Method for measuring modulation of G protein-coupled receptor activation using GTP analogs

文档序号：1894915 发布日期：2021-11-26 浏览：12次中文

阅读说明：本技术 用gtp类似物测量对g蛋白偶联受体激活的调控的方法 (Method for measuring modulation of G protein-coupled receptor activation using GTP analogs ) 是由托马斯·鲁克斯埃里克·特里奎特埃洛迪·杜普伊斯萨拉·比迪奥伊珍-菲利普·珀菲利普于 2020-01-30 设计创作，主要内容包括：本发明涉及用于确定分子调控G蛋白偶联受体(GPCR)激活的能力的方法,所述方法包括以下步骤：a)在第一容器中引入以下物质：-携带一种或多种GPCR和一种或多种αG蛋白的膜制剂,-标记有RET伴侣对的第一成员的不可水解或可缓慢水解的GTP源,-标记有RET伴侣对的第二成员的G蛋白的α亚基(αG蛋白)的配体,所述配体能够结合至全αG蛋白,所述全αG蛋白结合至标记有RET伴侣对的第一成员的不可水解或可缓慢水解的GTP,-任选的GPCR激动剂；b)测量第一容器中发出的RET信号；c)(i)在第二容器中引入与步骤a)中相同的试剂和待测分子,或(ii)在第一容器中引入待测分子；d)测量在步骤c)中获得的第一容器中或第二容器中发出的RET信号；e)比较在步骤b)和步骤d)中获得的信号,在步骤d)中获得的信号相对于在步骤b)中获得的信号存在调控表明待测分子能够调控GPCR激活。(The present invention relates to a method for determining the ability of a molecule to modulate the activation of a G protein-coupled receptor (GPCR), said method comprising the steps of: a) introducing into the first vessel: -a membrane preparation carrying one or more GPCRs and one or more α G proteins, -a non-hydrolysable or slowly hydrolysable source of GTP labelled with a first member of a RET chaperone pair, -a ligand labelled with an α subunit of a G protein (α G protein) of a second member of a RET chaperone pair, the ligand being capable of binding to an all α G protein bound to non-hydrolysable or slowly hydrolysable GTP labelled with a first member of a RET chaperone pair, -optionally a GPCR agonist; b) measuring an RET signal emitted in the first container; c) introducing (i) the same reagent and test molecule as in step a) into a second container, or (ii) a test molecule into a first container; d) measuring the RET signal emitted in the first container or in the second container obtained in step c); e) comparing the signals obtained in step b) and step d), the presence of a modulation in the signal obtained in step d) relative to the signal obtained in step b) indicating that the test molecule is capable of modulating GPCR activation.)

1. A method for determining the ability of a molecule to modulate G protein-coupled receptor (GPCR) activation, the method comprising the steps of:

a) Introducing into the first vessel:

-a membrane preparation carrying one or more GPCRs and one or more alpha G proteins,

-a non-hydrolysable or slowly hydrolysable GTP source labelled with a first member of a RET partner pair,

-a ligand of the alpha subunit of the G protein (alpha G protein) labelled with the second member of the RET partner pair, said ligand being capable of binding to the all-alpha G protein bound to the non-hydrolysable or slowly hydrolysable GTP of the first member of the labelled RET partner pair,

-optionally a GPCR agonist;

b) measuring the RET signal emitted in the first container;

c) (ii) introducing the same reagent and test molecule as in step a) into a second container, or (ii) introducing a test molecule into the first container;

d) measuring the RET signal emitted in the first container or in the second container obtained in step c);

e) comparing the signals obtained in step b) and step d), the presence of a modulation in the signal obtained in step d) relative to the signal obtained in step b) indicating that the test molecule is capable of modulating activation of the GPCR.

2. The method of claim 1, wherein the first vessel does not contain a GPCR agonist and in step e) the presence of a decrease in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is a GPCR agonist.

3. The method of claim 1, wherein the first vessel comprises a GPCR agonist and in step e):

-the presence of an increase in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is an antagonist or a negative allosteric modulator of the GPCR;

-a decrease in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is an agonist or positive allosteric modulator of the GPCR.

4. The method of claim 1, wherein the first vessel does not contain a GPCR agonist and in step e) the presence of an increase in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is a GPCR agonist.

5. The method of claim 1, wherein the first vessel comprises a GPCR agonist and in step e):

-a decrease in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is an antagonist or a negative allosteric modulator of the GPCR;

-the presence of an increase in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is an agonist or positive allosteric modulator of the GPCR.

6. The method according to any one of claims 1 to 5, wherein the α G protein is selected from the group consisting of proteins G- α i1, G- α i2, G- α i3, G- α o1, G- α o2, G- α q, G- α 12, G- α 13, G- α s, G- α z, G- α t1, G- α t2, G- α 11, G- α 14, G- α 15, G- α 16 and G- α gus, preferably from the group consisting of proteins G- α i1, G- α i2 and G- α i 3.

7. The method according to any one of the preceding claims, wherein the ligand is selected from an antibody, an antibody fragment, a peptide or an aptamer, preferably an antibody or an antibody fragment.

8. The method according to any of the preceding claims, wherein the non-hydrolysable or slowly hydrolysable GTP is selected from GTPgammaS (GTP γ S or GTPgS), GppNHp and GppCp.

9. The method of any of the preceding claims, wherein one member of the RET partner pair is a fluorescence donor compound or a luminescence donor compound and the other member of the RET partner pair is a fluorescence acceptor compound or a non-fluorescence acceptor compound (quencher).

10. The method of any one of claims 1-9, wherein the non-hydrolyzable or slowly hydrolyzable GTP is labeled with a fluorescent donor compound and the ligand of the α G protein is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher).

11. The method of any one of claims 1-9, wherein the non-hydrolyzable or slowly hydrolyzable GTP is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher), and the ligand of the α G protein is labeled with a fluorescent donor compound or a luminescent donor compound.

12. The method of any one of claims 9-11, wherein the fluorescence donor compound is a FRET partner selected from the group consisting of: europium cryptates, europium chelates, terbium cryptates, ruthenium chelates, quantum dots, allophycocyanins, rhodamines, anthocyanidins, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, and nitrobenzoxadiazoles.

13. The method of any one of claims 9-12, wherein the fluorescence acceptor compound is a FRET partner selected from the group consisting of: allophycocyanin, rhodamine, anthocyanin, squaraine, coumarin, proflavine, acridine, fluorescein, boron-dipyrromethene derivative, nitrobenzoxadiazole and quantum dots; GFP, GFP variants selected from GFP10, GFP2 and eGFP; YFP, a YFP variant selected from the group consisting of eYFP, YFP topaz, YFP citrine, YFP venus, and YPet; mOrange; DsRed.

14. The method of any one of claims 9-11, wherein the luminescent donor compound is a BRET partner selected from: luciferase (luc), Renilla luciferase (Rluc), Renilla luciferase variants (Rluc8), and firefly luciferase.

15. The method of any one of claims 9-11 and 14, wherein the fluorescence acceptor compound is a BRET partner selected from the group consisting of: allophycocyanin, rhodamine, anthocyanin, squaraine, coumarin, proflavine, acridine, fluorescein, boron-dipyrromethene derivative, nitrobenzoxadiazole and quantum dots; GFP, GFP variants selected from GFP10, GFP2 and eGFP; YFP, a YFP variant selected from the group consisting of eYFP, YFP topaz, YFP citrine, YFP venus, and YPet; mOrange; DsRed.

16. The method of any one of claims 1-10 and 12-15, wherein the non-hydrolyzable or slowly hydrolyzable GTP labeled with the first member of the RET partner pair is a fluorescent donor compound of general formula (I):

wherein:

x is O, NH or CH₂；

Y is O, NH or CH₂；

L is a bivalent linker;

Ln³⁺being a lanthanide complex, optionally carrying a reactive group G₃。

17. The method of any one of claims 1-9 and 11-15, wherein the non-hydrolyzable or slowly hydrolyzable GTP labeled with the first member of the RET partner pair is selected from GTPgN-octyl-Cy 5, GTPgN-octyl-AF 488, GTPgN-L15-fluorescein, GTPgO-linker-Cy 5(P), or GTPgS-linker-Cy 5 (R).

18. The method of any one of the preceding claims, wherein the non-hydrolyzable or slowly hydrolyzable GTP labeled with the first member of the RET partner pair is selected from GTPgN-C2(GTP- γ -N-C2), GTPgN-C3(GTP- γ -N-C3), GTPgN-octyl-C2 (GTP- γ -N-octyl-C2), GTPgN-octyl-C11 (GTP- γ -N-octyl-C11), GTPgN-octyl-C3 (GTP- γ -N-octyl-C3), GTPgO-hexyl-C2 (GTP- γ -O-hexyl-C2), GTPgO-hexyl-C3 (GTP- γ -O-hexyl-C3) or GTP-gN-octyl-thiosuccinimidyl-C2 (GTP- γ -N-octyl-C2) -thiosuccinimidyl-C2), preferably GTP-gN-octyl-thiosuccinimidyl-C2.

19. The method according to any of the preceding claims, wherein the ligand of the α G protein is an antibody or antibody fragment that specifically binds to the SwitchII domain of the α i G protein, preferably to peptide 215-294 in the α i G protein.

20. The method according to any one of the preceding claims, wherein the α G protein is selected from the group consisting of G- α i1, G- α i2 and G- α i3, and the ligand of the α G protein competes for binding to the α G protein with a peptide of sequence Ser-Arg-Gly-Tyr-His-Gly-Ile-Trp-Val-Gly-Glu-Gly-Arg-Leu-Ser-Arg (SEQ ID NO: 1).

21. The method of any one of the preceding claims, wherein the ligand of the α G protein is an antibody selected from the group consisting of:

(i) an antibody or antibody fragment capable of binding to the α G protein, the antibody or antibody fragment comprising:

-a heavy chain variable domain comprising the CDR1 of amino acid sequence SEQ ID NO 2, the CDR2 of amino acid sequence SEQ ID NO 3 and the CDR3 of amino acid sequence SEQ ID NO 4, and

-a light chain variable domain comprising the CDR1 of amino acid sequence SEQ ID No. 5, the CDR2 of amino acid sequence DTS and the CDR3 of the light chain variable domain consisting of amino acid sequence SEQ ID No. 6; or

(ii) (ii) an antibody or antibody fragment that competes with the antibody or antibody fragment according to (i) for binding to the α G protein.

Technical Field

The present invention relates to novel methods for measuring modulation of G protein-coupled receptor (RCPG or GPCR in english) activation, for example methods for determining the ability of a molecule to modulate GPCR activation. The method according to the invention particularly enables the detection of the presence or absence of a full G protein (full G protein) bound to a GTP analogue in a GPCR preparation.

Background

G protein-coupled receptors (RCPG or GPCRs in english) are a family of membrane receptors in the mammalian as well as throughout the animal kingdom. The G protein is a heterotrimeric protein activated by the GPCR (3 subunits: α, β, and γ). Through GPCRs, G proteins play a role in transducing signals from outside the cell to inside the cell (i.e., cellular responses to external stimuli). Their mechanisms of action, described generally, are presented in fig. 1 and summarized below:

in its inactive state (resting state), the alpha subunit of the G protein binds to the nucleotide GDP (the full G protein bound to GDP);

after GPCR activation, the latter binds to the α subunit of the G protein and triggers the activation process of the G protein, which consists of two steps: 1) (ii) GDP is removed from the G protein to produce empty G protein and form an inactive GPCR/empty G protein complex; and 2) immobilization of GTP, which leads to the formation of active G-proteins in the form of GTP (all G-proteins bound to GTP). In the first step, the G protein bound to the receptor is in a form called the "empty form". This state is described in the literature as transient, since it is described that the nucleotide GTP binds rapidly to the α subunit of the G protein. In addition, the β/γ subunit of the activated G protein dissociates from the α subunit;

The alpha subunit of the whole G protein bound to GTP then binds to the effector, activating them. The effector in turn activates a signaling pathway, causing a cellular response;

GTP is then hydrolyzed by the α subunit of the G protein to GDP, and the α subunit re-associates with the β/γ subunit, reforming the full G protein bound to GDP (inactive state).

There are several subtypes of α G proteins that have different selectivity profiles for different effectors and thus induce activation of preferential signaling pathways.

GPCRs are associated with many important physiological functions and are considered to be one of the preferred therapeutic targets for a large number of pathologies. Thus, a number of in vitro screening assays have been developed to identify molecules capable of modulating GPCRs. Assays have been developed that take advantage of various mechanisms of G protein activation and employ various techniques (Zhang et al; Tools for GPCR Drug Discovery; Acta Pharmacological Sinica,2012,33, 372).

Mention may in particular be made of affinity assays using radiolabeled ligands to measure the affinity of the ligand for the GPCR, proximity scintillation assays using scintillant beads on which the GPCR is immobilized (scintillation proximity assays), or functional assays using weakly or non-hydrolyzable GTP, such as GTP γ S. However, these assays are difficult to implement and sometimes require a membrane filtration step, which may limit their use as assays for High Throughput Screening (HTS).

Other assays for detecting GPCR activation have been developed. These assays are based in particular on energy transfer techniques (resonance energy transfer, RET), such as FRET (fluorescence resonance energy transfer) (Clinical Chemistry,1995,41,1391) or BRET (bioluminescence resonance energy transfer) (Proceedings of the National Academy of Sciences,1999,96(1), 151). Both of these techniques adopt the concept of molecules capable of supplying energy (called donors) or molecules capable of accepting energy (called acceptors) (Physical Chemistry Chemical Physics,2007,9, 5847). Energy transfer techniques for detecting the interaction between a GPCR and a G protein using, for example, a donor fused to a GPCR and an acceptor fused to a G protein (WO 2006/086883 and WO 2003/008435) or an acceptor fused to an α subunit of a G protein and a donor fused to a β and/or γ subunit of a G protein (Bunemann et al, proc.natl.acad.sci.,2003,26,16077-16082) may be mentioned. However, these techniques are limiting because they require the preparation of fusion proteins and they do not allow for the study of GPCRs and G proteins expressed endogenously in the cell (i.e., unmodified and not overexpressed). Furthermore, these techniques require the preparation of multiple membrane samples (specific preparations for each α G protein subtype) in order to distinguish between different subtypes of α G protein that can be activated by the receptor.

Energy transfer techniques have also been used to develop assays aimed at visualizing the regulation of the (active) GTP form of the G protein or the (inactive) GDP form of the G protein. Mention may be made first of the use, for example, of the following format (format): the G protein is fused to a biotin label to bind to a donor, which is itself coupled to streptavidin; the acceptors are bound to non-hydrolyzable or slowly hydrolyzable GTP analogs (WO 2006/035208). In addition, another format uses biotinylationA peptide (KaroBio) that distinguishes the GTP form from the GDP form and is bound to the donor by streptavidin coupled to the donor. Using anti-6 HIS antibody, receivingThe body binds to a GPCR fused to a 6HIS tag (WO 2004/035614). These techniques are also limiting, as they also require the preparation of fusion proteins and they do not allow for the study of GPCRs and G proteins expressed endogenously by the cell. Similarly, these techniques require the preparation of multiple membrane samples in order to distinguish between different subtypes of α G protein that can be activated by receptors.

Thus, there is a real need for a sensitive and reliable method that allows for easy determination of the modulation of GPCR activation, e.g. for easy determination of the ability of a molecule to modulate GPCR activation and/or for determining which subset of G proteins are activated by GPCRs.

Disclosure of Invention

The present invention aims to propose a novel method for screening molecules capable of modulating GPCRs in vitro. This new approach is based in particular on the ability to distinguish between: (i) a full form α G protein and an empty form G protein bound to non-hydrolysable or slowly hydrolysable GTP labeled with a member of the RET partner pair; or (ii) a full form α G protein bound to non-hydrolysable or slowly hydrolysable GTP labeled with a member of the RET partner pair and a full form α G protein bound to GDP.

In particular, the advantages of the invention are: 1) it uses a fluorescence-based detection method and therefore does not have radioactivity; 2) it does not require a washing step, thus simplifying its application, in particular for the activity of high-throughput screening of compounds; 3) it enables action in particular on unmodified G proteins and GPCRs; 4) it allows the differentiation of different isoforms of the α G protein activated by GPCRs in the same membrane preparation containing these different isoforms (the differentiation being provided by the use of a detector ligand that distinguishes the α G protein isoforms).

According to a first aspect, the present invention relates to a method for determining the ability of a molecule to modulate the activation of a G protein-coupled receptor (GPCR), said method comprising the steps of:

a) Introducing into the first vessel:

-a membrane preparation carrying one or more GPCRs and one or more alpha G proteins,

-a non-hydrolysable or slowly hydrolysable GTP source labelled with a first member of a RET partner pair,

-a ligand of the alpha subunit of the G protein (alpha G protein) labelled with the second member of the RET partner pair, said ligand being capable of binding to the all-alpha G protein bound to the non-hydrolysable or slowly hydrolysable GTP labelled with the first member of the RET partner pair,

-optionally a GPCR agonist;

b) measuring an RET signal emitted in the first container;

c) introducing (i) the same reagent and test molecule as in step a) into a second container, or (ii) a test molecule into a first container;

d) measuring the RET signal emitted in the first container or in the second container obtained in step c);

Drawings

FIG. 1 shows the mechanism of activation of G-proteins and GPCRs.

Fig. 2A to 2D illustrate 4 assay formats according to the present invention.

Figures 3A and 3B illustrate the activation test for delta opioid GPCRs using the assay GTPgN-octyl-C2 + DSV36S-d2 according to format 1A.

Figures 4A and 4B illustrate the activation test for delta opioid GPCRs using the assay GTPgN-octyl-C11 + DSV36S-d2 according to format 1A.

Figures 5A and 5B illustrate the activation test for the delta opioid GPCR using the assay GTPgO-hexyl-C2 + DSV36S-d2 according to format 1A.

Figures 6A and 6B illustrate the activation test of the delta opioid GPCR using the assay GTPgN-C2+ DSV36S-d2 according to format 1A.

Figures 7A and 7B illustrate the activation test of the delta opioid GPCR using the assay GTPgN-C3+ DSV36S-d2 according to format 1A.

FIG. 8 illustrates the binding assay for delta opioid GPCRs using the assay on GTPgN-octyl-C3 + DSV36S-d 2.

Figure 9 illustrates the binding assay for delta opioid GPCRs using the assay according to format 1A with GTPgO-hexyl-C3 + DSV36S-d 2.

Figures 10A and 10B illustrate the effect of membrane and GTP donor concentration on the activation assay performed on GTPgN-octyl-C2 + DSV36S-d2 on the delta opiate GPCR using the assay according to format 1A.

FIGS. 11A and 11B illustrate activation testing of GPCR dopamine D2 using the assay GTPgN-octyl-C2 + DSV36S-D2 according to Format 1A.

FIGS. 12A and 12B illustrate activation testing of GPCR dopamine D2 using the assay GTPgN-octyl-C11 + DSV36S-D2 according to Format 1A.

Fig. 13A-13B fig. 13A and 13B illustrate activation tests performed on the delta opioid GPCR using the assay according to format 1B for GTPgO-linker-Cy 5(P) + DSV36S-Lumi4 Tb.

Fig. 14A and 14B illustrate activation tests performed on the delta opioid GPCR using the assay according to format 1B on GTPgS-linker-Cy 5(R) + DSV36S-Lumi4 Tb.

Fig. 15A and 15B illustrate activation testing of the delta opioid GPCR using the assay GTPgN-L18-fluorescein + DSV36S-Lumi4Tb according to format 1B.

Figures 16A and 16B illustrate the activation test for the delta opioid GPCR using the assay GTPgN-octyl-C2 + DSV36S-d2 according to format 2A.

Figures 17A and 17B illustrate the activation test for the delta opioid GPCR using the assay GTPgN-octyl-C2 + DSV36S-d2 according to format 2A.

Figures 18A and 18B illustrate the activation test for the delta opioid GPCR using the assay GTPgN-octyl-C2 + DSV38S-d2 according to format 2A.

Figures 19A and 19B illustrate the activation test for delta opioid GPCRs using the assay GTPgN-octyl-C11 + DSV36S-d2 according to format 2A.

Figures 20A and 20B illustrate activation testing of the delta opioid GPCR using the assay GTPgO-hexyl-C2 + DSV36S-d2 according to format 2A.

Figures 21A and 21B illustrate the activation assay for delta opioid GPCRs using the assay GTPgN-C2+ DSV36S-d2 according to format 2A.

FIGS. 22A and 22B illustrate activation testing of the GPCR dopamine D2S using the assay GTPgN-octyl-C2 + DSV36S-D2 according to Format 2A.

FIGS. 23A and 23B illustrate activation testing of GPCR dopamine D2S using the assay GTPgN-octyl-C2 + DSV36S-D2 according to Format 2A.

FIGS. 24A and 24B illustrate activation testing of the GPCR dopamine D2S using the assay GTPgN-octyl-C2 + DSV36S-D2 according to Format 2A.

Fig. 25A and 25B illustrate activation testing of the delta opioid GPCR using the assay GTPgN-octyl-Cy 5+ DSV36S-Lumi4Tb according to format 2B.

Fig. 26A and 26B illustrate the activation test of the delta opioid GPCR using the assay GTPgN-octyl-AF 488+ DSV36S-Lumi4Tb according to format 2B.

Figures 27A and 27B illustrate activation assays for delta opioid GPCRs using the assay for GTP-gN-octyl-sulfosuccinimidyl-C2 + DSV36S-d2 according to format 2A.

Detailed Description

Definition of

In the sense of the present invention, the term "G protein" denotes a heterotrimeric protein consisting of three subunits, termed α G protein, β G protein and γ G protein.

In the sense of the present invention, the term "α G protein" or "G- α" denotes the α subunit of a G protein. The α G protein has two domains, a GTPase domain and an α helical domain. There are at least 20 different α G proteins, which can be classified into the following major protein families: g- α s (known to activate adenylate cyclase to increase cAMP synthesis), G- α i (known to inhibit adenylate cyclase), G- α olf (associated with olfactory receptors), G- α t (known to transduce visual signals in the retina in conjunction with rhodopsin), G- α q (known to stimulate phospholipase C), or the G- α 12/13 family (known to regulate the cytoskeleton, cellular junctions, and other processes associated with cellular motility). In a preferred embodiment of the invention, the α G protein is selected from the group consisting of proteins G- α i1, G- α i2, G- α i3, G- α o1, G- α o2, G- α q, G- α 12, G- α 13, G- α s, G- α z, G- α t1, G- α t2, G- α 11, G- α 14, G- α 15, G- α 16 and G- α gus, preferably from the group consisting of proteins G- α i1, G- α i2 and G- α i 3.

In the sense of the present invention, the term "all α G protein" denotes an α G protein which is bound to GTP or to non-or slowly hydrolysable GTP (labelled or unlabelled according to the invention) or to GDP. It is then referred to as "whole α G protein bound to GTP", "whole α G protein bound to non-hydrolysable or slowly hydrolysable GTP" or "whole α G protein bound to GDP". The whole α G protein (bound to GDP or to GTP) is shown in figure 1. In the context of the present invention, non-hydrolysable or slowly hydrolysable GTP labelled with the first member of the RET partner pair is used, which is capable of binding to the α G protein, which enables the obtaining of the whole α G protein bound to the non-hydrolysable or slowly hydrolysable GTP labelled with the first member of the RET partner pair.

The term "GDP" denotes guanosine diphosphate.

The term "GTP" denotes guanosine triphosphate.

The term "non-hydrolyzable or slowly hydrolyzable GTP" denotes GTP analogs that do not hydrolyze or hydrolyze to a very small amount to GDP. Mention may be made, for example, of GTP γ S (CAS No.37589-80-3), GppNHp (CAS No.148892-91-5) or GppCp (CAS No. 10470-57-2).

The term "non-hydrolysable or slowly hydrolysable GTP" or "labeled GTP analog" labeled with a member of a RET partner pair means a non-hydrolysable or slowly hydrolysable donor GTP ("GTP-donor") labeled with a member of a RET partner pair or a non-hydrolysable or slowly hydrolysable acceptor GTP ("GTP-acceptor") labeled with a member of a RET partner pair.

In the sense of the present invention, the term "empty α G protein" denotes an α G protein which is not bound to GTP or GDP or to non-hydrolysable or slowly hydrolysable GTP (modified or unmodified according to the present invention), in particular to non-hydrolysable or slowly hydrolysable GTP which is labeled with a member of the RET partner pair. Empty α G proteins are described in the literature as being in a transitional state between the full form bound to GDP and the full form bound to GTP or non-or slowly-hydrolysable GTP. The empty α G protein is shown in figure 1.

In the sense of the present invention, the term "membrane preparation" denotes a preparation comprising a cell membrane or cell membrane fragment or an artificial system mimicking a cell membrane carrying (or expressing on its surface) one or more GPCRs and one or more α G proteins. Thus, the term "membrane preparation" includes whole cells carrying (or expressing on their surface) one or more GPCRs and one or more α G proteins, permeabilized whole cells, lysed cells, purified cell membranes, and GPCR/α G protein complexes purified and reconstituted in nanodiscs (also known as "nanoscale phospholipid bilayers") or detergent mixtures.

The term "antibody", also known as "immunoglobulin", denotes a heterotetramer consisting of two heavy chains of about 50-70kDa each (called H chains) and two light chains of about 25kDa each (called L chains) joined to each other by intra-and inter-chain disulfide bonds. Each chain is composed of a variable region or domain at the N-terminal position (light chain called VL, heavy chain called VH) and a constant region at the C-terminal position (consisting of a single domain called CL for the light chain and three or four domains called CH1, CH2, CH3, CH4 for the heavy chain). Each variable domain typically comprises 4 "hinge regions" (designated FR1, FR2, FR3, FR4) and 3 regions directly responsible for binding to antigen, designated "CDRs" (designated CDR1, CDR2, CDR 3).

An "antibody" according to the invention may be of mammalian origin (e.g. human or mouse or camelidae), humanized, chimeric, recombinant. It is preferably a monoclonal antibody recombinantly produced by cells genetically modified according to techniques familiar to those skilled in the art. The antibody may be of any isotype, for example IgG, IgM, IgA, IgD or IgE, preferably IgG.

By "chimeric antibody" is meant an antibody whose heavy and light chain variable region sequences belong to a species different from the light and heavy chain constant region sequences. For the purposes of the present invention, it is preferred that the heavy and light chain variable region sequences be murine, while the heavy and light chain constant region sequences belong to a non-murine species. In this respect, for the constant region, all non-murine mammalian species are useful, in particular human, monkey, pig, cow, horse, cat, dog or bird, this list is not exhaustive. Preferably, the chimeric antibody according to the invention comprises human heavy and light chain constant region sequences and murine heavy and light chain variable region sequences.

By "humanized antibody" is meant an antibody that: for the antibody, some or all of the sequences of the regions involved in antigen recognition (hypervariable regions or CDRs: complementarity determining regions) and sometimes some of the amino acids of the FR regions (framework regions) are of non-human origin, while the sequences of the variable and constant regions not involved in antigen recognition are of human origin.

"human antibody" refers to an antibody comprising only human sequences (both light chain variable and constant regions and heavy chain variable and constant regions).

"antibody fragment" means any part of an immunoglobulin comprising at least one disulfide bond and capable of binding to an antigen recognized by an intact antibody, obtained by enzymatic digestion or by biological production, e.g., Fv, Fab '-SH, F (ab')²Diabodies, linear antibodies (also referred to as "single domain antibodies" or sdabs, or nanobodies), antibodies with single chains (e.g., scFv). Enzymatic digestion of immunoglobulins by pepsin produces an Fc fragment and F (ab')2 fragment that are split into several peptides. F (ab ')2 is formed from two Fab' fragments which are bound by interchain disulfide bonds. The Fab part consists of the variable region as well as the domains CH1 and CL. The Fab' fragment consists of the Fab region and the hinge region. Fab '-SH refers to a Fab' fragment in which cysteine residues of the hinge region carry free thiol groups.

The term "affinity" refers to the strength of all non-covalent interactions between a molecule (e.g., an antibody or antibody fragment) and an antigen being recognized (e.g., an antigen such as an α G protein). Affinity is usually expressed by the dissociation constant (Kd). The dissociation constant (Kd) can be measured by well-known methods (e.g. by FRET or SPR).

In the sense of the present invention, "identity" or "homology" is calculated by comparing two sequences aligned in a comparison window. The alignment of the sequences allows the number of common positions (nucleotides or amino acids) of the two sequences to be determined over the window of comparison. Thus, the number of common positions is divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of identity. The determination of percent sequence identity may be performed manually or using well-known software.

In a particular embodiment of the invention, the identity or homology corresponds to at least one substitution of the amino acid residue, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions, preferably at least one substitution of the amino acid residue that is made conservatively. "conservative substitution of an amino acid residue" consists of replacing an amino acid residue with another amino acid residue having similar properties in the side chain. Families of amino acids whose side chains have similar properties are well known, and mention may be made, for example, of basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), polar and uncharged side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine tryptophan, histidine).

Thus, a homologous antibody or antibody fragment or "antibody or antibody fragment variant" (i.e., an antibody or antibody fragment having the same function) has certain amino acids that can be substituted at the constant and/or variable region level with other amino acids without losing antigen binding ability. Preferably, such substitutions are made within the DNA sequence encoding the antibody or antibody fragment, i.e., the substitutions are substantially conservative. The skilled person uses his general knowledge to determine the number of substitutions that can be made and their location so as to be able to retain the function of the antibody or antibody fragment. To determine the ability of an antibody or one or more variants of an antibody fragment to specifically bind to an antigen, several suitable methods familiar to those skilled in the art and described in the prior art may be used. Thus, the antibody or antibody fragment can be measured by a binding method (e.g., an ELISA method), an affinity chromatography method, or the like. Variants of antibodies or antibody fragments can be generated, for example, by "phage display" methods, enabling the generation of phage libraries. A number of methods are known for generating "phage display" libraries and for targeting variants of antibodies or antibody fragments with desired functional properties.

Advantageously, the antibody or antibody fragment used in the context of the present invention binds to protein G α i, G α o and/or G α z, e.g. it binds to protein G α i1, protein G α i2 and/or protein G α i 3. The identifier of isoform 1 of the human α i 1G protein is UniProt P63096-1, and the identifier of isoform 2 is UniProt P63096-2. The gene encoding the human α i 1G protein is known under the name "GNAI 1" (Gene ID:2770, NCBI).

In the sense of the present invention, the term "molecule capable of modulating GPCR activation" denotes a molecule capable of activating or capable of inhibiting GPCR and thus capable of inducing or capable of preventing transduction of a signal from outside the cell to the inside of the cell through GPCR. It may be an agonist, antagonist, inverse agonist, positive allosteric modulator (allosteric modulator) or negative allosteric modulator.

In the sense of the present invention, the term "test molecule" is a molecule that is likely to modulate GPCR activation.

In the present specification, the term "molecule" not further specified denotes both the terms "molecule capable of modulating GPCR activation" and "test molecule".

The term "RET" (from the english term "resonant energy transfer") denotes an energy transfer technique.

The term "FRET" (from the English language "fluorescence resonance energy transfer") refers to the transfer of energy between two fluorescent molecules. FRET is defined as the non-radiative energy transfer caused by a dipole-dipole interaction between an energy donor and an energy acceptor. This physical phenomenon requires energy compatibility between these molecules. This means that the emission spectrum of the donor must at least partially cover the absorption spectrum of the acceptor. And In accordance with theory, FRET is a process that depends on the distance separating two molecules, the donor and acceptor: when these molecules are brought into close proximity to each other, FRE will be emittedAnd (4) a T signal.

The term "BRET" ("bioluminescence resonance energy transfer") refers to the transfer of energy between a bioluminescent molecule and a fluorescent molecule.

In the sense of the present invention, the term "ligand" denotes a molecule capable of binding to a target molecule. In the context of the present invention, the target molecule is an all α G protein bound to non-hydrolysable or slowly hydrolysable GTP labeled with the first member of the RET partner pair. In the context of the present invention, the ligand must be capable of binding to the all- α G protein bound to non-hydrolysable or slowly hydrolysable GTP labeled with the first member of the RET partner pair. However, the ligand need not be specific for the whole α G protein bound to non-hydrolysable or slowly hydrolysable GTP labeled with the first member of the RET partner pair. Thus, the ligands used in the context of the present invention are also capable of binding to the following proteins: α G protein bound to GDP, α G protein bound to GTP, α G protein bound to unlabelled non-or slowly hydrolysable GTP, or even capable of binding to empty α G protein. The ligand may be proteinaceous (e.g. protein or peptide) or nucleotide (e.g. DNA or RNA). In the context of the present invention, the ligand is advantageously selected from an antibody, an antibody fragment, a peptide or an aptamer, preferably an antibody or an antibody fragment. In the context of the present invention, the ligand may be directly or indirectly labelled by methods familiar to those skilled in the art, for example as described below, but preferably the ligand is directly labelled by covalent binding to a member of the RET partner pair.

The term "RET partner pair" denotes a pair consisting of an energy donor compound (hereinafter referred to as "donor compound") and an energy acceptor compound (hereinafter referred to as "acceptor compound"); these compounds emit a RET signal when they are close to each other and excited at the excitation wavelength of the donor compound. It is known that for two compounds to become RET partners, the emission spectrum of the donor compound must partially overlap the excitation spectrum of the acceptor compound. For example, when a fluorescent donor compound and acceptor compound are used, they are referred to as "FRET partner pairs"; alternatively, where a bioluminescent donor compound and an acceptor compound are used, they are referred to as a "BRET partner pair".

The term "RET signal" means any measurable signal representing RET between a donor compound and an acceptor compound. For example, the FRET signal may thus be a change in the intensity or luminescence lifetime of a fluorescent donor compound or acceptor compound (when the latter is fluorescent).

The term "container" denotes a well of a plate, a test tube or any other container suitable for mixing a membrane preparation with the reagents required for performing the method according to the invention.

The present invention relates to a method for determining the ability of a molecule to modulate the activation of a G protein-coupled receptor (GPCR), said method comprising the steps of:

a) Introducing into the first vessel:

-a membrane preparation carrying one or more GPCRs and one or more alpha G proteins,

-a non-hydrolysable or slowly hydrolysable GTP source labelled with a first member of a RET partner pair,

-a ligand of the alpha subunit of the G protein (alpha G protein) labelled with the second member of the RET partner pair, said ligand being capable of binding to the all-alpha G protein bound to the non-hydrolysable or slowly hydrolysable GTP labelled with the first member of the RET partner pair,

-optionally a GPCR agonist;

b) measuring an RET signal emitted in the first container;

c) introducing (i) the same reagent and test molecule as in step a) into a second container, or (ii) a test molecule into a first container;

d) measuring the RET signal emitted in the first container or in the second container obtained in step c);

In carrying out the process according to the invention, it is not necessary to add a source of GTP other than labeled non-hydrolyzable or slowly hydrolyzable GTP. Advantageously, no other sources of GTP are added in addition to the labeled non-hydrolyzable or slowly hydrolyzable GTP in performing the method according to the present invention.

It is also not necessary to add a GDP source when carrying out the method according to the invention. However, in carrying out the process according to the invention, for example in step a), small amounts of GDP can be tolerated. In formats 1A and 1B (fig. 2A and 2B), advantageously, no GDP source is added when performing the method according to the invention. In formats 2A and 2B (fig. 2C and 2D), the addition of GDP may allow for better discrimination of signal between the conditions without agonist and those with agonist.

Step a)

Step a) comprises introducing into the first vessel three or four of the following elements:

-a membrane preparation carrying one or more GPCRs and one or more alpha G proteins,

-a non-hydrolysable or slowly hydrolysable GTP source labelled with a first member of a RET partner pair,

-a ligand of the alpha subunit of the G protein (α G protein) labelled with the second member of the RET chaperone pair, said ligand being capable of binding to the all- α G protein bound to the non-hydrolysable or slowly hydrolysable GTP labelled with the first member of the RET chaperone pair, and

-optionally a GPCR agonist.

Advantageously, the non-hydrolyzable or slowly hydrolyzable GTP is selected from GTPgamma aS (GTP γ S or GTPgS), GppNHp and GppCp. GTP γ S must be labeled at a position other than the third phosphate (γ phosphate). The three or four elements may be introduced into the vessel sequentially in any order, or simultaneously or quasi-simultaneously (quasi-simultaneously). Mixing the three elements makes it possible to obtain a reaction solution suitable for carrying out RET. Other elements may also be added to the vessel to adjust the solution used to perform RET. For example, coelenterazine h (benzyl coelenterazine) or dideoxy coelenterazine (DeepBlueC) may be added ^TM) Or didehydrocoelenterazine (coelenterazine-400 a) or D-luciferin.

In a first embodiment, the ligand of the α G protein specifically binds to the SwitchII domain of the α G protein, in particular to peptide 215-294 of the α G protein. For example, the α G protein is selected from G- α i1, G- α i2 and G- α i3, and the ligand of the α G protein is the peptide KB1753 of the sequence Ser-Ser-Arg-Gly-Tyr-Tyr-His-Gly-lle-Trp-Val-Gly-Glu-Glu-Gly-Arg-Leu-Ser-Arg (SEQ ID No: 1).

In a second embodiment, the α G protein is selected from the group consisting of G- α i1, G- α i2 and G- α i3, and the ligand of the α G protein competes with peptide KB1753(SEQ ID No:1) for binding to said α G protein. The ability of ligands to compete with peptide KB1753(SEQ ID No:1) for binding to the α G protein can be tested by competition methods.

The "competition method" consists of: the ligands of the α G protein were tested for their ability to block the binding between peptide KB1753(SEQ ID No:1) and the α G protein, or to compete with peptide KB1753(SEQ ID No:1) for binding to the α G protein. In other words, the ligand of the α G protein that competes with peptide KB1753(SEQ ID No:1) for binding to the α G protein binds to the same epitope as peptide KB1753(SEQ ID No:1) or to an epitope sufficiently close to the epitope recognized by peptide KB1753(SEQ ID No:1) to prevent binding of the ligand of the α G protein due to steric hindrance. A number of types of competition methods are available for determining whether a ligand for the α G protein competes with peptide KB1753(SEQ ID No:1), for example by an ELISA assay. For example, ELISA competition methods involve the use of purified α G protein bound to a solid surface or cells, a ligand for the test α G protein bound to the α G protein, and the labeled peptide KB 1753. In general, the reference peptide KB1753(SEQ ID No:1) is used in unsaturated concentrations (relative to its dissociation constant Kd for the. alpha.G protein) and the signal is measured in the absence or presence of an increased concentration of the ligand of the α G protein to be detected. When the ligand of the α G protein of interest is present in excess, it can block specific binding of peptide KB1753(SEQ ID No:1) to the α G protein by at least 40% -45%, 45% -50%, 50% -55%, 55% -60%, 60% -65%, 65% -70%, 70% -75%, or more than 75%. In some cases, binding is blocked by at least 80% -85%, 85% -90%, 90% -95%, 95% -97%, or more than 97%.

Another example of a competition method may use TR-FRET. For example, TR-FRET involves the use of a purified and tagged α G protein, an anti-tag ligand (advantageously an antibody) labelled with a first member of a TR-FRET partner pair (advantageously a donor) capable of binding to the tag of the α G protein, a peptide KB1753(SEQ ID NO:1) labelled with a second member of the TR-FRET partner pair (advantageously an acceptor, by a method of indirect labelling with biotin on the peptide and a streptavidin-acceptor), and a ligand of the α G protein to be detected. In general, peptide KB1753(SEQ ID NO:1) is used at a non-saturating concentration (relative to its dissociation constant Kd for the α G protein) and the TR-FRET signal is measured in the absence or presence of an increased concentration of the ligand of the α G protein to be detected. When a ligand for the α G protein of interest is determined, it can block specific binding of peptide KB1753(SEQ ID No:1) to the α G protein by at least 40% -45%, 45% -50%, 50% -55%, 55% -60%, 60% -65%, 65% -70%, 70% -75%, or more than 75%. In some cases, binding is blocked by at least 80% -85%, 85% -90%, 90% -95%, 95% -97%, or more than 97%. If the ligands of the alpha G proteins tested inhibit the binding of the peptide KB1753(SEQ ID No:1), the positive (orthosteric) properties of the inhibition (as opposed to allosteric inhibition) can be confirmed by the Schild-Plot experiment, which consists of: the dissociation constant (Kd) of the labeled peptide KB1753(SEQ ID No:1) for the α G protein was measured in the absence or presence of increased concentrations of ligand for the α G protein. Inhibition is orthosteric (i.e., peptide KB1753(SEQ ID No:1) and the ligand for the α G protein bind to the same epitope of the α G protein) if the Kd changes in a linear and non-saturating manner as the concentration of the ligand for the α G protein increases. Inhibition is allosteric (i.e., peptide KB1753(SEQ ID No:1) and ligand do not bind to the same epitope of the α G protein) if the Kd changes in a nonlinear and saturable manner as the concentration of ligand in the α G protein increases.

The ligand for the α G protein may be an antibody or antibody fragment.

Thus, in a third embodiment, the ligand of the α G protein is an antibody or antibody fragment capable of binding to the α G protein, said antibody or antibody fragment comprising:

-a heavy chain variable domain comprising the CDR1 of amino acid sequence SEQ ID No. 2, CDR2 of amino acid sequence SEQ ID No. 3 and CDR3 of amino acid sequence SEQ ID No. 4; and

a light chain variable domain comprising the CDR1 of amino acid sequence SEQ ID NO:5, the CDR2 of amino acid sequence DTS (i.e. the three amino acids "Asp Thr Ser", i.e. the three amino acids aspartic acid, threonine, serine) and the CDR3 of amino acid sequence SEQ ID NO: 6.

The heavy chain variable domain of an antibody or antibody fragment capable of binding to an α G protein may comprise:

-FR 1 having at least 80% homology, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% homology with the amino acid sequence SEQ ID No. 7;

-FR 2 having at least 80% homology, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% homology with the amino acid sequence SEQ ID No. 8;

-FR 3 having at least 80% homology, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% homology with the amino acid sequence SEQ ID No. 9; and/or

FR4 that has at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology or even 100% homology with the amino acid sequence SEQ ID NO 10.

The light chain variable domain of an antibody or antibody fragment capable of binding to an α G protein may comprise:

FR4 having at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology or even 100% homology with the amino acid sequence SEQ ID No. 14.

In particular embodiments of the antibody or antibody fragment capable of binding to α G protein:

the heavy chain variable domain comprises:

FR1 of amino acid sequence SEQ ID NO. 7 (i.e. having 100% homology with the amino acid sequence SEQ ID NO. 7),

FR2 of amino acid sequence SEQ ID NO. 8,

FR3 of amino acid sequence SEQ ID NO 9, and

-FR 4 of amino acid sequence SEQ ID No. 10; and

the light chain variable domain comprises:

FR1 of amino acid sequence SEQ ID NO. 11,

FR2 of amino acid sequence SEQ ID NO. 12,

FR3 of amino acid sequence SEQ ID NO 13, and

FR4 of amino acid sequence SEQ ID NO. 14.

In particular embodiments of the antibody or antibody fragment capable of binding to α G protein, the heavy chain variable domain may have at least 80% homology, preferably at least 90% homology, e.g. at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homology with the amino acid sequence SEQ ID No. 15; and the light chain variable domain may have at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology or 100% homology with the amino acid sequence SEQ ID No. 16.

Thus, the ligand of the α G protein may be an antibody or antibody fragment, wherein:

the heavy chain variable domain has at least 80% homology, preferably at least 90% homology, for example at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology or 100% homology with the amino acid sequence SEQ ID No. 15;

-the light chain variable domain has at least 80% homology, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homology with the amino acid sequence SEQ ID NO 16; and-the CDR1 of the variable domain of the heavy chain consists of the amino acid sequence SEQ ID No. 2, the CDR2 of the variable domain of the heavy chain consists of the amino acid sequence SEQ ID No. 3, the CDR3 of the variable domain of the heavy chain consists of the amino acid sequence SEQ ID No. 4, the CDR1 of the variable domain of the light chain consists of the amino acid sequence SEQ ID No. 5, the CDR2 of the variable domain of the light chain consists of the amino acid sequence DTS and the CDR3 of the variable domain of the light chain consists of the amino acid sequence SEQ ID No. 6.

Advantageously, the ligand of the α G protein is an antibody or antibody fragment, wherein the heavy chain variable domain consists of the amino acid sequence SEQ ID NO:15 (i.e.the heavy chain variable domain has 100% homology with the amino acid sequence SEQ ID NO: 15) and the light chain variable domain consists of the amino acid sequence SEQ ID NO: 16.

The antibodies described in the examples under reference DSV36S (DSV antibodies are available on demand from Cisbio Bioassays) comprise a heavy chain variable domain consisting of the amino acid sequence SEQ ID NO:15 and a light chain variable domain consisting of the amino acid sequence SEQ ID NO: 16.

An antibody or antibody fragment capable of binding to α G protein according to the above-described third embodiment is referred to as "reference antibody or antibody fragment" in the fourth embodiment below.

In a fourth embodiment, the ligand of the α G protein is an antibody or antibody fragment that competes for binding to the α G protein with a reference antibody or antibody fragment, hereinafter referred to as "competing antibody or antibody fragment".

The ability of an antibody or antibody fragment to compete with a reference antibody or antibody fragment for binding to α G protein can be tested by competition methods. The "competition method" consists of: the test antibody (or antibody fragment) blocks the ability of the reference antibody or antibody fragment to bind to the antigen, or competes with the reference antibody or antibody fragment for binding to the antigen. In other words, an antibody that competes with the reference antibody or antibody fragment binds to the same epitope as the reference antibody or antibody fragment or to an epitope sufficiently close to the epitope recognized by the reference antibody or antibody fragment to prevent binding of the reference antibody or antibody fragment due to steric hindrance.

Many types of competition methods are available for determining whether an antibody or antibody fragment competes with a reference antibody or antibody fragment, such as: by competitive ELISA assays, by direct or indirect sandwich methods, by direct or indirect solid phase Radioimmunoassay (RIA), by direct or indirect solid phase Enzyme Immunoassay (EIA), and the like. For example, competition ELISA methods involve the use of purified antigen bound to a solid surface or cells, a test antibody bound to an unlabeled antigen, and a labeled reference antibody or antibody fragment. Typically, the reference antibody or antibody fragment is present at a non-saturating concentration (relative to its dissociation constant Kd for α G protein) and the signal is measured at increasing concentrations of the test antibody or antibody fragment. When the antibody is present in excess, it may block or inhibit (e.g., reduce) specific binding of the reference antibody or antibody fragment to the antigen by at least 40% -45%, 45% -50%, 50% -55%, 55% -60%, 60% -65%, 65% -70%, 70% -75%, or more than 75%. In certain instances, binding is inhibited by at least 80% -85%, 85% -90%, 90% -95%, 95% -97%, or more than 97%.

The competing antibody or antibody fragment used in the method according to the invention may be obtained, for example, by performing the protocol described in example 26 together with a reference antibody or antibody fragment. The antibodies described in the examples under reference DSV38S (DSV antibodies are available on demand from Cisbio Bioassays) are competing antibodies that can be used in the method according to the invention.

The reference antibody or antibody fragment and the competing antibody or antibody fragment defined above are hereinafter collectively referred to as "antibodies or antibody fragments used in the method according to the invention".

The antibody or antibody fragment used in the method according to the invention may bind to the α G protein in isolated form and/or present in the membrane environment, e.g. it may bind to α G protein present in a preparation made from a membrane carrying one or more GPCRs and one or more α G proteins. Because the antibodies according to the invention are capable of binding to α G protein, α G protein does not have to complex with GPCR.

The antibody or antibody fragment used in the method according to the invention may bind to the α G protein with a dissociation constant (Kd) of less than or equal to 20nM as measured in FRET. Dissociation constants below 20nM are preferred for proper performance of RET. Advantageously, the antibody or antibody fragment used in the method according to the invention can bind to α G protein with a dissociation constant (Kd) of less than or equal to 20nM, e.g. less than or equal to 10nM or less than or equal to 5nM affinity constants, e.g. 0-20nM (excluding 0), 0-10nM (excluding 0), 0-5nM (excluding 0), measured in FRET. A method of measuring Kd of an antibody or antibody fragment according to the invention in FRET is described in example 27.

The antibodies or antibody fragments used in the method according to the invention are particularly advantageous for carrying out the method according to the invention.

For example, an antibody or antibody fragment used in the method according to the present invention can be obtained by performing the protocol described in example 26.

The inventors also show that the antibodies or antibody fragments used in the method according to the invention bind to the SwitchII domain of the α G protein, more specifically to the peptide 215-294 of the α G protein.

The first container may optionally comprise a GPCR agonist. GPCR agonists are widely described in the literature, for example in table 1 of application WO 2011/018586.

Step b)

Step b) comprises measuring the RET signal emitted in the first container (i.e. the container obtained in step a)). The measured signal corresponds to the signal obtained in the container in the absence of the molecule to be detected. The measurement can be performed by a conventional method familiar to those skilled in the art and does not cause any particular problem. Devices capable of detecting and measuring RET signals are commonly used, such as the pheasar FS microplate reader (BMG Labtech) in TR-FRET or bioluminescent reading mode.

Step c)

In one embodiment, step c) comprises introducing the same reagent and test molecule as in step a) into a second container. Advantageously, a second container is prepared in the same way as the first container, except that the molecule to be tested is present in the second container. This embodiment is advantageous because it enables the simultaneous measurement of RET signals emitted in the first container and in the second container. This embodiment also enables simultaneous measurement of RET signals emitted in one or more second containers. This embodiment is therefore particularly advantageous, since it enables several different molecules to be tested in parallel.

In another embodiment, step c) comprises introducing the molecule to be tested in a first container. This embodiment has the advantage that only one vessel is used for carrying out the method according to the invention.

Step d)

Step d) comprises measuring the RET signal emitted in the first receptacle or in the second receptacle obtained in step c). The measured signal corresponds to the signal obtained in the container in the presence of the molecule to be detected. As in step b), the measurement can be carried out by conventional methods familiar to the person skilled in the art and does not pose any particular problem. Devices capable of detecting and measuring RET signals are commonly used, such as the pheasar FS microplate reader (BMG Labtech) in TR-FRET or bioluminescent reading mode.

Step e)

Step e) comprises comparing the signals obtained in step b) and step d), the presence of modulation of the signal obtained in step d) relative to the signal obtained in step b) indicating that the test molecule is capable of modulating GPCR activation. The modulation of the signal may be an increase in the signal or a decrease in the signal.

One skilled in the art can easily compare the signals in step b) and step d) and define a threshold that allows it to assess the regulation (qualify), for example a difference between the signals of more than 5%, more than 10%, more than 15%, more than 20% or more than 25%. For example, the ratio between the signals in step b) and step d) may be calculated. Generally, for a given pair of RET partners, the greater the difference between the signals, the greater the ratio between the signals, and the greater the modulation (e.g., activation or inhibition) of GPCR activation. However, the difference between the signals may vary depending on the RET chaperone pair used to perform the method according to the invention. The regulatory level of GPCR activation enables the identification of molecules that are to some extent agonists, antagonists, inverse agonists, positive allosteric modulators or negative allosteric modulators.

In a first embodiment, the first container does not comprise a GPCR agonist and in step e) the presence of a decrease in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is a GPCR agonist.

In a second embodiment, the first vessel comprises a GPCR agonist and in step e):

-a decrease in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is an agonist or a positive allosteric modulator of the GPCR.

In a third embodiment, the first container does not contain a GPCR agonist and in step e) the presence of an increase in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is a GPCR agonist.

In a fourth embodiment, the first vessel comprises a GPCR agonist and in step e):

-a decrease in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is an antagonist or a negative allosteric modulator of the GPCR;

-the presence of an increase in the signal obtained in step d) relative to the signal obtained in step b) indicates that the test molecule is an agonist or positive allosteric modulator of the GPCR.

Labelling of ligands with members of RET partner pairs

The ligand may be directly or indirectly labelled.

The ligand may be directly labelled with a member of the RET partner pair (e.g. a fluorescent compound when performing FRET) by conventional methods known to those skilled in the art based on the presence of reactive groups on the ligand. For example, when the ligand is an antibody or antibody fragment, the following reactive groups may be used: terminal amino groups, carboxylic acid groups of aspartic and glutamic acids, amine groups of lysine, guanidine groups of arginine, thiol groups of cysteine, phenolic groups of tyrosine, indole rings of tryptophan, thioether groups of methionine, imidazole groups of histidine.

The reactive group may form a covalent bond with a reactive group carried by the ligand. Suitable reactive groups carried by the ligand are familiar to the person skilled in the art, e.g. a donor compound or an acceptor compound functionalized with a maleimide group will be able to, e.g., covalently bind to a thiol group carried by a cysteine carried by a protein or peptide (e.g. an antibody or antibody fragment). Similarly, a donor/acceptor compound carrying an N-hydroxysuccinimide ester will be able to covalently bind to an amine present in a protein or peptide.

The ligand may also be indirectly labeled with a fluorescent or bioluminescent compound, for example by introducing into the measurement medium an antibody or antibody fragment that is itself covalently bound to the acceptor compound/donor compound, which second antibody or antibody fragment specifically recognizes the ligand.

Another very classical indirect labeling method involves immobilization of biotin on the ligand to be labeled and then incubation of the biotinylated ligand in the presence of streptavidin labeled with an acceptor/donor compound. Suitable biotinylated ligands can be prepared by techniques familiar to those skilled in the art; the company Cisbio Bioassays, for example, markets streptavidin labeled with a fluorophore under the trade name "d 2" (ref.610SADLA).

In the context of the present invention, the ligand is labeled with (i) a fluorescence donor compound or a luminescence donor compound; or (ii) a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher). Preferably, the ligand is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher).

Labelling non-or slowly-hydrolysable GTP with a member of the RET partner pair

Non-hydrolyzable or slowly hydrolyzable GTP can be labeled directly or indirectly. Preferably, the non-hydrolyzable or slowly hydrolyzable GTP is directly labeled.

Direct labeling of non-hydrolyzable or slowly hydrolyzable GTP with a member of the RET partner pair (e.g., a fluorescent compound when performing FRET) can be performed by methods based on the presence of reactive groups on the non-hydrolyzable or slowly hydrolyzable GTP.

The reactive group may form a covalent bond with a reactive group carried by a member of the RET partner pair. Suitable reactive groups carried by members of the RET partner pair are familiar to the person skilled in the art, e.g. donor compounds or acceptor compounds functionalized with maleimide groups will be able to bind e.g. covalently to thiol groups. Similarly, a donor/acceptor compound bearing an N-hydroxysuccinimide ester would be able to covalently bind to an amine.

In the context of the present invention, non-hydrolyzable or slowly hydrolyzable GTP is labeled with (i) a fluorescence donor compound; or (ii) a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher). Preferably, the non-hydrolyzable or slowly hydrolyzable GTP is labeled with a fluorescent donor compound.

In a specific embodiment, the non-hydrolyzable or slowly hydrolyzable GTP is labeled with a fluorescent donor compound and the ligand for the α G protein is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher). In another embodiment, the non-hydrolyzable or slowly hydrolyzable GTP is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher), and the ligand of the α G protein is labeled with a fluorescent donor compound or a luminescent donor compound.

Labels for performing FRET

In a specific embodiment, the ligand and the non-hydrolyzable or slowly hydrolyzable GTP are each labeled with a member of a FRET partner pair, i.e., a fluorescent energy donating compound or a fluorescent energy accepting compound.

It is within the ability of those skilled in the art to select a pair of FRET partners for obtaining a FRET signal. For example, donor-acceptor pairs useful for studying FRET phenomena are specifically described in the work of Joseph r.lakowicz (Principles of fluorescence spectroscopy, 2 nd edition, 338), to which reference may be made by those skilled in the art.

Fluorescence donor compounds

Fluorescent energy-donating compounds with a long lifetime (>0.1ms, preferably in the range of 0.5ms to 6 ms), in particular lanthanide complexes (i.e. chelates of rare earth elements, macrocycles or cryptates), are advantageous because they enable time-resolved measurements, i.e. measurement of TR-FRET (time-resolved FRET) signals, eliminating most of the background noise emitted by the measurement medium. For this reason, they are generally preferred for carrying out the process according to the invention. Advantageously, these compounds are lanthanide complexes. These complexes (e.g., chelates or cryptates) are particularly suitable as members of energy donating FRET pairs.

Europium (Eu)³⁺) Terbium (Tb)³⁺) Dysprosium (Dy)³⁺) Samarium (Sm)³⁺) Neodymium (Nd)³⁺) Ytterbium (Yb)³⁺) Or erbium (Er)³⁺) The complexes of (b) are also suitable rare earth element complexes for the purposes of the present invention, particular preference being given to europium (Eu)³⁺) And terbium (Tb)³⁺) The complex of (1).

Numerous rare earth element complexes have been described and several are currently marketed by Perkin Elmer, Invitrogen and Cisbio Bioassays.

Examples of rare earth element chelates or cryptates suitable for the purposes of the present invention are:

-a lanthanide cryptate comprising one or more pyridine units. Such cryptates of rare earth elements are described, for example, in patents EP 0180492, EP 0321353, EP 0601113 and international application WO 01/96877. Terbium (Tb)³⁺) And europium (Eu)³⁺) The cryptates of (a) are particularly suitable for the purposes of the present invention. Lanthanide cryptates are sold by Cisbio Bioassays. As a non-limiting example, mention may be made of europium cryptates of formula (which can be coupled to the compound to be labelled via a reactive group, here for example NH)₂Group):

lanthanide chelates as described in detail in patents US 4761481, US 5032677, US 5055578, US 5106957, US 5116989, US 4761481, US 4801722, US 4794191, US 4637988, US 4670572, US 4837169, US 4859777. Patents EP 0403593, US 5324825, US 5202423, US 5316909 describe chelates formed by a nonadentate (e.g. terpyridine) ligand. Lanthanide chelates are sold by the company Perkin Elmer.

Lanthanide complexes formed by chelating agents (e.g. tetraazacyclododecane) which are substituted by aromatic ring-containing chromophores may also be used, such as those described by Poole r. et al in biomol. chem,2005,3,1013-1024 "Synthesis and catalysis of high elevation and catalysis stable lanthanide complexes capable of use in a cell". The complexes described in application WO 2009/10580 can also be used.

The lanthanide cryptates described in patents EP 1154991 and EP 1154990 may also be used.

Terbium cryptate of formula (I), which can be reactedCoupled to the compound to be labelled, where the reactive group is, for example, NH₂Group):

the synthesis of which is described in international application WO 2008/063721 (compound 6a, page 89).

The terbium cryptate Lumi4-Tb from Lumiphore, sold by Cisbio Bioassays.

Quantum dyes from Research Organics company of formula (which can be coupled to the compound to be labelled via a reactive group, here NCS):

ruthenium chelates, in particular complexes formed from ruthenium ions and several bipyridines, such as ruthenium (II) tris (2,2' -bipyridine).

Terbium chelate DTPA-cs124 Tb of formula, marketed by Life Technologies (which can be coupled to the compound to be labelled via the reactive group R), the synthesis of which is described in US patent US 5622821.

Terbium chelates of the formula described by Latva et al (Journal of luminescences 1997,75(2): 149-169):

advantageously, the fluorescence donor compound is a FRET partner selected from the following compounds: europium cryptates, europium chelates, terbium cryptates, ruthenium chelates, quantum dots, allophycocyanins, rhodamines, anthocyanins (cyanins), squaraines (squaraines), coumarins, proflavins (proflavins), acridines, fluoresceins (fluorosceins), boron-dipyrromethene derivatives, and nitrobenzoxadiazoles.

Particularly advantageously, the fluorescence donor compound is a FRET partner selected from the following compounds: europium cryptate, europium chelate, terbium cryptate, ruthenium chelate and quantum dots; europium and terbium chelates and cryptates are particularly preferred.

The non-hydrolyzable or slowly hydrolyzable GTP labeled with a fluorescent donor compound, which can be used in the FRET method of the present invention, is represented by the following general formulae (1) and (2a, 2b, 2 c):

labeling of GTP analogs can be performed at different positions of GTP:

in the gamma position (O, NH, CH) of the phosphate₂) (ii) a Or

At the 2 'and 3' positions of the ribose

In a specific embodiment, the non-hydrolyzable or slowly hydrolyzable GTP labeled with a fluorescent donor compound that can be used in the FRET method of the present invention is represented by the general formula (I):

Wherein:

x is O, NH or CH₂；

Y is O, NH or CH₂；

L is a bivalent linker;

Ln³⁺being a lanthanide complex, optionally carrying a reactive group G₃。

"lanthanide complex" means a chelate, macrocycle, cryptate or any organic species capable of complexing an atom of the lanthanide family, lanthanide (Ln) being selected from: eu, Sm, Tb, Gd, Dy, Nd and Er; preferably the lanthanide is Tb, Sm or Eu; even more preferably Eu or Tb.

Compounds where Y ═ O are referred to as GTP- γ -O analogs. Compounds with Y ═ NH are referred to as GTP- γ -N analogs. Y is CH₂The compounds of (a) are referred to as GTP-gamma-C analogues.

The divalent linker L is advantageously selected from:

-direct bonding;

-straight or branched C₁-C₂₀(preferably C)₁-C₈) An alkylene group, optionally containing one or more double or triple bonds;

-C₅-C₈a cycloalkylene group; or

-C₆-C₁₄An arylene group;

the alkylene, cycloalkylene or arylene groups optionally containing one or more heteroatoms (e.g. oxygen, nitrogen, sulphur, phosphorus) or one or more carbamoyl or carboxamido (carboxamido) groups, the alkylene, cycloalkylene or arylene groups optionally being substituted by 1 to 5, preferably 1 to 3C₁-C₈Alkyl radical, C₆-C₁₄Aryl, sulfonate or oxo (oxo) groups.

Even more advantageously, the divalent linker L is chosen from the following groups:

wherein n, m, p, q are integers from 1 to 16, preferably from 1 to 5; and e is an integer from 1 to 6, preferably from 1 to 4.

Quite advantageously, the divalent linker L is chosen from a direct bond, a linear or branched C₁-C₈An alkylene group or a group of the formula:

the divalent linker L is preferably selected from:

-(CH₂)_n-；-(CH₂)_n-O-(CH₂)_m-O-(CH₂)_p-；

group- (CH)₂) n-is quite particularly preferred.

The group L may also advantageously be a group of formula:

wherein m, n and p are integers from 1 to 16, preferably from 1 to 5.

Reactive group G₃Selected from one of the following groups: acrylamide, optionally activated amines (e.g. cadaverine or ethylenediamine), activated esters, aldehydes, alkyl halides, anhydrides, anilines, azides, aziridines, carboxylic acids, diazoalkanes, haloacetamides, halotriazines (e.g. monochlorotriazine, dichlorotriazine), hydrazines (including hydrazides), imidoesters, isocyanates, isothiocyanates, maleimides, sulfonyl halides, thiols, ketones, acid halides, succinimidyl esters, hydroxysuccinimidyl esters, hydroxysulfosuccinimidyl esters, azidonitrophenyl, azidophenyl, 3- (2-pyridyldithio) -propionamide, glyoxal, triazines, acetylene groups, in particular groups selected from the group having the formula:

Wherein w varies from 0 to 8; v is equal to 0 or 1; ar is a saturated or unsaturated heterocyclic ring having 5 or 6 ring members, containing 1 to 3 heteroatoms, optionally substituted with halogen atoms.

Preferably, the reactive group G₃Selected from the group consisting of amines (optionally protected in the form of-NHBoc), succinimidyl esters, hydroxysuccinimidyl esters, haloacetamides, hydrazines, halotriazines, isothiocyanates, maleimido groups or carboxylic acids (optionally in the group-CO)₂Me、-CO₂the form of tBu is protected). In the latter case, the acid must be activated to the ester form to be able to react with the nucleophile. Lanthanide complexes Ln³⁺Advantageously selected from one of the complexes given below:

advantageously, the lanthanide complex Ln³⁺Is selected from one of complexes C1 to C17, C24 to C32 and C36 to C44. More advantageously, the lanthanide complex Ln³⁺Is selected from one of complexes C1 to C17 and C36 to C44. Even more advantageously, lanthanide complexes Ln³⁺Is selected from the formulaOne of compounds C1 to C17. Even more advantageously, lanthanide complexes Ln³⁺Is selected from one of complexes C1 to C4 and C11 to C17. Even more advantageously, lanthanide complexes Ln³⁺Is selected from one of complexes C1 to C4 and C11. Very advantageously, the lanthanide complexes Ln ³⁺Is complex C2 or complex C3.

The preparation of the above GTP analogs is described in the literature or in french patent application No. FR 1900856 filed on 30/1/2019.

Advantageously, the GTP analogue labelled with a fluorescence donor compound may be selected from GTPgN-C2(GTP- γ -N-C2), GTPgN-C3(GTP- γ -N-C3), GTPgN-octyl-C2 (GTP- γ -N-octyl-C2), GTPgN-octyl-C11 (GTP- γ -N-octyl-C11) as shown below, GTPgN-octyl-C3 (GTP- γ -N-octyl-C3), GTPgO-hexyl-C2 (GTP- γ -O-hexyl-C2), GTPgO-hexyl-C3 (GTP- γ -O-hexyl-C3) or GTP-gN-octyl-thiosuccinimidyl-C2 (GTP- γ -N-octyl-thiosuccinimidyl-C2).

In a particularly preferred embodiment, the GTP analog labeled with a fluorescence donor compound is GTP-gN-octyl-thiosuccinimidyl-C2.

Fluorescent acceptor compounds

The fluorescent acceptor compound may be selected from the group consisting of: allophycocyanin, in particular allophycocyanin known under the trade name XL 665; luminescent organic molecules, such as rhodamine, anthocyanins (e.g. Cy5), squaraines, coumarins, proflavins, acridines, fluorescein, boron-dipyrromethene derivatives (sold under the name "Bodipy"), fluorophores named "Atto", fluorophores named "DY", compounds named "Alexa", nitrobenzoxadiazoles. Advantageously, the fluorescent acceptor compound is selected from the group consisting of allophycocyanins, rhodamines, anthocyanins, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, nitrobenzoxadiazoles.

The expressions "anthocyanidin" and "rhodamine" are understood as "anthocyanidin derivatives" and "rhodamine derivatives", respectively. Those skilled in the art are familiar with these various commercially available fluorophores.

The "Alexa" compound is sold by Invitrogen corporation; "Atto" compounds are sold by Atotec corporation; "DY" compounds are sold by Dyomics; "Cy" compounds are sold by Amersham Biosciences; other compounds are sold by various chemical reagent suppliers (e.g., Sigma Aldrich or Acros).

The following fluorescent proteins can also be used as fluorescent acceptor compounds: cyan fluorescent protein (AmCyan1, Midori-Ishi Cyan, mTFP1), Green fluorescent protein (EGFP, AcGFP, TurboGFP, Emerald, Azami Green, ZsGreen), yellow fluorescent protein (EYFP, Topaz, Venus, mCitrine, YPet, PhiYFP, ZsYellow1, mBanna), Orange and red fluorescent protein (Orange kussibir, mOrange, tdtomato, DsRed2, DsRed-Express, DsRed-Monomer, mTangerine, AsRed2, mRFP1, JRed, mCherry, mRed 1, HcRed-Tandem, mPelim 143), far infrared fluorescent protein (KamSho 2, KamKa 2).

Advantageously, for the ligand of the α G protein, the fluorescent acceptor compound is a FRET partner selected from the following compounds: allophycocyanin, rhodamine, anthocyanins, squaraines, coumarins, proflavins, acridines, fluorescein, boron-dipyrromethene derivatives, nitrobenzoxadiazoles and quantum dots; GFP, GFP variants selected from GFP10, GFP2 and eGFP; YFP, a YFP variant selected from the group consisting of eYFP, YFP topaz, YFP citrine, YFP venus, and YPet; mOrange; DsRed.

Advantageously, for labeled GTP analogs, the fluorescent acceptor compound is a FRET partner selected from the group consisting of: rhodamine, anthocyanins, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, and nitrobenzoxadiazoles.

The non-hydrolyzable or slowly hydrolyzable GTP labeled with a fluorescent acceptor compound that can be used in the FRET method of the present invention is represented by the following general formulae (3) and (4a, 4b, 4 c):

labeling of GTP analogs can be performed at different positions of GTP:

in the gamma position (O, NH, CH) of the phosphate₂) (ii) a Or

-at the 2 'and 3' positions of the ribose.

In a specific embodiment, the non-hydrolyzable or slowly hydrolyzable GTP labeled fluorescent acceptor compounds useful in the methods of the present invention may be selected from GTPgO-linker-Cy 5(P) (GTP-gO-hexyl-Cy 5 disSO 3-) (Jena Bioscience-NU-834-CY5), GTPgS-linker-Cy 5(R) (GTP-gS-EDA-Cy5) (Jena Bioscience-NU-1610-CY5), GTPgN-octyl-AF (GTP-gN-octyl-AF 488) (Cisbio Bioassays), GTPgN-L18-fluorescein (GTP-gN-EDA-pentyl-fluorescein) (Cisbio bioays) and GTPgN-octyl-CY 5 (GTP-gN-octyl-5) (Cisbio biosystems) represented by the following formula:

Labelling for BRET

In particular embodiments, the ligand is labeled with a member of a BRET partner pair, i.e., an energy donating luminescent compound or an energy accepting fluorescent compound.

Direct labeling of ligands with luminescent donor compounds or fluorescent acceptor compounds of the proteinaceous type (members of the BRET partner pair) can be carried out by the conventional Methods known to the person skilled in the art and described in particular in the articles by Tarik Issad and Ralf couplers (Bioluminescence response Energy Transfer to Monitor Protein-Protein Interactions, Transmembrane signalling Protocols pp 195. 209, Methods in part of the MIMB Molecular biology series, Vol. 332), to which the person skilled in the art refers.

Direct labeling of the ligand or non-hydrolyzable or slowly hydrolyzable GTP with an organic molecular fluorescent acceptor compound (member of a BRET partner pair) can be carried out by conventional methods known to those skilled in the art based on the presence of a reactive group on the ligand as described above.

The reactive group may form a covalent bond with a reactive group carried by a member of the BRET partner pair. Suitable reactive groups carried by members of a BRET partner pair are familiar to those skilled in the art, e.g. an acceptor compound functionalized with a maleimide group will be able to, for example, covalently bind to a thiol group carried by a cysteine carried by a protein or peptide (e.g. an antibody or antibody fragment). Similarly, an acceptor compound carrying an N-hydroxysuccinimide ester will be able to covalently bind to an amine present in a protein or peptide.

It is within the ability of those skilled in the art to select BRET partner pairs for obtaining BRET signals. For example, donor-acceptor pairs useful for studying BRET phenomenon are specifically described in Dasiel O.Borroto-Eschela (BIOLUMINESCENCE RESONANCE ENERGY TRANSFER (BRET) METHOD TO STRUDY G PROTEIN-COUPLED RECEPTOR-RECEPTOR TYRINE KINASE HETERORECEPTOR COMPLEXES, METHODS Cell biol.2013; 117: 141-164), TO which reference may be made by a person skilled in the art.

Luminescent donor compounds

In a specific embodiment, the luminescent donor compound is a BRET partner selected from: luciferase (luc), Renilla Luciferase (Rluc), Renilla Luciferase variant (Rluc8), and firefly Luciferase.

Fluorescent acceptor compounds

In a specific embodiment, the fluorescence acceptor compound is a BRET partner selected from the group consisting of: allophycocyanin, rhodamine, anthocyanin, squaraine, coumarin, proflavine, acridine, fluorescein, boron-dipyrromethene derivative, nitrobenzoxadiazole and quantum dots; GFP, GFP variants (GFP10, GFP2, eGFP); YFP, YFP variant (eYFP, YFP topaz, YFP citrine, YFP venus, YPet); mOrange; DsRed.

Examples

Material

Cell membrane preparations expressing the receptor under study and the α i G protein were purchased from Perkin Elmer or Euroscreen. The following table lists the reference numbers and basal cells (base cells) of the various samples used:

[ Table 1]

	Basal cell	Suppliers of goods	Reference numerals
				Delta opiates	HEK293	Perkin Elmer	6110549400UA
Delta opiates	CHO-K1	Euroscreen	Service
				Dopamine D2S	CHO-K1	Euroscreen	Service

the-DSV 36S and DSV38S antibodies were produced by Cisbio Bioassays and may be obtained from Cisbio Bioassays on demand (under respective reference numbers DSV36S and DSV 38S). The DSV36S antibody comprises a heavy chain variable domain consisting of the amino acid sequence SEQ ID NO. 14 and a light chain variable domain consisting of the amino acid sequence SEQ ID NO. 15. The antibody is labeled with a fluorescent probe (acceptor red-d 2 or donor Lumi4Tb) compatible with TR-FRET detection. Two antibodies, DSV36S and DSV38S, bind at the level of the switch II domain of the α i G protein.

Nucleotides GTP, GDP and GTP γ S were purchased from Sigma Aldrich (catalog references G8877, G7127 and G8634, respectively).

GPCR delta opioid agonists (SNC162) and dopamine D2S agonists (PPHT) and GPCR delta opioid antagonists (Naltrindole) were purchased from Tocris (catalog references 1529 and 0740, respectively).

Low volume 384 well plates, white background, purchased from Greiner Bio One (catalog reference 784075).

Non-hydrolyzable/slowly hydrolyzable analogs of GTP labeled with donor or acceptor fluorophores (GTPgN-C2; GTPgN-C3; GTPgN-octyl-C2; GTPgN-octyl-C11; GTPgN-octyl-C3; GTPgO-hexyl-C2; GTPgO-hexyl-C3; GTP-gN-octyl-thiosuccinimidyl-C2; GTPgN-octyl-Cy 5; GTPgN-octyl-AF 488) are synthesized in Cisbio Bioassays.

Non-hydrolyzable/slowly hydrolyzable analogues of GTP labeled with acceptor fluorophores GTPgO-linker-Cy 5(P) and GTPgS-linker-Cy 5(R) are purchased from Jena Bioscience under the respective reference numbers NU-834-CY5 and NU-1610-CY 5.

Method

Preparation of reagents

All reagents were diluted in TrisHCl buffer 50mM pH7.4, MgCl₂10mM, BSA 0.1%, NaCl 10mM or 100mM or 300mM or 500mM (concentrations are indicated in the legend for each graph), 0. mu.M or 0.5. mu.M or 1. mu.M GDP (concentrations are indicated in the legend for each graph). Membranes were prepared 4 x for distribution at 1 μ g/well or 10 μ g/well (amounts are specified in the legend for each figure). The nucleotide GTPgS (non-specific signal conditions) was prepared as 6.67 ×, to obtain a final concentration in the wells of 100 μ M. Test compounds (agonists or antagonists) were prepared as 10 ×, to obtain the values mentioned in the figureAnd the final concentration in the well. anti-G- α i antibody for detection was prepared as 4 ×, for final concentrations in wells as follows: antibody DSV36S-d2(10 nM); antibody DSV36S-Lumi4Tb (0.5nM or 1 nM); antibody DSV38S-d2(10 nM). Non-hydrolyzable/slowly hydrolyzable GTP analogs labeled with donor or acceptor fluorescent probes were prepared 4 x for the final concentration in the wells mentioned in the legend for each graph.

Distribution of reagents in 384-well plates

Membranes expressing GPCRs and G proteins: 5 μ L

Buffer or GTPgS nucleotides (for non-specific signal conditions): 3 μ L

Non-hydrolyzable/slowly hydrolyzable GTP analog-donor or acceptor: 5 μ L

anti-G- α i-donor or acceptor ligand: 5 μ L

Buffer or test compound (agonist and/or antagonist): 2 μ L.

Nonspecific signal (fluorescence background noise) was measured using wells containing excess GTPgS (100 μ M).

Reading HTRF signals

The plates were incubated at 21 ℃ for 20h (unless otherwise indicated in the figure), and then HTRF signals were measured on a pheasar reader (BMG Labtech) using the following configuration:

a module: HTRF (excitation 337 nm; emission 665nm and 620nm)

Excitation: laser, 40 flashes; or lamp, flash 100 times

Read window: time: 60 μ s-integration: 400 μ s.

Signal processing

From the raw signals at 665nm (for red acceptor-Cy 5) or 520nm (for green acceptor-AF 488 or fluorescein) and 620nm, the HTRF ratio was calculated by the following formula: HTRF ratio ═ signal at 665nm or signal at 520 nm/signal at 620nm 10,000.

Test format

Figure 2A shows the principle of an assay using a non-hydrolyzable/slowly hydrolyzable GTP analog labeled with a RET donor partner and an anti-G protein ligand labeled with a RET acceptor partner, where activation of the GPCR with an agonist compound induces a decrease in binding of the donor GTP analog to the G protein and thus a decrease in RET signal (format 1A).

Figure 2B shows the assay principle using a non-hydrolyzable/slowly hydrolyzable GTP analog labeled with a RET acceptor partner and an anti-G protein ligand labeled with a RET donor partner, where activation of the GPCR with an agonist compound induces a decrease in the binding of the acceptor GTP analog to the G protein and thus a decrease in the RET signal (format 1B).

Figure 2C shows the assay principle using a non-hydrolyzable/slowly hydrolyzable GTP analog labeled with a RET donor partner and an anti-G protein ligand labeled with a RET acceptor partner, where activation of the GPCR with an agonist compound induces an increase in binding of the donor GTP analog to the G protein and thus an increase in RET signal (format 2A).

Figure 2D shows the assay principle using a non-hydrolyzable/slowly hydrolyzable GTP analog labeled with a RET acceptor partner and an anti-G protein ligand labeled with a RET donor partner, where activation of the GPCR with an agonist compound induces an increase in the binding of the acceptor GTP analog to the G protein and thus an increase in the RET signal (format 2B).

Example 1 to example 7: activation assay for delta opiate gpcr (dor) according to format 1A: reduction of TR-FRET signal between GTP-donor and anti-G-alphai protein antibody acceptor under agonist stimulation

First, HEK293 or CHO-K1 cell membrane preparations expressing delta opiate GPCRs and G α i proteins were used to test the ability of the GTP-donor/anti-G- α i antibody acceptor pair to generate specific TR-FRET signals when bound to G proteins. The following experimental conditions were used:

example 1/fig. 3A: GTPgN-octyl-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 10mM；BSA0.1％。

Example 2/figure 4A: GTPgN-octyl-C11 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 10mM；BSA 0.1％。

Example 3/figure 5A: GTPgO-hexyl-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 10mM；BSA 0.1％。

Example 4/figure 6A: GTPgN-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 10mM；BSA 0.1％。

Example 5/figure 7A: GTPgN-C3 (final 6nM in well); DSV36S-d2 (final 10nM in well); 1 μ g HEK-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂10mM；NaCl 10mM；BSA 0.1％。

Example 6/figure 8: GTPgN-octyl-C3 (final 6nM in well); DSV36S-d2 (final 10nM in well); 1 μ g HEK-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl ₂ 10mM；NaCl 10mM；BSA 0.1％。

Example 7/figure 9: GTPgO-hexyl-C3 (final 6nM in well); DSV36S-d2 (final 10nM in well); 1 μ g HEK-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 10mM；BSA 0.1％。

The membranes were incubated in the absence or presence of a large excess of GTPgS (100 μ M). The observed difference in TR-FRET signals (HTRF ratio) between these two conditions indicates that the analogs GTPgN-octyl-C2, GTPgN-octyl-C11, GTPgO-hexyl-C2, GTPgN-C2, GTPgN-C3, GTPgN-octyl-C3, GTPgO-hexyl-C3 are able to bind to the α i G protein and produce TR-FRET signals with anti-G- α i antibody acceptors (fig. 3A to fig. 9).

Second, GPCR agonists were tested for their ability to modulate the proportion of α G protein bound to the GTP-donor using the same membranes and experimental conditions described above. A decrease in the TR-FRET signal (HTRF ratio) produced by agonist stimulation indicates a decrease in the proportion of α G protein forms bound to the GTP-donor (i.e., an increase in empty α G protein forms). Thus, GPCR receptors activated by their agonists cause the GTP-donor to leave the G protein, which then becomes empty and causes a decrease in the TR-FRET signal. These results are shown in fig. 3B (example 1), fig. 4B (example 2), fig. 5B (example 3), fig. 6B (example 4), and fig. 7B (example 5). This modulation of signal by agonists was not tested for the analogues GTPgN-octyl-C3 (example 6) and GTPgO-hexyl-C3 (example 7).

Example 8: effect of membrane and GTP-donor concentrations on activation assays performed on delta opiate gpcrs (dor) according to format 1A: reduction of TR-FRET signal between GTP-donor and anti-G-alphai protein antibody acceptor under agonist stimulation

First, CHO-K1 cell membrane preparations (1 μ G/well or 10 μ G/well) expressing delta opiate GPCRs and G α i proteins were used to examine the ability of GTPgN-octyl-C2/anti-G- α i antibody DSV36S-d2 to generate specific TR-FRET signals upon binding to G proteins. GTPgN-octyl-C2 was used at a final 2nM or 6nM in the wells. The membranes were incubated in the absence or presence of a large excess of GTPgS (100 μ M). The difference in TR-FRET signals (HTRF ratio) observed between these two conditions indicates that the GTPgN-octyl-C2 analog is able to bind to α i G protein and generate TR-FRET signals with anti-G- α i antibody DSV36S-d2 (fig. 10A). Furthermore, the left panel shows the increase in signal amplitude (S/B ═ total signal/non-specific signal) as the amount of membrane increases from 1 μ g to 10 μ g per well. The right panel shows the increase in signal amplitude (S/B) as the concentration of GTPgN-octyl-C2 was increased from 2nM to 6 nM.

Second, GPCR agonists were tested for their ability to modulate the proportion of α G protein bound to the GTP-donor using the same membranes and experimental conditions described above. A decrease in the TR-FRET signal (HTRF ratio) produced by agonist stimulation indicates a decrease in the proportion of α G protein forms bound to the GTP-donor (i.e., an increase in empty α G protein forms). Thus, GPCR receptors activated by their agonists cause the GTP-donor to leave the G protein, which then becomes empty and causes a decrease in the TR-FRET signal. These results are shown in fig. 10B. In addition, the left panel shows the increase in signal amplitude (S/B-signal of medium without agonist/agonist signal) as the amount of membrane increases from 1 μ g to 10 μ g per well. The right panel shows the increase in signal amplitude (S/B) as the concentration of GTPgN-octyl-C2 was increased from 2nM to 6 nM.

Example 9 and example 10: activation assay for GPCR dopamine D2S (D2S) according to format 1A: reduction of TR-FRET signal between GTP-donor and anti-G-alphai protein antibody acceptor under agonist stimulation

First, CHO-K1 cell membrane preparations expressing the GPCR dopamine D2S and α i G proteins were used to test the ability of the GTP-donor/anti-G- α i antibody acceptor pair to generate a specific TR-FRET signal when bound to G proteins. The following experimental conditions were used:

example 9/fig. 11A: GTPgN-octyl-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-D2S membranes/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 10mM；BSA 0.1％。

Example 10/fig. 12A: GTPgN-octyl-C11 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-D2S membranes/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 10mM；BSA 0.1％。

The membranes were incubated in the absence or presence of a large excess of GTPgS (100 μ M). The observed difference in TR-FRET signals (HTRF ratio) between the two conditions indicates that the analogs GTPgN-octyl-C2 and GTPgN-octyl-C11 are able to bind to α i G protein and generate TR-FRET signals with anti-G- α i antibody acceptors (fig. 11A to fig. 12A).

Example 11 to example 13: activation assay for delta opiate gpcr (dor) according to format 1B: reduction of TR-FRET signal between GTP-acceptor and anti-G-alphai protein antibody donor under agonist stimulation

First, HEK293 cell membrane preparations expressing delta opiate GPCRs and G α i proteins were used to test the ability of GTP-acceptor/anti-G- α i antibody donor pairs to generate specific TR-FRET signals when bound to G proteins. The following experimental conditions were used:

example 11/fig. 13A: GTPgO-linker-Cy 5(P) (final 250nM in well); DSV36S-Lumi4Tb (final 0.25nM in well); 1 μ g HEK-DOR membrane/well; buffer solution: TrisHCl50mM pH7.4; MgCl₂10 mM; 10mM NaCl; BSA 0.1%. Read after incubation for 3h at 21 ℃.

Example 12/fig. 14A: GTPgS-linker-Cy 5(R) (final 250nM in well); DSV36S-Lumi4Tb (final 0.25nM in well); 10 μ g HEK-DOR membrane/well; buffer solution: TrisHCl50mM pH7.4; MgCl₂10 mM; 10mM NaCl; BSA 0.1%. Read after incubation at 21 ℃ for 1 h.

Example 13/fig. 15A: GTPgN-L18-fluorescein (final 31nM in well); DSV36S-Lumi4Tb (final 0.25nM in well); 1 μ g HEK-DOR membrane/well; buffer solution: TrisHCl50mM pH7.4; MgCl₂ 10mM；NaCl 10mM；BSA 0.1％。

The membranes were incubated in the absence or presence of a large excess of GTPgS (100 μ M). The observed difference in TR-FRET signals (HTRF ratio) between these two conditions indicates that the analogs GTPgO-linker-Cy 5(P), GTPgS-linker-Cy 5(R) and GTPgN-L18-fluorescein are able to bind to α i G protein and generate TR-FRET signals with the anti-G- α i antibody donor (fig. 13A to fig. 15A).

Second, GPCR agonists were tested for their ability to modulate the proportion of α G protein bound to the GTP-acceptor using the same membranes and experimental conditions described above. A decrease in the TR-FRET signal (HTRF ratio) produced by agonist stimulation indicates a decrease in the proportion of α G protein forms bound to the GTP-acceptor (i.e., an increase in empty α G protein forms). Thus, GPCR receptors activated by their agonists cause the GTP-acceptor to leave the G protein, which then becomes empty and causes a decrease in the TR-FRET signal. These results are shown in fig. 13B (example 11), fig. 14B (example 12), and fig. 15B (example 13). For the analog GTPgS-linker-Cy 5(R) (example 12), this modulation of signal by the agonist was very slight.

Example 14 to example 19: activation assay for delta opiate gpcr (dor) according to format 2A: increase in TR-FRET signal between GTP-donor and anti-G-alphai protein antibody acceptor under agonist stimulation

First, CHO-K1 cell membrane preparations expressing delta opiate GPCRs and G α i proteins were used to test the ability of the GTP-donor/anti-G α i antibody acceptor pair to generate a specific TR-FRET signal when bound to G proteins. The following experimental conditions were used:

example 14/fig. 16A: GTPgN-octyl-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl ₂ 10mM；NaCl 500mM；BSA 0.1％。

Example 15/figure 17A: GTPgN-octyl-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 300mM；GDP 0.5μM；BSA 0.1％。

Example 16/fig. 18A: GTPgN-octyl-C2 (final 6nM in well); DSV38S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 300mM；GDP 0.5μM；BSA0.1％。

Example 17/fig. 19A: GTPgN-octyl-C11 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 300mM；GDP 0.5μM；BSA0.1％。

Example 18/fig. 20A: GTPgO-hexyl-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 300mM；GDP 0.5μM；BSA 0.1％。

Example 19/fig. 21A: GTPgN-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 300mM；GDP 0.5μM；BSA0.1％。

The membranes were incubated in the absence or presence of a large excess of GTPgS (100 μ M). The observed difference in TR-FRET signals (HTRF ratio) between these two conditions indicates that the analogs GTPgN-octyl-C2, GTPgN-octyl-C11, GTPgO-hexyl-C2, GTPgN-C2 are able to bind to α i G protein and produce TR-FRET signals with anti-G- α i antibody acceptors (fig. 16A to fig. 21A).

Second, GPCR agonists were tested for their ability to modulate the proportion of α G protein bound to the GTP-donor using the same membranes and experimental conditions described above. An increase in the TR-FRET signal (HTRF ratio) generated by agonist stimulation indicates an increase in the proportion of α G protein forms bound to the GTP-donor (i.e., a decrease in empty α G protein forms). Thus, GPCR receptors activated by their agonists cause GTP-donor binding to G proteins which then become GTP-donor form and cause an increase in TR-FRET signal. These results are shown in fig. 16B (example 14), fig. 17B (example 15), fig. 18B (example 16), fig. 19B (example 17), fig. 20B (example 18), and fig. 21B (example 19). Furthermore, figure 17B (example 15) shows a second condition in which activation by a fixed concentration of the GPCR agonist SNC162(200nM) is inhibited by an increased concentration of the GPCR antagonist (naltrexone). This inhibition of activation is observed from the decrease in the TR-FRET signal (HTRF ratio).

Example 20 to example 22: activation assay for GPCR dopamine D2S (D2S) according to format 2A: increase in TR-FRET signal between GTP-donor and anti-G-alphai protein antibody acceptor under agonist stimulation

Example 20/figure 22A: GTPgN-octyl-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-D2S membranes/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 10mM；GDP 1μM；BSA 0.1％。

Example 21/fig. 23A: GTPgN-octyl-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-D2S membranes/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 100mM；BSA 0.1％。

Example 22/fig. 24A: GTPgN-octyl-C2 (final 6nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-D2S membranes/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂ 10mM；NaCl 100mM；GDP 1μM；BSA 0.1％。

The membranes were incubated in the absence or presence of a large excess of GTPgS (100 μ M). The difference in TR-FRET signals (HTRF ratio) observed between these two conditions indicates that the analog GTPgN-octyl-C2 is able to bind to α i G protein and generate TR-FRET signals with anti-G- α i antibody acceptors (fig. 22A to fig. 24A).

Example 23 and example 24: activation assay for delta opiate gpcr (dor) according to format 2B: increase in TR-FRET signal between GTP-acceptor and anti-G-alphai protein antibody donor under agonist stimulation

First, CHO-K1 cell membrane preparations expressing delta opiate GPCRs and G α i proteins were used to test the ability of the GTP-acceptor/anti-G- α i antibody donor pair to generate a specific TR-FRET signal when bound to G proteins. The following experimental conditions were used:

example 23/figure 25A: GTPgN-octyl-Cy 5 (final 50nM in well); DSV36S-Lumi4Tb (final 1nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂10 mM; NaCl 300 mM; GDP 0.5. mu.M; BSA 0.1%. Read after incubation for 3h at 21 ℃.

Example 24/fig. 26A: GTPgN-octyl-AF 488 (final 50nM in well); DSV36S-Lumi4Tb (final 1nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl₂10 mM; NaCl 300 mM; GDP 0.5. mu.M; BSA 0.1%. Read after incubation for 3h at 21 ℃.

The membranes were incubated in the absence or presence of a large excess of GTPgS (100 μ M). The observed difference in TR-FRET signals (HTRF ratio) between the two conditions indicates that the analogs GTPgN-octyl-Cy 5 and GTPgN-octyl-AF 488 are able to bind to the α i G protein and generate TR-FRET signals with the anti-G- α i antibody donor (fig. 25A to fig. 26A).

Second, GPCR agonists were tested for their ability to modulate the proportion of α G protein bound to the GTP-acceptor using the same membranes and experimental conditions described above. An increase in the TR-FRET signal (HTRF ratio) produced by agonist stimulation indicates an increase in the proportion of α G protein forms bound to the GTP-acceptor (i.e., a decrease in empty α G protein forms). Thus, GPCR receptors activated by their agonists cause GTP-acceptor binding to G proteins which then become GTP-acceptor form and cause an increase in TR-FRET signal. These results are shown in fig. 25B (example 23) and fig. 26B (example 24).

Example 25: activation assay for delta opiate gpcr (dor) according to format 2A: increase in TR-FRET signal between GTP-donor and anti-G-alphai protein antibody acceptor under agonist stimulation

example 25/fig. 27A: GTP-gN n-octyl-thiosuccinimidyl-C2 (final 7.5nM in well); DSV36S-d2 (final 10nM in well); 10 μ g CHO-DOR membrane/well; buffer solution: TrisHCl 50mM pH7.4; MgCl ₂60mM；NaCl 150mM；BSA 0.1％。

The membranes were incubated in the absence or presence of a large excess of GTPgS (100 μ M). The difference in TR-FRET signals (HTRF ratio) observed between these two conditions indicates that the analog GTP-gN-octyl-thiosuccinimidyl-C2 is capable of binding to α i G protein and generating TR-FRET signals with anti-G- α i antibody acceptors (fig. 27A).

Example 26: protocol for obtaining anti-G α i1 protein antibodies for use in the method according to the invention

Immunization of mice

TST-G.alpha.i 1 recombinant protein (alpha.i 1G protein of the sequence UniProt P63096-1, with the tag TwinStreptag (TST) at the N-terminus by means of a TEV linker (IBA)) was produced in Sf9 insect cells (infected with baculovirus encoding said protein) and then purified on an affinity column by means of the tag TwinStreptag (TST) (Strep-Tactin Superflow high capacity resin (IBA, Cat: 2-1208-) -002)).

It is pre-treated by injection in GTPgS-containing buffer (HEPES 20mM pH8, NaCl100mM, MgCl)₂BALB/c mice were immunized with TST-G.alpha.i 1 protein diluted in 3mM, CHAPS 11mM, GTPgS 100. mu.M). Three booster injections (three boost) were performed at one month intervals after the initial injection.

Blood punctures of mice 15 days after each injection allowed to verify the presence of immune responses.

For this purpose, an ELISA-type assay was established. The buffer solution (Tris HCl 20mM pH8.5, NaCl 140mM, EDTA 2mM, MgCl) containing GTPgS was preliminarily added₂10mM, BSA 0.1%, GTPgS 1. mu.M) diluted to 20. mu.g/mL of TST-G.alpha.i 1 protein was adsorbed by the tag TwinStreptagXT (IBA, Cat. No: 2-4101-001) in 96-well plates. For this, 100 μ L of protein was added to each well, followed by incubation at 37 ℃ for 2h, followed by 3 washes in 1 × PBS buffer, 0.05% Tween 20.

Then, serial dilutions of blood punctures with dilution factors of 10 to 100 million were added at the level of 100 μ L/well and incubated for 2h at 37 ℃. Antibodies not immobilized to the protein were removed by three washing steps in 1 × PBS buffer, 0.05% Tween20, and the immobilized antibodies were then detected using an anti-mouse Fc secondary antibody (Sigma # a0168, diluted to 1/10000 in PBS, BSA 0.1%) conjugated to HRP (horseradish peroxidase). After incubation for 1h at 37 ℃ and then 3 washes in 1 XPBS buffer, 0.05% Tween20, development of HRP was performed by colorimetric assay at 450nm after incubation of the HRP substrate TMB (3,3',5,5' -tetramethylbenzidine, Sigma # T0440) for 20min at room temperature with stirring.

To ensure that the antibodies detected by the ELISA assay were indeed directed against the α i 1G protein and not against the tag TwinStrepTag, the same punctures were tested by the ELISA assay after pre-incubation with an excess of another orthogonal protein (SNAPTag-TwinStrepTag) bearing the tag TwinStrepTag. Thus, the anti-tag antibody binds to the tagged orthogonal protein and therefore does not bind to the α i 1G protein attached to the bottom of the well; in this case, no HRP signal or a decrease in HRP signal was detected.

Mice with minimal signal reduction and with optimal antibody titers in the case of anti-tag controls were selected for lymphocyte hybridization (also referred to as fusion) in the next step. The spleens of the mice were recovered and a mixture of lymphocytes and plasma cells obtained from the spleens was fused in vitro with a myeloma cell line in the presence of a cell fusion catalyst of the polyethylene glycol type. Mutant myeloma cell lines lacking the HGPRT enzyme (hypoxanthine guanine phosphoribosyl transferase) are used to select the hybrid cells (called hybridomas). These cells are cultured in a medium containing hypoxanthine, aminopterin (methotrexate), and thymidine (HAT medium) to enable the removal of unfused myeloma cells, thereby selecting hybridomas of interest. Unfused splenocytes die because they cannot proliferate in vitro. Thus, only hybridomas survive.

These hybridomas are then cultured in a culture plate. Supernatants from these hybridomas were then tested to assess their ability to produce anti- α i 1G protein antibodies. For this purpose, an ELISA assay as described above was performed.

To evaluate the selectivity of the antibodies in the different forms of α i 1G protein (full form vs bound to GDP to full form vs empty form of GTPgS), assays were performed in parallel under conditions where TST-G α i1 protein was preincubated in buffer containing 1 μ M GDP or 1 μ M GTPgS or no nucleotides. The optimal hybridoma was then cloned using a limiting dilution procedure to obtain hybridoma clones.

Then, the hybridoma clone of interest was injected into mice (intraperitoneal injection) to allow the antibody to be produced in large amounts in ascites.

The antibody is then purified by affinity chromatography on a column with protein a resin.

The antibody purified as above competes with the DSV36S antibody for the ability to bind to the α G protein

All reagents were diluted in TrisHCl buffer 50mM pH7.4, MgCl₂10mM, BSA 0.1%, NaCl 10 mM. The G α i1 protein was prepared as 2 ×, to obtain a final concentration in the wells of 2.5 nM. Nucleotide GTPgS was prepared as 2 ×, to obtain a final concentration in wells of 10 μ M. Both reagents were prepared in the same solution and pre-incubated at room temperature for 30 minutes before being re-distributed into the wells. Antibodies purified as above were prepared 4 x to obtain a final concentration in wells between 0.01 μ M and 1 μ M. Antibody DSV36S-d2 was prepared 4X to achieve a final concentration of 10 nM. The anti-Twin-Strep-tag-Lumi 4Tb antibody was prepared 4 x to obtain a final concentration in the well of 0.5 nM.

Reagents were distributed in 384-well plates as follows:

1. add 10. mu.L of preincubation mixture of α i 1G protein + GTPgS per well;

2. add 5. mu.L of purified antibody per well;

3. incubate the plate at room temperature for 30 minutes;

4. each well was added 5. mu.L of a mixture of anti-Twin-Strep-tag-Lumi 4 Tb antibody and antibody DSV36S-d 2.

Plates were incubated at room temperature for 1 hour before reading HTRF signal.

The antibodies according to the invention are capable of inhibiting the HTRF signal obtained with antibody DSV36S-d 2. In contrast, antibodies not according to the invention were unable to inhibit the signal generated by DSV36S-d 2.

Sequence listing

[ Table 3]

Sequence listing

<110> CISBIO BIOASSAY

CNRS

<120> method for measuring modulation of activation of G protein-coupled receptor using GTP analog

<130> 1H305110 - 0016

<150> FR1900880

<151> 2019-01-30

<160> 16

<170> BiSSAP 1.3.6

<210> 1

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> peptide KB1753

<400> 1

Ser Ser Arg Gly Tyr Tyr His Gly Ile Trp Val Gly Glu Glu Gly Arg

1 5 10 15

Leu Ser Arg

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> VH-CDR1

<400> 2

Gly Phe Asn Ile Lys Asp Tyr Tyr

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> VH-CDR2

<400> 3

Ile Asp Pro Glu Asn Gly Asn Thr

1 5

<210> 4

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> VH-CDR3

<400> 4

Thr Arg Gly Gly Gly Tyr Tyr Ser Asp Trp Tyr Phe Asp Val

1 5 10

<210> 5

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> VL-CDR1

<400> 5

Ser Ser Val Ser Tyr

1 5

<210> 6

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> VL-CDR3

<400> 6

Gln Gln Trp Ser Ser Asn Pro Pro Ile Thr

1 5 10

<210> 7

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> VH-FR1

<400> 7

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Leu Val Lys Leu Ser Cys Lys Ala Ser

20 25

<210> 8

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> VH-FR2

<400> 8

Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly

1 5 10 15

Trp

<210> 9

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> VH-FR3

<400> 9

Ile Tyr Asp Pro Lys Phe Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr

1 5 10 15

Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

<210> 10

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> VH-FR4

<400> 10

Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 11

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> VL-FR1

<400> 11

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser

20 25

<210> 12

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> VL-FR2

<400> 12

Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

1 5 10 15

Tyr

<210> 13

<211> 36

<212> PRT

<213> Artificial sequence

<220>

<223> VL-FR3

<400> 13

Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala

20 25 30

Thr Tyr Tyr Cys

<210> 14

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> VL-FR4

<400> 14

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

1 5 10

<210> 15

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable Domain (VH)

<400> 15

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Leu Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Gly Gly Tyr Tyr Ser Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 16

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable Domain (VL)

<400> 16

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Ile

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

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