CN104293877B - A kind of rapid screening method of GPR120 activator - Google Patents
A kind of rapid screening method of GPR120 activator Download PDFInfo
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- CN104293877B CN104293877B CN201410508486.XA CN201410508486A CN104293877B CN 104293877 B CN104293877 B CN 104293877B CN 201410508486 A CN201410508486 A CN 201410508486A CN 104293877 B CN104293877 B CN 104293877B
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Abstract
The invention discloses a kind of rapid screening method of GPR120 activator, use Computer-Aided Drug Design, to carrying out the compound of conformation search, do you use Discovery? Studio? 2.5 hypogen carries out the Pharmacophore Model configuration of 3D-QSAR, do you use Fischer simultaneously? 90% checking, obtains the linear regression of substantial activity and calculated activity. Draw according to the structural relation of the structure-activity relationship of GPR120 activator and endogenic ligand aliphatic acid: carboxyl is the necessary group of GPR120 activator, and rider is selected the candidate compound that contains carboxyl. Verify with the high flux screening of cell platform again, thus the candidate molecules of acquisition GPR120 activator. By the virtual screening of medicine, obtain at short notice the clue of reactive compound, goal in research is focused on to tens compounds from millions of compounds. And then from the compound screening, utilize cell Platform Screening to go out lead compound, and improve speed and the efficiency of screening compounds, shorten the cycle of new drug research.
Description
Technical field
The present invention relates to drug screening method, be specifically related to a kind of rapid screening method of GPR120 activator.
Background technology
GPR120 is nearest found g protein coupled receptor, belongs to the rhodopsin sample acceptor man in GPCRs superfamilyFamily. GPR120 all has expression in Various Tissues, has higher expression in colon. And GPR120 is moving at different generaIn the cells such as the taste buds cell of thing, intestines endocrine L cell, adipocyte, macrophage, all there is expression (HirasawaetAl.2005), GPR120 may with fat, type II diabetes is relevant.
Diabetes have become a difficult problem for the global people's health of impact as the metabolic disease taking hyperglycaemia as feature.GLP-1 is a kind of hormone of intestinal secretion, and it can promote blood sugar to rely on the secretion of insulin, and aliphatic acid comes as energy simultaneouslySource provides important condition for secreting intestines polypeptide. Have been reported and claim that in small intestine, expressing very high GPR120 is long-chain unsaturated lipidFat acid acceptor, and have the results show long-chain unsaturated fatty acid to stimulate GPR120 can increase the secretion of GLP-1, promote pancreasThe circulation (Hirasawaetal.2005) of island element. Therefore GPR120 is for treatment diabetes and other feeding desorder disease (examplesAs baulimia) be a very promising acceptor.
GPR120 ω-3 (such as DHA and the EPA) FAAs (Ohetal.2010) that are otherwise known as. Have been reported ω-3 aliphatic acid and little molecule activator stimulate GPR120 can produce antiinflammatory action widely, and the principle of antiinflammatory action is shown in Fig. 1, andWhat macrophage regulated organizes antiinflammatory action can improve the insulin resistance producing because of diabetes. This experimental result has illustratedAnti-inflammatory and insulin sensitizing agent are reacted the tie connecting by GPR120. Therefore for treatment insulin resistance, GPR120 doesBe that a novel targets is very promising.
Important function (Wuetal.2013) has also been played in the conduction of GPR120 signal in Human colon cancer. In colon cancerTissue and cell line in, the expression of GPR120 significantly raises. Activating the PI3K/Akt – NF-kB signal path of GPR120 can urgeEntering secretion and the expression of angiogenic factors in colon cancer cell, accelerate the Angiogenesis in colon cancer cell, is cancer cellBreeding provides nutritional support. And exciting GPR120 promotes tumor cell migration and the conduction of tumour cell interstitial. Therefore,GPR120 can be used as the brand-new target spot for the treatment of colon cancer.
Until 2005, the people such as Hirasawa have found the endogenic ligand (Hirasawaetal.2005) of GPR120,GPR120 is considered to orphan receptor always. They from 1000 multiple compounds, with quantitative Flow Cytometry at stable tableReach the fluorescence labeling acceptor quantity that detects endocytosis in the HEK293 cell of GPR120-EGFP, the part that filters out GPR120 isThe unrighted acid of the saturated fatty acid of C14-C18 chain length and C16-C22 chain length. After this, non-endogenic little molecule excitementAgent is also found, GW9508, NCG21, TUG-891 (Sunetal.2010, Hudsonetal.2013, ShimpukadeEtal.2012) similar at effect and the endogenous LCFA of activation GPR120, effectively in activating cellCa in ERK, cell2+The secretion of release reaction and GLP-1.
Summary of the invention
The object of the invention is to set up a kind of rapid screening method of GPR120 activator, the method is from knownGPR120 activator sets out, and sets up the screening technique of a set of high efficient and reliable, finds novel little molecule activator by contributing to, and hasEffect improves the speed of screening, reduces screening cost.
First the present invention uses Computer-Aided Drug Design technology, with reference to previous work and pertinent literature, to enteringThe compound of row conformation search, uses the hypogen of DiscoveryStudio2.5 to carry out the Pharmacophore Model structure of 3D-QSARType uses Fischer90% checking simultaneously, obtains the linear regression of substantial activity and calculated activity. According to the GPR120 of reportThe structural relation of the structure-activity relationship of activator and endogenic ligand aliphatic acid draws: carboxyl is the necessary group of GPR120 activator,Therefore rider is selected the candidate compound that contains carboxyl. Further verify with the high flux screening of cell platform again, therebyObtain the candidate molecules of GPR120 activator.
To achieve these goals, the present invention adopts following technical scheme:
A rapid screening method for GPR120 activator, comprises the following steps:
(1) pick out pEC50 value and be greater than 4 20-50 GPR120 activator as candidate compound;
(2) compound in step (1) use DiscoveryStudio2.5 hypogen module construction pharmacophoreModel: use Caesar method to carry out conformation search (employing default setting); Then isomorphic map not being produced to Pharmacophore Model entersRow is superimposed, obtains altogether 5-15 Pharmacophore Model; Obtain substantial activity and the calculated activity of candidate compound at report interfaceLinear regression, we determine regression coefficient R2Be worth the highest model as optimum Pharmacophore Model;
(3) (it is one that Dutch SPECS company is found in 1987 to SPECS database to adopt in step (2) Pharmacophore ModelIndividual medium scale compound library) carry out virtual screening, obtain marking value front 500-1000 compound from high to low, and pressMarking value is arranged as list from high to low;
(4) according to screening marking value with becomes five times of the property of medicine to restrain principles, manually in order selecting step (3) with carboxyl50-100 compound;
(5) compound of step (4) screening is carried out to active primary dcreening operation, concentration is that 10 μ M stimulate after STC-1 cell, filters outThe compound that causes higher or close ERK phosphorylation degree than positive control drug GW9508 carries out next step EC50Mensuration;
(6) compound that pEC50 value is greater than positive control drug GW9508 is as GPR120 activator.
The invention has the beneficial effects as follows:
The present invention, by the virtual screening of medicine, can obtain the clue of reactive compound at short notice, will study orderMark focuses on tens compounds from millions of compounds. And then in compound from virtual screening, utilize cell flatPlatform filters out lead compound, has therefore greatly improved speed and the efficiency of screening compounds, shortens the cycle of new drug research.
Brief description of the drawings
Fig. 1 omega-fatty acid and GPR120 effect produce the principle of antiinflammatory action;
The GPR120 Pharmacophore Model schematic diagram that Fig. 2 builds, wherein 1 is hydrogen bond receptor, the 2nd, aromatic core, the 3rd, hydrophobicCenter;
Fig. 3 is as the structural formula of compound schematic diagram that builds pharmacophore;
Compound (table 1) substantial activity that Fig. 4 has selected and the linear regression of calculated activity.
Detailed description of the invention
Below in conjunction with accompanying drawing and embodiment, the invention will be further described.
A rapid screening method for GPR120 activator, step is as follows:
(1), with reference to previous work and pertinent literature, from the GPR120 activator of having reported, select pEC50 value and be greater than 427 (Shimpukadeetal., 2012) compounds as the Pharmacophore Model configuration candidate compound of 3D-QSAR (table1), be used as the structural formula of compound schematic diagram that builds pharmacophore as Fig. 3.
(2) Caesar method (J.Chem.Inf.Model.47, the 1923-32 of use DiscoveryStudio2.5(2007)) 27 candidate compounds are carried out to conformation search according to default setting, use afterwards hypogen to not isomorphic map generationPharmacophore Model is carried out superimposed, obtains altogether 10 Pharmacophore Model, and obtains the reality of candidate compound at report interfaceThe linear regression of activity and calculated activity, determines regression coefficient R2Be worth the highest model as optimum Pharmacophore Model (Fig. 2), R2Be 0.888 (Fig. 4).
(3) (it is one that Dutch SPECS company is found in 1987 to SPECS database to adopt in step (2) Pharmacophore ModelIndividual medium scale compound library) carry out virtual screening based on Pharmacophore Model (Bioorg.Med.Chem.Lett.2010,20,5130-2), obtain marking value 500 compounds from high to low, and be arranged as from high to low list by marking value;
(4) according to screening marking value and becoming five times of rule principles of the property of medicine, manually choose in order 50 chemical combination with carboxylThing (table 2);
(5) compound of step (4) screening is carried out to active primary dcreening operation, concentration is that 10 μ M stimulate after STC-1 cell, filters outThe compound that causes higher or close ERK phosphorylation degree than positive control drug GW9508 carries out next step EC50Mensuration, liveProperty primary dcreening operation the results are shown in Table 2;
(6) adopt bioluminescence resonance energy to shift (BRET) to sifting out 13 compound determination EC50，pEC50Value is greater thanThe compound of positive control drug GW9508 is as GPR120 activator, and activity the results are shown in Table 3.
Biological activity determination:
First adopt the STC-1 cell of high expressed GPR120, select concentration 10uM to be primary dcreening operation concentration, positive with GW9508Contrast medicine, using GAPDH or actin as interior mark, uses immunoblotting (Hirasawaetal., 2005), measures table 2In after 50 compound irritation cell 10min in born of the same parents ERK phosphorylation degree change (table 2). There are 13 compounds (table 3) to drawPlay the STC-1 cell line ERK phosphorylation of relatively high (ERK phosphorylation degree is higher than GW9508 or close with it), further surveyDetermine the EC of compound in table 350。
Adopt bioluminescence resonance energy to shift (BRET) and further determine the compd E C in table 350Method is: adopt winkTime transfection method (Shimpukadeetal., 2012), by GPR120-YFP and two kinds of plasmids of RLuc-β-arrestin2 withTime transfection (Shimpukadeetal., 2012) enter in HEKC (HEK293), transfection 24h-48h, adds concentration oceanRenilla luciferase (final concentration is 3 μ M) is hatched 5-15min, with the compound stimulation 5-in variable concentrations (1nM-10uM) table 3After 10min, use ELIASA (POLARstarOmega) fluorescence intensity than (460nm/520nm) change detection compd E C50。
It is further true that luciferase reporter gene detection kit (Promega, E1910) detects intracellular free calcium levelDetermine compd E C50Method be: adopt the method (Shimpukadeetal., 2012) of transient transfection, NFAT-FLuc, TK-This three kinds of plasmids transfection simultaneously of RLuc and GPR120-Gq is entered in HEK293 cell, after transfection 24h-48h, with the compound in table 3Stimulate, utilize Dual-Luciferase detection kit to detect (concrete grammar is according to the operation of kit description) intracellular calcium denseThereby the variation of degree obtains the EC of compound50(table 3).
The compound that table 1 builds for pharmacophore
Table 2 compound is to GPR120 agonist activity primary dcreening operation result
The active better EC of compound of table 350
Experimental result shows: the aromatic acids compound of selecting according to area of computer aided drug sieve has good activationThe activity of GPR120, shows that the compound in the present invention has higher using value in the diseases such as treatment diabetes, obesity.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned, not the present invention is protected to modelThe restriction of enclosing, one of ordinary skill in the art should be understood that, on the basis of technical scheme of the present invention, those skilled in the art are notNeed to pay various amendments that creative work can make or distortion still in protection scope of the present invention.
Claims (5)
1. a rapid screening method for GPR120 activator, is characterized in that, comprises the following steps:
(1) pick out pEC50 value and be greater than 4 20-50 GPR120 activator as candidate compound;
(2) compound in step (1) uses the hypogen module construction Pharmacophore Model of DiscoveryStudio2.5, makesCarry out conformation search by Caesar method, then isomorphic map is not produced to Pharmacophore Model and carry out superimposedly, obtain 5-15 drug effectGroup's model, obtains the substantial activity of candidate compound and the linear regression of calculated activity at report interface, determine regression coefficientR2Be worth the highest model as optimum Pharmacophore Model;
(3) adopt optimum Pharmacophore Model in step (2) to carry out virtual screening to SPECS database, obtain marking value from height toFront 500-1000 compound of low arrangement, and be arranged as from high to low list by marking value;
(4) according to screening marking value and becoming five times of rule principles of the property of medicine, manually choose in order 50-100 the chemical combination with carboxylThing;
(5) compound of step (4) screening is carried out to active primary dcreening operation, concentration is that 10 μ M stimulate after STC-1 cell, filters out than sunProperty contrast medicine GW9508 causes that the compound of higher or close ERK phosphorylation degree carries out next step pEC50Mensuration;
(6)pEC50Value is greater than the compound of positive control drug GW9508 as GPR120 activator.
2. the rapid screening method of GPR120 activator as claimed in claim 1, is characterized in that, described step 1) in selectGo out pEC50 value and be greater than 27 compounds of 4 as candidate compound.
3. the rapid screening method of GPR120 activator as claimed in claim 1, is characterized in that, described step 2) in altogetherTo 10 Pharmacophore Model.
4. the rapid screening method of GPR120 activator as claimed in claim 1, is characterized in that, described step 3) obtain beating500 compounds that score value is higher.
5. the rapid screening method of GPR120 activator as claimed in claim 1, is characterized in that, described step 3) manually pressOrder is chosen 50 compounds with carboxyl.
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| CN108701171B (en) * | 2015-10-22 | 2022-06-10 | 马古苏托科技大学 | Pharmacophores, compounds and methods having application in the treatment of cancer by inhibition of CYP17a1 and CYP19a1 |
| CN106701061B (en) * | 2016-11-15 | 2019-01-29 | 山东大学 | A kind of GPR120 small-molecule fluorescent probe and its application |
| US11230526B1 (en) | 2018-03-06 | 2022-01-25 | Janssen Pharmaceutica Nv | Cylcoalkenyl derivatives useful as agonists of the GPR120 and/or GPR40 receptors |
| WO2019171278A1 (en) * | 2018-03-06 | 2019-09-12 | Janssen Pharmaceutica Nv | Heterocylcoalkenyl derivatives useful as agonists of the gpr120 and / or gpr40 |
| CN111321194A (en) * | 2018-12-14 | 2020-06-23 | 中国科学院大连化学物理研究所 | Label-free screening model for FFA4 ligand |
| CN112786122B (en) * | 2021-01-21 | 2023-12-29 | 北京晶泰科技有限公司 | Molecular screening method and computing equipment |
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| US20140206714A1 (en) * | 2012-08-27 | 2014-07-24 | University Of Tennessee Research Foundation | Lpa2 receptor-specific benzoic acid derivatives |
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| WO2003038442A2 (en) * | 2001-10-29 | 2003-05-08 | Vertex Pharmaceuticals Incorporated | Processes for producing optimized pharmacophores |
| WO2003063784A2 (en) * | 2002-01-28 | 2003-08-07 | Bristol-Myers Squibb Company | MOLECULES THAT MODULATE GαQ ACTIVITY AND METHODS OF TREATING URINARY INCONTINENCE |
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