CN104293877A - Rapid screening method for GPR120 agonist - Google Patents
Rapid screening method for GPR120 agonist Download PDFInfo
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- CN104293877A CN104293877A CN201410508486.XA CN201410508486A CN104293877A CN 104293877 A CN104293877 A CN 104293877A CN 201410508486 A CN201410508486 A CN 201410508486A CN 104293877 A CN104293877 A CN 104293877A
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Abstract
The invention discloses a rapid screening method for a GPR120 agonist. The method comprises the following steps: assisting drug design by using a computer, carrying out 3D-QSAR pharmacophore model configuration on a compound which is subjected to conformation searching by using hypogen of Discovery Studio 2.5, and simultaneously validating by using Fischer 90% to obtain linear regression of actual activity and calculation activity; drawing a conclusion that carboxyl is a necessary group of the GPR120 agonist according to the structure-function relationship of the GPR120 agonist and the structural relationship of endogenous ligand fatty acids; manually screening out a candidate compound containing carboxyl; and screening and validating at high flux of a cell platform, so as to obtain candidate molecules of the GPR120 agonist. Through virtual screening of drugs, a clue of an active compound is obtained within a short period of time; research targets are concentrated into dozens of compounds from millions of compounds; and a lead compound is screened out from the screened compounds by virtue of the cell platform, so that the rate and efficiency of screening the compounds are increased, and the novel drug research period is shortened.
Description
Technical field
The present invention relates to drug screening method, be specifically related to a kind of rapid screening method of GPR120 agonist.
Background technology
GPR120 is the g protein coupled receptor be found recently, belongs to the Visual purple sample receptor family in GPCRs superfamily.GPR120 all has expression in Various Tissues, has higher expression level in colon.And GPR120 all has expression (Hirasawa et al.2005) in the cell such as taste buds cell, enteroendocrine L cell, adipocyte, scavenger cell of different genera animal, GPR120 may with fat, type II diabetes is relevant.
Diabetes have become the difficult problem affecting global people's health as the metabolic disease taking hyperglycemia as feature.GLP-1 is a kind of hormone of intestinal secretion, and it can promote that blood sugar relies on the secretion of Regular Insulin, and lipid acid provides important condition as energy derive for secreting intestines polypeptide simultaneously.Have been reported and claim that in small intestine, express very high GPR120 be long-chain unsaturated fatty acid acceptor, and have the results show long-chain unsaturated fatty acid to stimulate GPR120 can increase the secretion of GLP-1, promote the circulation (Hirasawa et al.2005) of Regular Insulin.Therefore GPR120 is a very promising acceptor for treatment diabetes and other drinking and eating irregularly diseases (such as exessive appetite).
GPR120 is otherwise known as ω-3 (such as DHA and EPA) Fatty Acid Receptors (Oh et al.2010).Having been reported omega-fatty acid and small molecule agonist stimulates GPR120 can produce anti-inflammatory action widely, and the principle of anti-inflammatory action is shown in Fig. 1, and the anti-inflammatory action of organizing that scavenger cell regulates can improve insulin resistance because diabetes produce.This experimental result describes GPR120 and anti-inflammatory and insulin sensitizing agent is reacted the tie connected.Therefore for treatment insulin resistance, GPR120 is very promising as a novel targets.
GPR120 intracellular signaling also serves vital role (Wu et al.2013) in Human colon cancer.In the tissue and cell strain of colorectal carcinoma, the expression of GPR120 significantly raises.The PI3K/Akt – NF-kB signal path activating GPR120 can promote secretion and the expression of angiogenic factors in colon cancer cell, accelerates the vasculogenesis in colon cancer cell, for propagation of cancer cells provides nutritional support.And exciting GPR120 promotes tumor cell migration and the conduction of tumour cell interstitial.Therefore, GPR120 can as the brand-new target spot for the treatment of colorectal carcinoma.
Until 2005, the people such as Hirasawa have found the endogenic ligand (Hirasawa et al.2005) of GPR120, and GPR120 is considered to orphan receptor always.They are from 1000 multiple compounds, in the HEK293 cell of stably express GPR120-EGFP, detect the fluorescent mark acceptor quantity of endocytosis by quantitative flow cell technology, the part filtering out GPR120 is the saturated fatty acid of C14-C18 chain length and the unsaturated fatty acids of C16-C22 chain length.After this, non-endogenic small molecule agonist is also found, GW9508, NCG21, TUG-891 (Sun et al.2010, Hudson et al.2013, Shimpukade et al.2012) the activation effect of GPR120 and endogenous longer chain fatty acid similar, can Ca in ERK, cell in active cells effectively
2+the secretion of release reaction and GLP-1.
Summary of the invention
The object of the invention is to the rapid screening method setting up a kind of GPR120 agonist, the method, from known GPR120 agonist, sets up the screening method of a set of high efficient and reliable, finds novel small molecule agonist by contributing to, effective speed improving screening, reduces screening cost.
First the present invention uses Computer-Aided Drug Design technology, with reference to previous work and pertinent literature, to the compound carrying out stable conformation, the hypogen of Discovery Studio 2.5 is used to carry out the Pharmacophore Model configuration of 3D-QSAR, use Fischer 90% to verify simultaneously, obtain the linear regression of substantial activity and calculated activity.Draw according to the structure activity relationship of GPR120 agonist reported and the structural relation of endogenic ligand lipid acid: carboxyl is the necessary group of GPR120 agonist, therefore the candidate compound containing carboxyl selected by rider.Verify further with the high flux screening of cell platform again, thus obtain the candidate molecules of GPR120 agonist.
To achieve these goals, the present invention adopts following technical scheme:
A rapid screening method for GPR120 agonist, comprises the following steps:
(1) 20-50 the GPR120 agonist alternatively compound that pEC50 value is greater than 4 is picked out;
(2) compound in step (1) uses the hypogen module construction of Discovery Studio 2.5 Pharmacophore Model: use Caesar method to carry out stable conformation (employing default setting); Then producing Pharmacophore Model to different conformation carries out superimposed, obtains 5-15 Pharmacophore Model altogether; Obtain the substantial activity of candidate compound and the linear regression of calculated activity at report interface, we determine regression coefficient R
2be worth the highest model as optimum Pharmacophore Model;
(3) Pharmacophore Model in step (2) is adopted to carry out virtual screening to SPECS database (it is a medium scale compound library that Dutch SPECS company is found in 1987), obtain marking value front 500-1000 compound from high to low, and be arranged as list from high to low by marking value;
(4) according to screening marking value and druggability five times rule principle, manually in order selecting step (3) with 50-100 compound of carboxyl;
(5) carry out active primary dcreening operation to the compound that step (4) is screened, concentration is after 10 μMs of stimulation STC-1 cells, filters out and causes the compound of higher or close ERK phosphorylation degree to carry out next step EC than positive control drug GW9508
50mensuration;
(6) pEC50 value is greater than the compound of positive control drug GW9508 as GPR120 agonist.
The invention has the beneficial effects as follows:
The present invention, by the virtual screening of medicine, can obtain the clue of active compound at short notice, and goal in research is focused on tens compounds from millions of compounds.And then from the compound after virtual screening, utilize cell Platform Screening to go out lead compound, therefore substantially increase speed and the efficiency of SCREENED COMPOUND, shorten the cycle of new drug research.
Accompanying drawing explanation
Fig. 1 omega-fatty acid and GPR120 effect produce the principle of anti-inflammatory action;
The GPR120 Pharmacophore Model schematic diagram that Fig. 2 builds, wherein 1 for hydrogen bond receptor, 2 are aromatic core, 3 is hydrophobic centers;
Fig. 3 is used as the structural formula of compound schematic diagram building pharmacophore;
Compound (table 1) substantial activity that Fig. 4 has selected and the linear regression of calculated activity.
Embodiment
Below in conjunction with accompanying drawing and embodiment, the invention will be further described.
A rapid screening method for GPR120 agonist, step is as follows:
(1) with reference to previous work and pertinent literature, 27 (Shimpukade et al. that pEC50 value is greater than 4 are selected from the GPR120 agonist reported, 2012) compound is as Pharmacophore Model configuration candidate compound (table 1) of 3D-QSAR, is used as the structural formula of compound schematic diagram of structure pharmacophore as Fig. 3.
(2) the Caesar method (J.Chem.Inf.Model.47 of Discovery Studio 2.5 is used, 1923-32 (2007)) according to default setting, stable conformation is carried out to 27 candidate compounds, using hypogen to produce Pharmacophore Model to different conformation afterwards carries out superimposed, obtain 10 Pharmacophore Model altogether, and obtain the substantial activity of candidate compound and the linear regression of calculated activity at report interface, determine regression coefficient R
2be worth the highest model as optimum Pharmacophore Model (Fig. 2), R
2be 0.888 (Fig. 4).
(3) the middle Pharmacophore Model of step (2) is adopted to carry out the virtual screening (Bioorg.Med.Chem.Lett.2010 based on Pharmacophore Model to SPECS database (it 1987 is a medium scale compound library that Dutch SPECS company is found in), 20,5130-2), obtain marking value 500 compounds from high to low, and be arranged as list from high to low by marking value;
(4) according to screening marking value and druggability five times rule principle, 50 compounds (table 2) with carboxyl are manually chosen in order;
(5) carry out active primary dcreening operation to the compound that step (4) is screened, concentration is after 10 μMs of stimulation STC-1 cells, filters out and causes the compound of higher or close ERK phosphorylation degree to carry out next step EC than positive control drug GW9508
50mensuration, active primary dcreening operation the results are shown in Table 2;
(6) adopt Bioluminescence Resonance Energy transfer (BRET) to sifting out 13 compound determination EC
50, pEC
50value is greater than the compound of positive control drug GW9508 as GPR120 agonist, and Activity Results is in table 3.
Biological activity determination:
First the STC-1 cell of high expression level GPR120 is adopted, concentration 10uM is selected to be primary dcreening operation concentration, take GW9508 as positive control drug, using GAPDH or actin as interior mark, use Western blotting (Hirasawa et al., 2005), in chart 2 after 50 compound irritation cell 10min in born of the same parents ERK phosphorylation degree change (table 2).There are 13 compounds (table 3) the STC-1 cell strain ERK phosphorylation of relatively high (ERK phosphorylation degree is higher than GW9508 or close with it) can be caused, the EC of compound in further chart 3
50.
The compd E C in table 3 is determined in employing Bioluminescence Resonance Energy transfer (BRET) further
50method is: method (the Shimpukade et al. adopting transient transfection, 2012), by GPR120-YFP and RLuc-β-arrestin2 two kinds of plasmids transfection simultaneously (Shimpukade et al., 2012) enter in HEKC (HEK293), transfection 24h-48h, add concentration ocean renilla luciferase (final concentration is 3 μMs) and hatch 5-15min, microplate reader (POLARstar Omega) fluorescence intensity ratio (460nm/520nm) change detection compd E C is used after stimulating 5-10min with the compound in different concns (1nM-10uM) table 3
50.
Luciferase reporter gene detection kit (Promega, E1910) detects intracellular free calcium level further deterministic compound EC
50method be: adopt method (the Shimpukade et al. of transient transfection, 2012), this three kinds of plasmids transfection simultaneously of NFAT-FLuc, TK-RLuc and GPR120-Gq is entered in HEK293 cell, after transfection 24h-48h, stimulate with the compound in table 3, utilize Dual-Luciferase detection kit detect the change of (concrete grammar according to test kit specification sheets operation) intracellular calcium concentration thus obtain the EC of compound
50(table 3).
The compound that table 1 builds for pharmacophore
Table 2 compound is to GPR120 agonist activity primary dcreening operation result
The EC of the active better compound of table 3
50
Experimental result shows: have according to the aromatic acids compound that area of computer aided drug sieve is selected the activity activating GPR120 preferably, shows that the compound in the present invention has higher using value in disease such as treatment diabetes, obesity etc.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; 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 do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (5)
1. a rapid screening method for GPR120 agonist, is characterized in that, comprises the following steps:
(1) 20-50 the GPR120 agonist alternatively compound that pEC50 value is greater than 4 is picked out;
(2) compound in step (1) uses the hypogen module construction Pharmacophore Model of Discovery Studio2.5, Caesar method is used to carry out stable conformation, then producing Pharmacophore Model to different conformation carries out superimposed, obtain 5-15 Pharmacophore Model, obtain the substantial activity of candidate compound and the linear regression of calculated activity at report interface, determine regression coefficient R
2be 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 front 500-1000 the compound that marking value arranges from high to low, and be arranged as list from high to low by marking value;
(4) according to screening marking value and druggability five times rule principle, 50-100 the compound with carboxyl is manually chosen in order;
(5) carry out active primary dcreening operation to the compound that step (4) is screened, concentration is after 10 μMs of stimulation STC-1 cells, filters out and causes the compound of higher or close ERK phosphorylation degree to carry out next step pEC than positive control drug GW9508
50mensuration;
(6) pEC
50value is greater than the compound of positive control drug GW9508 as GPR120 agonist.
2. the rapid screening method of GPR120 agonist as claimed in claim 1, is characterized in that, described step 1) in pick out 27 compounds alternatively compound that pEC50 value is greater than 4.
3. the rapid screening method of GPR120 agonist as claimed in claim 1, is characterized in that, described step 2) in obtain 10 Pharmacophore Model altogether.
4. the rapid screening method of GPR120 agonist as claimed in claim 1, is characterized in that, described step 3) obtain 500 higher compounds of marking value.
5. the rapid screening method of GPR120 agonist as claimed in claim 1, is characterized in that, described step 3) manual 50 compounds chosen in order with carboxyl.
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| CN108701171A (en) * | 2015-10-22 | 2018-10-23 | 马古苏托科技大学 | In pharmacophore, the Compounds and methods for by inhibiting that there is application in CYP17A1 and CYP19A1 treating cancers |
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| 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 |
| CN112786122A (en) * | 2021-01-21 | 2021-05-11 | 北京晶派科技有限公司 | Molecular screening method and computing equipment |
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| CN108701171A (en) * | 2015-10-22 | 2018-10-23 | 马古苏托科技大学 | In pharmacophore, the Compounds and methods for by inhibiting that there is application in CYP17A1 and CYP19A1 treating cancers |
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| CN106701061B (en) * | 2016-11-15 | 2019-01-29 | 山东大学 | A kind of GPR120 small-molecule fluorescent probe and its application |
| WO2019171277A1 (en) * | 2018-03-06 | 2019-09-12 | 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 |
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| 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 |
| CN112786122A (en) * | 2021-01-21 | 2021-05-11 | 北京晶派科技有限公司 | Molecular screening method and computing equipment |
| 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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