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Method for detecting compounds modulating dimers of VFT domain membrane proteins

一种二聚体、化合物的技术，应用在调节膜受体的化合物领域，能够解决没有观察到FRET信号等问题

Active Publication Date: 2012-08-15
CIS BIO INT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These studies did not observe any changes in the FRET signal when the receptor was activated by its agonist GABA, suggesting that the method is not suitable for studying these dimers

Method used

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  • Method for detecting compounds modulating dimers of VFT domain membrane proteins
  • Method for detecting compounds modulating dimers of VFT domain membrane proteins
  • Method for detecting compounds modulating dimers of VFT domain membrane proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-12

[0201] Reagent:

[0202] Modulators (agonists, antagonists, allosteric modulators):

[0203] DCG IV, L-glutamic acid and LY341495 are available in the Tocris Biosciences product catalog. 4-MPPTS (Positive Allosteric Modulator of the mGluR2 Receptor, also known as LY487379) was a gift from Addex Pharmaceuticals, the synthesis of which is described in US Patent US 6 800 651 . Medium:

[0204] DMEM, DMEM+Glutamax, MEM-NEAA, MEM-penicillin / streptomycin, trypsin / EDTA are available in the Gibco catalog. The fetal bovine serum used was Lonza brand, South American type (batch 7SB0017).

[0205] Fluorescent Conjugates:

[0206] BG-Lumi4-Tb: Benzylguanine-Lumi4-Tb conjugate, sold by Cisbio Bioassays ( SNAP-Lumi4Tb). BG is the substrate of the Snap-tagase.

[0207] BC-Lucifer Yellow: benzylcytosine-Lucifer Yellow conjugate, sold by NEB Company, the product name is CLIP-Vista Green. BC is the substrate of the Clip-tagase.

[0208] BG-fluorescein yellow: benzylguanine-fluorescein...

Embodiment 1

[0235] Example 1: To mGluR 2 -mGluR 2 Display of dimer activated compounds with agonist effects (R of the FRET partner pair used 0 : )

[0236] Program:

[0237] Transfection by electroporation:

[0238] Incubate Cos-7 cells at 37 °C and 5% CO 2 cultured in complete medium. At confluence, wash once with PBS, and detach cells with trypsin-EDTA solution at 37° C. for 10 min. Mix a suspension of 10 trillion cells with 5 μg total DNA (3.5 μg pK5 Plasmid, 1.2 μg prK5 Flag-Cliptag-mGluR2 Plasmid and 0.3 μg pK5 HA-Snaptag-mGluR2 Plasmids) were mixed in electroporation buffer with a final volume of 300 μl, and then a 280V, 900μF electric shock was applied in a 4mm electroporation cuvette (Eurogentec) using a Gene-Pulser II electroporator (BIO-RAD). These cells were then inoculated into 96-well plates (Greiner CellStar 96-well plates) at an amount of 150000 cells / well, and the final volume of complete medium in each well was 100 μl, and then incubated for 24 h.

[0239...

Embodiment 2

[0253] Example 2: Display of deactivating compounds with competitive antagonist effects on mGluR2-mGluR2 dimers (R of the FRET partner pair used 0 : )

[0254] Reagents and plasmids

[0255] The same reagents and plasmids as described in Example 1 were used. The known antagonist compound LY341495 (Tocris Bioscience) was used.

[0256] Program

[0257] The same protocol as described in Example 1 was used. Final concentrations were [glutamate] = 100 μM (in all wells); and [LY341495] = 1000 nM, 316 nM, 100 nM, 31.6 nM, 10 nM, 3.16 nM or 1 nM.

[0258] result

[0259] figure 2 Represents the TRF520 signal as a function of the concentration of the antagonist LY341495 in the presence of a fixed concentration of glutamate (control curve: in the absence of glutamate).

[0260] In the presence of glutamate in the absence of the competitive antagonist LY341495, the receptor was activated and a 50% change in TRF520 was observed. The presence of the antagonist prevented this ef...

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PUM

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Abstract

The invention relates to a method for selecting compounds having a modulating effect on the activation state of a dimer of VFT domain proteins expressed in cellular membranes in a measuring medium, said dimer including a first protein and a second protein which are identical or different, wherein the method includes the following steps: (a) marking the first and second proteins in the N-terminal portion of the VFT domain by members of a FRET partner pair, the Forster radius (R0) of said pair ranging between 20 and 55 Angstrom; (b) measuring the FRET signal in the absence and in the presence of the compound to be tested within a predetermined time window; (c) selecting the compound to be tested as a modulating compound if a difference in the FRET signal in the absence and in the presence of the compound to be tested is measured during step (b). The invention can be used in the research for new drugs and new taste modulators.

Description

technical field [0001] The present invention relates to compounds that modulate membrane receptors that are useful in the search for new drugs and new taste modulators, as well as in agriculture. Background technique [0002] G-protein coupled receptors (GPCRs) represent 3.4% of the genome and thus constitute the largest family of mammalian membrane receptors. The isolation and subsequent cloning of GPCRs made it possible to identify about 900 genes in humans, of which about 500 correspond to olfactory and taste receptors and about 400 correspond to genes that bind endogenous ligands. receptor. The heterogeneity of these receptors thus provides a very broad recognition of external signals (odor, light, taste molecules) and internal signals (hormones and neurotransmitters). There are three main classes of the GPCRs: [0003] Class A, based on rhodopsin, is the most broadly represented class. The ligand-binding sites of these receptors mainly involve the transmembrane doma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/542
CPCG01N33/542G01N33/9426G01N2333/726G01N2500/04
Inventor 艾蒂安·杜玛扎纳于里安·兹维尔埃里克·特兰凯特让-菲利普·宾
Owner CIS BIO INT
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