze: contain typical α in the Ligustilide structure, β-unsaturated structural unit, its Autodock molecular docking result is shown in Figure 14 A and Figure 14 B.With 15D-PGJ2 to dock the result similar, the α of Ligustilide, β-unsaturated group also all is on the position relative with Cys273 (Figure 14 A) and Cys288 (Figure 14 B) from the space respectively, and its binding energy all reaches about-5, fully guaranteed the stability of this kind mode of action, thereby made Ligustilide become the optimal drug that further carries out the cell platform validation.
2, by other candidate compound behind the virtual screening, can proceed the experiment of cell high flux, thereby determine whether it is II phase enzyme inducer medicine according to the method for Ligustilide, finally determine whether it is medicine with cancer-resisting.
31 kinds of cysteine residues Cys257 with keap1 albumen of embodiment are avtive spot, carry out the virtual screening of cancer chemoprotective cancer therapy drug.
Method is identical with embodiment 1, and difference is: when active pocket is set: the centre coordinate with Cys257 is the center of active pocket.
41 kinds of cysteine residues Cys297 with keap1 albumen of embodiment are avtive spot, carry out the virtual screening of cancer chemoprotective cancer therapy drug.
Method is identical with embodiment 1, and difference is: when active pocket is set: the centre coordinate with Cys297 is the center of active pocket.
Claims (3)
1. one kind is the virtual screening method of the novel anticancer medicine of target spot with Keap1, it is characterized in that: may further comprise the steps:
1) determines the PDB structured data of Keap1 protein structure;
2) adopt molecular docking software, determine the activated centre, set active pocket according to the active cysteine residues site of keap1;
3) the micromolecule part is screened, the screening project is the screening that contains that there is something special of screening of toxicity screening, quasi-medicated property and pharmacophore;
4) set up butt joint micromolecule part database;
5) according to the active pocket of setting, utilize molecular docking software, butt joint is made with active pocket one by one of the micromolecule part in the micromolecule part database docked;
6), tentatively determine to have the lead drug of cancer chemoprotective or anticancer effect according to butt joint result's ordering.
According to claim 1 described a kind of be the virtual screening method of the novel anticancer medicine of target spot with Keap1, it is characterized in that: step 3) can place step 1) to any one step of step 6).
According to claim 1 described a kind of be the virtual screening method of the novel anticancer medicine of target spot with Keap1, it is characterized in that: step 6) is: according to butt joint result's ordering, micromolecule part pharmacophore contains that there is something special and the interaction mode in micromolecule part and activated centre, comprehensive evaluation tentatively determines to have the lead drug of cancer chemoprotective or anticancer effect.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1725222A (en) * | 2004-07-23 | 2006-01-25 | 中国科学院上海药物研究所 | Combinatorial chemistry centralized repository design and optimization method |
| CN1730665A (en) * | 2005-08-03 | 2006-02-08 | 华东理工大学 | PI3K enzyme inhibitor quick-speed screening method |
| WO2006107719A2 (en) * | 2005-04-01 | 2006-10-12 | Enzo Biochem, Inc. | Compositions and methods for the inhibition of dishevelled proteins |
| CN101089245A (en) * | 2006-12-30 | 2007-12-19 | 中国医学科学院放射医学研究所 | Virtual screening method for compound pesticide effect matter base of traditional chinese medicine |
| CN101736406A (en) * | 2009-11-04 | 2010-06-16 | 西华大学 | Method for discovering active components of traditional Chinese medicine by metabolic forecast and virtual sieving |
-
2010
- 2010-08-06 CN CN2010102464498A patent/CN101916330B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1725222A (en) * | 2004-07-23 | 2006-01-25 | 中国科学院上海药物研究所 | Combinatorial chemistry centralized repository design and optimization method |
| WO2006107719A2 (en) * | 2005-04-01 | 2006-10-12 | Enzo Biochem, Inc. | Compositions and methods for the inhibition of dishevelled proteins |
| CN1730665A (en) * | 2005-08-03 | 2006-02-08 | 华东理工大学 | PI3K enzyme inhibitor quick-speed screening method |
| CN101089245A (en) * | 2006-12-30 | 2007-12-19 | 中国医学科学院放射医学研究所 | Virtual screening method for compound pesticide effect matter base of traditional chinese medicine |
| CN101736406A (en) * | 2009-11-04 | 2010-06-16 | 西华大学 | Method for discovering active components of traditional Chinese medicine by metabolic forecast and virtual sieving |
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