阅读说明：本技术 一种基于增强采样分子动力学模拟的混合拟、抗糖皮质激素干扰物的识别方法 (It is a kind of that quasi-, Antiglucocorticoid chaff interferent recognition methods is mixed based on enhancing sample molecules dynamics simulation ) 是由 史薇 陈钦畅 于红霞 于 2019-09-24 设计创作，主要内容包括：本发明公开了一种基于增强采样分子动力学模拟的混合拟、抗糖皮质激素干扰物的识别方法,基于核受体变构和共调节因子调控机制,采用增强采样分子动力学模拟方法,通过识别局部和全局自由能最低点并进行动态轨迹聚类,提取配体化合物作用下糖皮质激素受体的稳态构象,并根据受体第12号螺旋稳定位置与共调节因子招募/抑制机制的关系,判断受测化合物的内分泌干扰效应,识别和预测拟性、抗性和混合拟、抗糖皮质激素干扰物。相比于传统体外实验方法,此方法成本低廉、效率更高,且避免了混合拟、抗性干扰物的细胞特异性问题；相比于已有的计算机辅助筛选方法,此方法能有效识别稳态构象,实现混合拟、抗糖皮质激素干扰效应的预测。(The invention discloses a kind of mixing based on enhancing sample molecules dynamics simulation is quasi-, the recognition methods of Antiglucocorticoid chaff interferent, based on nuclear receptor allosteric and total regulatory factor regulatory mechanism, using enhancing sample molecules dynamics simulation method, by identifying Mobile state trajectory clustering of locally going forward side by side with global free energy minimum point, extract the stable state conformation that ligand compound acts on lower glucocorticoid receptor, and according to No. 12 spiral settling positions of acceptor control regulatory factor recruitment/suppression mechanism relationship together, judge the endocrine disrupting of tested compounds, identification and the quasi- property of prediction, resistance and mixing are quasi-, Antiglucocorticoid chaff interferent.Compared to traditional ill vitro test method, the method is low in cost, more efficient, and avoids and mix quasi-, resistance chaff interferent cell-specific problem；Compared to existing computer-aided screening method, the method can effectively identify stable state conformation, realize and mix quasi-, Antiglucocorticoid disturbing effect prediction.)

1. a kind of mixing based on enhancing sample molecules dynamics simulation is intended, the recognition methods of Antiglucocorticoid chaff interferent, It is characterized in that, comprising the following steps:

(1) construct and optimize the structure of glucocorticoid receptor；Ligand compound structure is constructed and optimizes, by ligand compound pair It is connected in the hormone binding pocket of glucocorticoid receptor, obtains ligand-glucocorticoid receptor complex；Selection can be mentioned effectively Gained ligand-glucocorticoid receptor complex is carried out enhancing sampling by the set variable of No. 12 screws of high acceptor control Molecular dynamics simulation；

(2) the molecular motion track obtained according to enhancing sample molecules dynamics simulation, selection can effectively describe acceptor control 12 The set variable of spiral status draws free energy characteristic pattern and obtains free energy low spot；To enhancing sample molecules dynamics simulation Obtained molecular motion track carries out conformation cluster, obtains representative conformation；According to free energy low spot and corresponding representative structure As extracting representative stable state conformation；

(3) it is located at the stable state conformation for stopping the co-activation factor and co-suppression factor binding site for No. 12 spiral, is determined as Barrier type conformation；Exposure co-activation factor binding site is located at for No. 12 spiral and stops co-suppression factor binding site Stable state conformation is determined as activated form conformation；The exposure co-activation factor and co-suppression factor bound site are located at for No. 12 spiral The stable state conformation set, is determined as competitive type conformation；

(4) sentenced according to stable state conformations and its quantity for promoting receptor to form the ligand compound of single barrier type conformation It is set to Antiglucocorticoid chaff interferent；For promoting receptor to form the ligand compound of single activated form conformation, it is determined as quasi- sugar Cortin chaff interferent；For promote receptor formed singular competition type conformation or more than one type stable state conformation it is liganded Object is closed, is judged to mixing quasi-, Antiglucocorticoid chaff interferent.

2. the method according to claim 1, wherein receptor combines in Protein Data Bank in step (1) The glucocorticoid receptor of endogenous hormone or quasi- property drug, it is desirable that resolution ratio is less than 2.5 Ethylmercurichlorendimides, the optimization method of receptor are as follows: Incomplete amino acid residue is supplemented completely first, hydrogen atom then is added to receptor, finally assigns the field of force AMBER to receptor.

3. the method according to claim 1, wherein ligand-receptor complex carries out enhancing and adopts in step (1) The method of sample molecular dynamics simulation are as follows: ligand and receptor assign the field of force CHARMM respectively, and immerse in TIP3P model water, add After entering sodium ion or chloride equilibrium system charge, energy minimum is carried out；By two step of canonical assemblage and isothermal isobaric ensemble After balance, system is made to maintain 300K and 1 normal atmosphere pressure；The enhancing sample molecules for finally carrying out no less than 25 nanoseconds are dynamic Mechanical simulation, Gauss height and width are respectively set to 4.0 every mole of kilojoules and 0.2 nanometer.

4. the method according to claim 1, wherein No. 12 spiral fortune of acceptor control can be effectively improved in step (1) The selection method of the set variable of dynamic property are as follows: with glucocorticoid receptor I757 and L733, I757 and Q597 and K770 and V675 The distance between alpha-carbon atom carry out enhancing sample molecules dynamics simulation for set variable.

5. the method according to claim 1, wherein can effectively describe No. 12 helical forms of acceptor control in step (2) The selection method of the set variable of state are as follows: using the c-terminus and the 3rd, 4 and No. 5 spiral of No. 12 spiral of glucocorticoid receptor The distance between center be set variable 1, helicity, that is, α spiral beating score value of No. 12 spiral is set variable 2, draw from By energy characteristic pattern, the position of No. 12 spiral is described, wherein c-terminus, that is, 758-762 of No. 12 spiral of glucocorticoid receptor Number amino acid, No. 12 spiral, that is, 749-765 amino acid.

6. the method according to claim 1, wherein in step (2), the conformation clustering method of motion profile are as follows: It based on the track of No. 12 spiral, according to its root-mean-square-deviation, is clustered, is obtained each poly- for boundary with 0.16 nanometer The representative conformation of class.

7. the method according to claim 1, wherein in step (2), the judgment method of representative stable state conformation Are as follows: global and local free energy minimum point and its free energy are obtained according to free energy characteristic pattern first；Secondly it selects global free Minimum point and the local free energy minimum point within 20 every mole of kilojoules can be differed from it by, obtain corresponding set variate-value；Most Afterwards, corresponding cluster is positioned according to set variate-value, and obtains representative conformation, as representative stable state conformation.

8. the method according to claim 1, wherein in step (3), the judgment method of stable state conformations are as follows: If the alpha-carbon atom in No. 12 spiral and the minimum range between the midpoint of both I572 and Q597 alpha-carbon atom less than 6 Ethylmercurichlorendimides, Then think to block the co-activation factor and co-suppression factor binding site；If alpha-carbon atom and I572 in No. 12 spiral with Distance is greater than 6 Ethylmercurichlorendimides and less than 11 Ethylmercurichlorendimides between the midpoint of both Q597 alpha-carbon atom, then it is assumed that exposes the co-activation factor and leads Shelves co-suppression factor binding site；If the midpoint of alpha-carbon atom and both I572 and Q597 alpha-carbon atom in No. 12 spiral it Between distance be greater than 11 Ethylmercurichlorendimides, then it is assumed that expose the co-activation factor and co-suppression factor binding site.

9. the method according to claim 1, wherein step (1), (2), in (3), for No. 12 helix length Greater than the nuclear receptor of 12 amino acid, it can be based on the above method, it is corresponding with after glucocorticoid receptor overlapping according to receptor Amino acid setting set variable, carry out enhancing sample molecules dynamics simulation, judge the stable state type and predicted interference of receptor Type.

10. the method according to claim 1, wherein the molecular simulation that the molecular dynamics simulation uses is soft Part is gromacs and plumed software package.

Technical field

The invention belongs to use computer program to carry out prediction toxicology field, and in particular to a kind of to use computer software The nuclear receptor based on enhancing sample molecules the dynamics simulation quasi- property, resistance and the mixing that mediate be quasi-, resistance glucocorticoid is dry Disturb object recognition methods.

Background technique

Incretion interferent (endocrine disrupting chemicals, EDCs) refers to through disturbance endocrine system Cause the ligand compound of adverse effect.EDCs is in surrounding medium^1,2, food³Even blood of human body^4,5In have extensive detection, It is exposed can cause a series of influences to human health, increase reproductive disease⁶, birth defect⁷, prostate and breast cancer⁸, cardiopulmonary Disease⁹, obesity¹⁰And the nervous system disease¹¹Etc. risks, and cause huge economic loss, European Union is because of disease caused by EDCs Spending is 217,000,000,000 dollars, account for the 1.28% of GDP, and the U.S. reaches 340,000,000,000 dollars, and it is total to account for domestic production The 2.33% of value^12,13.Thus, identification and the control of EDCs are always the hot spot of whole world Environmental Health and security fields research.

The main function approach of EDCs be by with the nuclear receptor (nuclear receptor) in nucleus, including it is female swash Plain receptor (estrogen receptor, ER) (including ER α and ER β), androgen receptor (androgen receptor, AR), Thyroid Hormone Receptors (thyroid hormone receptor, TR) (including TR α and TR β) and glucocorticoid receptor (glucocorticoid receptor, GR) etc., in conjunction with and change its function, so as to cause disturbing effect.In recent years, GR is situated between The endocrine disrupting led is considered as the important mechanisms of environment incretion disturbing effect, glucocorticoid disturbing effect also by To more and more concerns¹⁴.EDCs can generate excitement or antagonistic effect (i.e. quasi- property or resistance effect) by nuclear receptor, There are a large amount of EDCs that can cause quasi- property and resistance simultaneously^15,16.The quasi- property and resistance effect of EDCs can activate different harmful final result roads Diameter (adverse outcome pathway, AOP) simultaneously leads to different harmful final results (adverse outcome, AO).Such as, intend Glucocorticoid efficiency can damage reproductive function¹⁷, and antiglucocorticoid effects can aggravate inflammatory reaction¹⁸.Mix quasi-, resistance Effect usually has cell, tissue specificity^19–21, it means that there is mixing to intend, the EDCs of resistance effect will affect bigger model The tissue enclosed²², and be by a kind of screening technique of cell line only it is incomplete, be easy to produce false negative^23,24.EDCs may Pure quasi- property effect is presented in a kind of cell line, and resistance effect is presented in another cell line^15,16, therefore, need to improve The existing screening technique based on cell intends property, resistance with effective screening and mixes quasi-, resisting substance.

The generation regulatory factor, including the co-activation factor together of quasi- property, resistance effect that nuclear receptor mediates (coactivator, CoA) and the co-suppression factor (corepressor, CoR), effect it is closely related.Existing research report CoA and CoR are illustrated to quasi- property, resistance and mixes quasi-, resistance effect importance^25,26.In fact, existing crystalline results hair Under mixing now is quasi-, resistance effector substance acts on, nuclear receptor can form the phenomenon that multistable conformation coexists, that is, be formed simultaneously No. 12 spiral (H12) of a variety of different types of conformations, receptor is stable at different positions, and respectively recruit CoA and CoR, to cause quasi- property and resistance effect respectively²⁷.Therefore, in red rosy clouds etc., (Yu Hongxia, Shi Wei, the small that enjoys of king are based on Molecule Motion The virtual screening method of the nuclear receptor mediating endocrine interfering substance of mechanical simulation: CN, CN103324861A [P] .2013.), (Zhang Aiqian, Lin Yuan, Peng Sufen, Liu Lei, Gao Changan, Han Shuo's Zhang Aiqian etc. go against a kind of organic estrogen receptor agonism of and short of money The recognition methods of anti-effect: CN, CN101381894A [P] .2009.) (Yu Hongxia, Shi Wei, Chen Qinchang, king are small for He Yuhong rosy clouds etc. Enjoy a kind of thyroid hormone replacement therapy virtual screening and its active quantitative calculating of interference that regulatory factor is total to based on nuclear receptor of Method: CN, CN105893759A [P] .2016) all once using molecular dynamics (molecular dynamics, MD) simulation etc. Computer assisted screening technique recruits situation by the stable case and/or common factor of nuclear receptor H12 and predicts ligand chemical combination The endocrine disrupting of object.However, existing computer assisted screening technique, all builds just for quasi- property or resistance effect Vertical prediction model, and without the allosteric and common factor recruitment process after considering EDCs in conjunction with receptor comprehensively and mix quasi-, resistance The phenomenon that multistable conformation that chaff interferent may cause coexists, therefore, it is impossible to distinguish quasi- property, resistance and the mixing of EDCs simultaneously Quasi-, resistance effect.Enhancing sample molecules dynamics simulation method can promote EDCs- receptor system to jump out local energy minimum point And global energy minimum point is searched for, Successful utilization is in the research of nuclear receptor conformation change^28,29, facilitating exploration EDCs can Phenomenon coexists for nuclear receptor multistable caused by energy and mixing resulting from is quasi-, resistance effect.Literature search the result shows that, In Before the present invention completes, do not find that phenomenon coexists in the nuclear receptor multistable obtained based on enhancing sample molecules dynamics simulation also Identification and prediction mix quasi-, resistance glucocorticoid chaff interferent report.

Bibliography

(1)Annamalai,Jayshree；Namasivayan,V.Endocrine disrupting chemical in the atmosphere:their effects on humans and wildlife.Environ.Int.2015,76,78– 97.

(2)Padhye,L.P.；Yao,H.；Kung'u,F.T.；Huang,C.-H.Year-long evaluation on the occurrence and fate of pharmaceuticals,personal care products,and endocrine disrupting chemicals in an urban drinking water treatment plant.Water Res.2014,51, 266–276.

(3)Mezcua,M.；Martínez-Uroz,M.A.；Gómez-Ramos,M.M.；Gómez,M.J.；Navas,J. M.；Fernández-Alba,A.R.Analysis of synthetic endocrine-disrupting chemicals in food:A review.Talanta 2012,100,90–106.

(4)Ballesteros,O.；Ballesteros,L.；Navalón,A.Talanta A multiclass method for the analysis of endocrine disrupting chemicals in human urine samples.Sample treatment by dispersive liquid–liquid microextraction.Talanta 2014,129,209–218.

(5)Xue,J.；Wu,Q.；Sakthivel,S.；Pavithran,P.V.；Vasukutty,J.R.；Kannan, K.Urinary levels of endocrine-disrupting chemicals,including bisphenols, bisphenol A diglycidyl ethers,benzophenones,parabens,and triclosan in obese and non-obese Indian children. Environ.Res.2015,137,120–128.

(6)Dickerson,S.M.；Gore,A.C.Estrogenic environmental endocrine- disrupting chemical effects on reproductive neuroendocrine function and dysfunction across the life cycle. Rev.Endocr.Metab.Disord.2007,8(2),143–159.

(7)Fernandez,M.F.；Olmos,B.；Granada,A.；López-Espinosa,M.J.；Molina- Molina,J. M.；Fernandez,J.M.；Cruz,M.；Olea-Serrano,F.；Olea,N.Human exposure to endocrine-disrupting chemicals and prenatal risk factors for cryptorchidism and hypospadias:A nested case-control study.Environ.Health Perspect.2007,115 (Suppl 1),8–14.

(8)Soto,A.M.；Sonnenschein,C.Environmental causes of cancer:Endocrine disruptors as carcinogens.Nat.Rev.Endocrinol.2010,6(7),363–370.

(9)Melzer,D.；Osborne,N.J.；Henley,W.E.；Cipelli,R.；Young,A.；Money,C.； McCormack,P.；Luben,R.；Khaw,K.T.；Wareham,N.J.；et al.Urinary bisphenol A concentration and risk of future coronary artery disease in apparently healthy men and women.Circulation 2012,125(12),1482–1490.

(10)Legler,J.；Fletcher,T.；Govarts,E.；Porta,M.；Blumberg,B.；Heindel, J.J.；Trasande,L. Obesity,diabetes,and associated costs of exposure to endocrine-disrupting chemicals in the European Union.J.Clin.Endocrinol.Metab .2015,100(4),1278–1288.

(11)Mustieles,V.；Pérez-Lobato,R.；Olea,N.；Fernández,M.F.Bisphenol A: Human exposure and neurobehavior.Neurotoxicology 2015,49,174–184.

(12)Attina,T.M.；Hauser,R.；Sathyanarayana,S.；Hunt,P.A.；Bourguignon, J.P.；Myers,J. P.；DiGangi,J.；Zoeller,R.T.；Trasande,L.Exposure to endocrine- disrupting chemicals in the USA:a population-based disease burden and cost analysis.Lancet Diabetes Endocrinol.2016,4(12),996–1003.

(13)Trasande,L.；Zoeller,R.T.；Hass,U.；Kortenkamp,A.；Grandjean,P.； Myers,J.P.；Digangi,J.；Bellanger,M.；Hauser,R.；Legler,J.；et al.Estimating burden and disease costs of exposure to endocrine-disrupting chemicals in the European Union.J.Clin. Endocrinol.Metab.2015,100(4),1245–1255.

(14)Sargis,R.M.；Johnson,D.N.；Choudhury,R.A.；Brady,M.J.Environmental endocrine disruptors promote adipogenesis in the 3T3-L1 cell line through glucocorticoid receptor activation.Obesity 2010,18(7),1283–1288.

(15)US EPA.iCSS ToxCast Dashboard https://actor.epa.gov/dashboard/.

(16)Kitamura,S.；Suzuki,T.；Sanoh,S.；Kohta,R.；Jinno,N.；Sugihara,K.； Yoshihara,S.；Fujimoto,N.；Watanabe,H.；Ohta,S.Comparative study of the endocrine-disrupting activity of bisphenol A and 19 related compounds.Toxicol.Sci.2005,84(2),249–259.

(17)M.,K.；J.A.,C.Glucocorticoid receptor signaling in health and disease.Trends Pharmacol.Sci.2013,34(9),518–530.

(18)Beck,K.R.；Sommer,T.J.；Schuster,D.；Odermatt,A.Evaluation of tetrabromobisphenol A effects on human glucocorticoid and androgen receptors: A comparison of results from human-with yeast-based in vitro assays.Toxicology 2016, 370,70–77.

(19)Crabtree,J.S.；Peano,B.J.；Zhang,X.；Komm,B.S.；Winneker,R.C.；Harris, H.A. Activity of three selective estrogen receptor modulators on hormone- dependent responses in the mouse uterus and mammary gland.Mol.Cell.Endocrinol.2008,287 (1–2),40–46.

(20)Safe,S.H.；Pallaroni,L.；Yoon,K.；Gaido,K.；Ross,S.；McDonnell, D.Problems for risk assessment of endocrine-active estrogenic compounds.Environ.Health Perspect. 2002,110(Suppl.6),925–929.

(21)Li,Y.；Perera,L.；Coons,L.A.；Burns,K.A.；Tyler Ramsey,J.；Pelch,K.E.； Houtman, R.；Van Beuningen,R.；Teng,C.T.；Korach,K.S.Differential in vitro biological action, coregulator interactions,and molecular dynamic analysis of bisphenol A(BPA),BPAF, and BPS ligand–ERαcomplexes.Environ.Health Perspect.2018,126(1),1–16.

(22)Vandenberg,L.N.；Maffini,M.V.；Sonnenschein,C.；Rubin,B.S.；Soto,A.M. Bisphenol-a and the great divide:A review of controversies in the field of endocrine disruption.Endocr.Rev.2009,30(1),75–95.

(23)Delfosse,V.；Grimaldi,M.；Cavaillès,V.；Balaguer,P.；Bourguet, W.Structural and functional profiling of environmental ligands for estrogen receptors.Environ.Health Perspect.2015,122(12),1306–1313.

(24)Wilson,V.S.；Bobseine,K.；Gray,L.E.Development and characterization of a cell line that stably expresses an estrogen-responsive luciferase reporter for the detection of estrogen receptor agonist and antagonists.Toxicol.Sci.2004,81(1),69–77.

(25)Liu,Z.；Auboeuf,D.；Wong,J.；Chen,J.D.；Tsai,S.Y.；Tsai,M.-J.；O' Malley,B.W. Coactivator/corepressor ratios modulate PR-mediated transcription by the selective receptor modulator RU486.Proc.Natl.Acad.Sci.2002,99(12), 7940–7944.

(26)Feng,Q.；O'Malley,B.W.Nuclear receptor modulation-Role of coregulators in selective estrogen receptor modulator(SERM)actions.Steroids 2014,90,39–43.

(27)Lusher,S.J.；Raaijmakers,H.C.A.；Vu-Pham,D.；Kazemier,B.；Bosch,R.； McGuire, R.；Azevedo,R.；Hamersma,H.；Dechering,K.；Oubrie,A.；et al.X-ray structures of progesterone receptor ligand binding domain in its agonist state reveal differing mechanisms for mixed profiles of 11β-substituted steroids.J.Biol.Chem.2012,287 (24),20333–20343.

(28)Duan,M.；Liu,N.；Zhou,W.；Li,D.；Yang,M.；Hou,T.Structural diversity of ligand-binding androgen receptors revealed by microsecond long molecular dynamics simulations and enhanced sampling.J.Chem.Theory Comput.2016,12(9), 4611–4619.

(29)Fratev,F.PPARγhelix 12exhibits an antagonist conformation.Phys.Chem.Chem. Phys.2016,18(13),9272–9280.

Summary of the invention

The technical problem to be solved by the present invention is to provide a kind of discovery sugar based on enhancing sample molecules dynamics simulation method Limitation in phenomenon coexists to solve existing method in discovery multistable conformation in the method for the stable state conformation of cortical hormone receptor Property.

The present invention also technical problems to be solved are to provide that quasi- property, resistance and mixing that a kind of nuclear receptor mediates be quasi-, resistance The screening of glucocorticoid chaff interferent and prediction technique, i.e., act on after nuclear receptor the function of leading to receptor according to ligand compound Property variation predict the quasi- property of ligand compound, resistance and mixing is quasi-, method of resistance disturbing effect, to solve the prior art The problems such as existing screening and true forecasting inaccuracy, while constructing a kind of rapid, efficient, accurately mixed based on computer program Close quasi-, antiestrogenic chaff interferent recognition methods.

In order to solve the above technical problems, The technical solution adopted by the invention is as follows:

It is a kind of that quasi-, Antiglucocorticoid chaff interferent recognition methods is mixed based on enhancing sample molecules dynamics simulation, The following steps are included:

(1) construct and optimize the structure of glucocorticoid receptor (glucocorticoid receptor, GR)；Building is simultaneously Optimize ligand compound structure, ligand compound is docked in the hormone binding pocket of GR, ligand-GR complex is obtained； The set variable that can effectively improve No. 12 spirals of acceptor control (helix 12, H12) motility is chosen, gained ligand-GR is compound Body carries out enhancing sample molecules dynamics simulation；

(2) the molecular motion track obtained according to enhancing sample molecules dynamics simulation, selection can effectively describe receptor H12 The set variable of state draws free energy characteristic pattern and obtains free energy low spot；Enhancing sample molecules dynamics simulation is obtained Molecular motion track carry out conformation cluster, obtain representative conformation；According to free energy low spot and corresponding representative conformation, Extract representative stable state conformation；

(3) it is located at the stable state conformation for stopping the co-activation factor and co-suppression factor binding site for H12, is judged to hindering Gear type conformation；It is located at the stable state conformation for exposing co-activation factor binding site and stopping co-suppression factor binding site to H12, It is determined as activated form conformation；It is located at the stable state conformation of the exposure co-activation factor and co-suppression factor binding site for H12, sentences It is set to competitive type conformation；

(4) according to stable state conformations and its quantity, for promoting receptor to form the ligand chemical combination of single barrier type conformation Object is determined as resistance chaff interferent；For promoting receptor to form the ligand compound of single activated form conformation, it is determined as that quasi- property is dry Disturb object；For promoting the ligand compound of the stable state conformation of receptor formation singular competition type conformation or more than one type, determine To mix quasi-, resistance chaff interferent.

In step (1), nuclear receptor is selected from Protein Data Bank (http://www.rcsb.org/pdb/home/ Home.do the crystal structure of GR is searched for and downloaded in), it is desirable that ligand is that endogenous hormone or quasi- property drug, resolution ratio are less than 2.5 Ethylmercurichlorendimides, the optimization method of receptor are as follows: the integrality of inspection structure first under Swiss-PdbViewer software, and will be residual Scarce amino acid residue supplement is complete, then adds hydrogen atom to receptor under SYBYL software, finally assigns to receptor The field of force AMBER.

In step (1), the building of ligand compound and optimization method are as follows: Chem3D software is used, by ligand compound point Minor structure first uses (Molecular Mechanics, the Allinger Force Field of molecular mechanics Allihn Gree field 2 Version 2, MM2) tentatively optimized, then Bao Weier (Powell) gradient algorithm and trie wave are used under SYBYL software (Tripos) field of force carries out suboptimization again.

In step (1), method that ligand-GR complex carries out enhancing sample molecules dynamics simulation are as follows: ligand and GR points Not Fu Yu the field of force CHARMM (Chemistry at HARvard Macromolecular Mechanics), and immerse TIP3P In model water, after sodium ion or chloride equilibrium system charge is added, energy minimum is carried out；By canonical assemblage and isothermal After isobaric two step of assemblage balance, system is made to maintain 300K and 1 normal atmosphere pressure；Finally carry out no less than 25 nanoseconds Enhance sample molecules dynamics simulation, Gauss height and width are respectively set to 4.0 every mole of kilojoules and 0.2 nanometer.

In step (1), can effectively improve receptor H12 motility set variable selection method are as follows: with the I757 of GR with The distance between alpha-carbon atom of L733, I757 and Q597 and K770 and V675 set variable carries out enhancing sample molecules dynamics Simulation.

In step (2), the selection method of the set variable of receptor H12 state can effectively be described are as follows: using the H12 carboxylic of GR The distance between center of cardinal extremity (758-762 amino acid) and H3, H4 and H5 is set variable 1, H12 (749-765 ammonia Base acid) helicity, that is, α spiral beating score value be set variable 2, the position of H12 is described.

In step (2), the conformation clustering method of motion profile are as follows: inclined according to its root mean square based on the track of H12 Difference is clustered for boundary with 0.16 nanometer, obtains the representative conformation of each cluster.

In step (2), the judgment method of representative stable state conformation are as follows: first according to free energy characteristic pattern obtain it is global and Local free energy minimum point and its free energy；Secondly select global free energy minimum point and differ from it by every mole of 20 kilojoule with Interior local free energy minimum point, obtains corresponding set variate-value；Finally, corresponding cluster is positioned according to set variate-value, And representative conformation is obtained, as representative stable state conformation.

In step (3), the judgment method of stable state conformations are as follows: if alpha-carbon atom and I572 in No. 12 spiral with Minimum range between the midpoint of both Q597 alpha-carbon atom is less than 6 Ethylmercurichlorendimides, then it is assumed that blocks the co-activation factor and co-suppression Factor binding site；If alpha-carbon atom in No. 12 spiral and distance is big between the midpoint of both I572 and Q597 alpha-carbon atom In 6 Ethylmercurichlorendimides less than 11 Ethylmercurichlorendimides, then it is assumed that expose the co-activation factor and main file co-suppression factor binding site；If the 12nd Alpha-carbon atom in number spiral and distance is greater than 11 Ethylmercurichlorendimides between the midpoint of both I572 and Q597 alpha-carbon atom, then it is assumed that exposure The co-activation factor and co-suppression factor binding site.

Step (1), in (3), is greater than No. 12 helix length the nuclear receptor of 12 amino acid, can be based on (2) The above method gathers variable with amino acid setting corresponding after glucocorticoid receptor overlapping according to receptor, enhance and adopt Sample molecular dynamics simulation, the stable state type and predicted interference type for judging receptor.

In the present invention, the molecular simulation software that the molecular dynamics simulation uses is gromacs and plumed software package.

Beneficial effect

The present invention is based on glucocorticoid receptor allosterics and total regulatory factor regulatory mechanism, using enhancing sample molecules power Analogy method is learned, is clustered by free energy low spot and dynamic trajectory, the stable state conformation that ligand compound acts on lower receptor is extracted, And according to H12 settling position regulatory factor recruitment/suppression mechanism relationship together, the sugared cortex of tested ligand compound is judged Hormone disturbing effect, screening and prediction intend property, resistance and mix quasi-, resistance glucocorticoid chaff interferent.

The present invention carries out the prediction of endocrine disrupting using enhancing sample molecules dynamics simulation method, establishes for the first time Multistable conformation identification and mixing is quasi-, resistance glucocorticoid chaff interferent prediction technique.

Compared with prior art, the present invention has the advantage that

(1) molecular simulation is carried out using enhancing sample molecules dynamics simulation method, more comprehensively finds nuclear receptor Global and local minimum energy point, and clustered by dynamic trajectory and obtain stable state conformation；

(2) according to nuclear receptor H12 settling position regulatory factor recruitment/suppression mechanism relationship together, by stable state conformation point For activated form, barrier type and competitive type three types, and the disturbing effect of ligand compound is judged accordingly: quasi- property, resistance and mixed Close quasi-, resistance；

(3) compared to traditional ill vitro test method, the method is low in cost, more efficient, and it is quasi-, anti-to avoid mixing The cell-specific problem of property chaff interferent；Compared to existing computer-aided screening method, the method more can be identified effectively surely State conformation is realized and mixes quasi-, resistance disturbing effect prediction.

Detailed description of the invention

Fig. 1 is quasi- quasi-, anti-and mixing of the invention, antiestrogenic chaff interferent virtual screening and prediction flow chart.

Fig. 2A is the free energy characteristic pattern of GR and corresponding stable state conformation under quasi- glucocorticoid standard substance acts on.

Fig. 2 B is the free energy characteristic pattern and corresponding stable state structure that mixing is quasi-, Antiglucocorticoid standard substance acts on lower GR As.

Fig. 3 A is the free energy characteristic pattern and corresponding stable state conformations that 12 ligand compounds act on lower GR.

Fig. 3 B is the free energy characteristic pattern and corresponding stable state conformations that 7 ligand compounds act on lower GR.

Fig. 4 is the regulatory factor recruitment/inhibition assay result comparison together of GR stable state conformations.

Specific embodiment

According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real It applies content described in example and is merely to illustrate the present invention, it is described in detail without that should will not limit in claims The present invention.

Below in conjunction with the embodiment of the present invention and attached drawing, technical solution of the present invention is clearly and completely described, it is clear that institute The embodiment of description is only a specific embodiment of the invention, rather than all.Based on the embodiments of the present invention, originally Research field other those of ordinary skill other embodiments obtained without making creative work, all locate In the scope of protection of the invention.

Following embodiment flow chart according to figure 1 is carried out, and used receptor is the GR of the mankind, ligand compound It selects quasi- glucocorticoid standard items dexamethasone (DEX), mixing to intend, is normal in Antiglucocorticoid standard items RU486 and environment The incretion interferent bisphenol seen.