Fluorescent GTP Analogues and Uses

Abstract

Provision of fluorescent GTP analogs and uses. The present invention provides a compound of the formula: [C1] TIFF2022523322000079.tif40156 (where X, Y, L, and Ln 3+ is as defined in the specification.

Description

[Technical field]

[0001] The present invention relates to fluorescent GTP analogues that are useful for detecting molecules capable of altering the activation of G protein-coupled receptors by energy transfer techniques. [Background technology]

[0002] G protein-coupled receptors (GPCRs) are a family of membrane receptors found in mammals and throughout the animal kingdom. G proteins are heterotrimeric proteins (three subunits: α, β, and γ) that are activated by GPCRs. Through GPCRs, G proteins are responsible for transmitting signals from outside the cell to inside the cell (i.e., the cellular response to external stimuli). Their commonly described mechanism of action is shown in Figure 1 and outlined below. In the resting, inactive state, the α subunit of the G protein is bound to the nucleotide GDP (intact G protein bound to GDP). After activation of the GPCR, the GPCR binds to the α subunit of the G protein, initiating the G protein activation process, which consists of two steps: 1) GDP is released from the G protein, leaving an empty G protein, and an inactive GPCR / empty G protein complex is formed. 2) GTP binds to form an active G protein (a complete G protein bound to GTP). In the first phase, the receptor-bound G protein is in what is called an "empty" form. In the literature, this state is considered transient because the nucleotide GTP is said to bind immediately to the α subunit of the G protein. Also, the β / γ subunits of the activated G protein dissociate from the α subunit. The intact GTP-bound α subunit of the G protein then binds to and activates the effector, which then activates a signaling pathway that results in a cellular response. ·GTP is then hydrolyzed by the α subunit of the G protein to form GDP, which recombines with the β / γ subunits to reform the complete G protein bound to GDP (inactive state).

[0003] There are several Gα protein subtypes that display distinct selectivity profiles towards different effectors (Non-Patent Document 1), thereby inducing the activation of preferential signaling pathways.

[0004] GPCRs are involved in many important physiological functions and are considered one of the preferred therapeutic targets for many pathologies. Therefore, many in vitro screening tests have been developed to identify molecules that can alter GPCRs. The tests developed exploit different G protein activation mechanisms and employ various techniques (Non-Patent Document 2). In particular, affinity tests using radiolabeled ligands to measure the affinity of ligands for GPCRs, proximity scintigraphy tests using scintigraphy beads bound to GPCRs, or functionality tests using poorly or non-hydrolyzable GTP such as GTPγS (GTP-gamma-S). However, these tests are difficult to perform and may require a membrane filtration step, which may limit their use as high-throughput screening (HTS) tests. Other tests have also been developed to confirm GPCR activation. These tests are in particular based on energy transfer techniques (resonance energy transfer (RET)), such as fluorescence resonance energy transfer (FRET) (see Non-Patent Document 3) or bioluminescence resonance energy transfer (BRET) (Non-Patent Document 4). These two techniques involve the concept of a molecule that can give energy (called a donor) or accept energy (called an acceptor) (see Non-Patent Document 5). For example, there are energy transfer techniques that reveal the interaction between GPCRs and G proteins using a donor coupled to a GPCR and a receptor coupled to a G protein (Patent Document 1 and Patent Document 2), or a receptor coupled to the α subunit of a G protein and a donor coupled to the β and / or γ subunit of a G protein (Non-Patent Document 6). However, these techniques are limited because they require the preparation of fusion proteins and do not allow the study of GPCRs and G proteins that are endogenously expressed by cells (i.e., unmodified and non-overexpressed). On the other hand, to distinguish the various subtypes of Gα proteins activated by the receptor, these techniques require the preparation of multiple membrane samples (specific preparations for each subtype of Gα protein).Energy transfer techniques have also been used to develop tests aimed at visualizing changes in the GTP (active) form of G proteins or the GDP (inactive) form of G proteins, see, for example, US Pat. No. 5,233,633 and US Pat. No. 5,233,633, which employ GTP analogs linked to cyanine-type molecules.

[0005] Patent Document 5 describes a method for detecting energy transfer signals using a labeled ATP derivative. However, the derivative cannot bind to G proteins. Non-Patent Document 7 describes that a GTP analog can be used in time-resolved fluorescence detection methods. The analog is composed of a europium chelate bound to the phosphate atom at the gamma position of GTP via a nitrogen atom. However, the structure of the europium chelate is not disclosed, nor is the method used to synthesize the analog. (2003) describes another GTP analogue obtained by coupling gamma-[(8-aminooctyl)imido]guanosine-5'-triphosphate with {2,2',2'',2'''-{[[2-(4-isothiocyanatophenyl)ethyl]imino]bis(methylene)bis{4-{[4-(RD-glucopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III), the synthesis of which is described in (2003)).

[0006] Thus, there is a real need to have available compounds capable of binding to G proteins and which can be practically used in methods for detecting molecules capable of altering the activation of G protein-coupled receptors by energy transfer techniques. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2006 / 086883 [Patent Document 2] International Publication No. 2003 / 008435 [Patent Document 3] International Publication No. 2006 / 035208 [Patent Document 4] Specification of US Patent Application Publication No. 2007 / 0287162 [Patent Document 5] International Publication No. 2009 / 068751 [Non-Patent Document]

[0008] [Non-Patent Document 1] Journal of Molecular Biology, 2016, 428, 3850 [Non-Patent Document 2] Zhang et al.; Tools for GPCR Drug Discovery; Acta Pharmacologica Sinica, 2012, 33, 372 [Non-Patent Document 3] Clinical Chemistry, 1995, 41, 1391 [Non-Patent Document 4] Proceedings of the National Academy of Sciences, 1999, 96(1), 151 [Non-Patent Document 5] Physical Chemistry Chemical Physics, 2007, 9, 5847 [Non-Patent Document 6] Bunemann et al., Proceedings of the National Academy of Sciences, 2003, 26, 16077 [Non-Patent Document 7] Drug Discovery Today, 2002, 7(18), S150 [Non-Patent Document 8] Analytical Chemistry, 2009, 81, 5033 [Non-Patent Document 9] Analytical Chemistry, 2003, 75, 3193 [Summary of the Invention] [Means for solving the problem]

[0009] The present invention relates to a compound of general formula (I): [ka] (In the formula, X is O, NH, or CH2; Y is O, NH, or CH2; L is a divalent linking group, Ln 3+ is a lanthanide complex which may carry a reactive group G3 (defined below), When X is NH and LY is an octylamino group, Ln 3+ is understood not to be {2,2',2'',2'''-{[[2-(4-isothiocyanatophenyl)ethyl]imino]bis(methylene)bis{4-{[4-(RD-glucopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III). [Brief description of the drawings]

[0010] [Figure 1] Represents the mechanism of action of G proteins. [Diagram 2] 1 depicts an assay format used to detect binding of the compounds of the invention to G proteins. [Diagram 3] 1 shows a test for binding the compounds of the present invention to Gαi proteins. [Figure 4] 1 shows a test for binding the compounds of the present invention to Gαi proteins. [Diagram 5] 1 shows a test for binding the compounds of the present invention to Gαi proteins. [Figure 6] 1 shows a test for binding the compounds of the present invention to Gαi proteins. [Figure 7] 1 shows a test for binding the compounds of the present invention to Gαi proteins. [Figure 8] 1 shows a test for binding the compounds of the present invention to Gαi proteins. [Figure 9] 1 shows a test for binding the compounds of the present invention to Gαi proteins. [Figure 10] 1 shows a test for binding the compounds of the present invention to Gαi proteins. [Figure 11] The effect of membranes and test compound concentration on the binding of the compound to Gαi protein is shown. [Figure 12] 1 shows a test for binding the compounds of the present invention to Gαi proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] According to one aspect, the present invention provides a compound of formula (I): [ka] (In the formula, X is O, NH, or CH2; Y is O, NH, or CH2; L is a divalent linking group, Ln 3+ is a lanthanide complex which may carry a reactive group G3 (defined below), However, when X is NH and LY is an octylamino group, Ln 3+ relates to a compound represented by the formula {2,2',2'',2'''-{[[2-(4-isothiocyanatophenyl)ethyl]imino]bis(methylene)bis{4-{[4-(RD-glucopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III)}.

[0012] The term "lanthanide complex" is understood to mean a chelate, macrocycle, cryptate or any organic species capable of complexing an atom of the lanthanide family, the lanthanide (Ln) being selected from Eu, Sm, Tb, Gd, Dy, Nd or Er. The lanthanide is preferably Tb, Sm or Eu, more preferably Eu or Tb.

[0013] The various families of compounds of formula (I) include those of formulas (Ia) to (Ig): [ka] (Wherein, L and Ln 3+ has the meaning given above).

[0014] The first family of compounds according to the invention is made up of compounds of formula (Ia), (Id) and (If). This family is called the GTP-gamma-O family (because the divalent linking group is attached to the phosphate at the gamma position of GTP via an oxygen atom). The second family of compounds according to the invention is made up of compounds of formula (Ib), (Ie) and (Ig). This family is called the GTP-gamma-N family (because the divalent linking group is attached to the phosphate at the gamma position of GTP via a nitrogen atom). The third family of compounds according to the invention is made up of compounds of formula (Ic). This family is called the GTP-gamma-C family (because the divalent linking group is attached to the phosphate at the gamma position of GTP via a carbon atom).

[0015] In one embodiment, X is O. In another embodiment, X is NH. In another embodiment, X is CH2.

[0016] In one embodiment, the divalent linking group L is ·Direct binding; a linear or branched alkylene group having 1 to 20 carbon atoms, preferably 1 to 8 carbon atoms, which may contain one or more double or triple bonds; a cycloalkylene group having 5 to 8 carbon atoms; or Arylene group with 6 to 14 carbon atoms is selected from The alkylene group, cycloalkylene group, or arylene group may contain one or more heteroatoms such as oxygen, nitrogen, sulfur, or phosphorus, or one or more carbamoyl groups or carboxamide groups, and the alkylene group, cycloalkylene group, or arylene group may be substituted with 1 to 5, and preferably 1 to 3, alkyl groups having 1 to 8 carbon atoms, aryl groups having 6 to 14 carbon atoms, sulfonate groups, or oxo groups.

[0017] The divalent linking group L is the following group: [ka] (wherein n, m, p and q are integers ranging from 1 to 16, preferably from 1 to 5, and e is an integer ranging from 1 to 6, preferably from 1 to 4).

[0018] In a particularly advantageous manner, the divalent linking group L is a direct bond, a linear or branched alkylene group having 1 to 8 carbon atoms, or a group of the formula: [ka] is selected from the group:

[0019] The divalent linking group L is: [ka] is preferably selected from -(CH2) n The - group is particularly preferred.

[0020] According to another embodiment, the divalent linking group L has the formula: [ka] (wherein m, n, and p are integers of 1 to 16, preferably 1 to 5).

[0021] In one embodiment, the lanthanide complex Ln 3+ is selected from one of the following complexes: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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[0022] Depending on the pH, the -SO3H, -CO2H, and -PO(OH)2 groups are in deprotonated or non-deprotonated form. - group, -CO2 - group, and -PO(OH)O - It is also represented as a group.

[0023] Lanthanide complexes Ln 3+ is advantageously selected from one of the complexes C1 to C17, C24 to C32 and C36 to C44. 3+ More advantageously, the lanthanide complex Ln is selected from one of the complexes C1 to C17 and C36 to C44. 3+ is further advantageously selected from one of the complexes C1 to C17. 3+ is further advantageously selected from one of the complexes C1 to C4 and C11 to C17. 3+ is further advantageously selected from one of the complexes C1 to C4 and C11. 3+ is very advantageously complex C2 or complex C3.

[0024] The lanthanide complexes C1 to C90 are described in the following publications: These complexes are either commercially available or can be obtained by the synthetic routes described in the following publications: C1: International Publication No. 03 / 076938 C2: European Patent Application Publication No. 0 203 047 C3-C4: International Publication No. 2008 / 025886 C5:Spectrochimica Acta;Part A,Molecular and Biomolecular Spectroscopy,2001,57(11),2197. C6:Spectrochimica Acta;Part A,Molecular and Biomolecular Spectroscopy,2001,57(11),2197. C7:Analytical Biochemistry,2000,286(1),17. C8: International Publication No. 01 / 96877 C9:Spectrochimica Acta;Part A,Molecular and Biomolecular Spectroscopy,2001,57(11),2197. C10: International Publication No. 01 / 96877 C11: International Publication No. 2008 / 063721 C12-C17: International Publication No. 2017 / 098180 C18~C20: International Publication No. 2013 / 011236 C21-C35: International Publication No. 2014 / 111661 C36~C44: International Publication No. 2018 / 229408 C45~C47:Organic&Biomolecular Chemistry,2012,10,8509. C48: International Publication No. 2010 / 084090 C49: International Publication No. 2009 / 010580 C50~C51:Chemical Communications,2007,3841 C52: International Publication No. 2006 / 120444 C53:European Journal of Inorganic Chemistry,2010,3961. C54:Bioconjugate Chemistry,2009,20(3),625. C55: European Patent Application Publication No. 0 770 610 C56: International Publication No. 2016 / 106241 C57-C58: European Patent Application Publication No. 0 770 610 C59~C60:Analytical Chemistry,2003,75,3193-3201 C61:Bioconjugate Chemistry,1997,8(2),127 C62: International Publication No. 93 / 11433 C63: U.S. Patent Application Publication No. 5,696,240 C64a~C81b: International Publication No. 2018 / 22932 C82: International Publication No. 2010 / 070232 C83~C85: International Publication No. 2018 / 045385 C86a~b:Analyst,2010,135(1),42 C87~C89:Chemistry-A European Journal,2011,17,9164 C90:Journal of the American Chemical Society,2004,126(15),4888

[0025] The following schemes 1-19 provide a more detailed description of the synthesis of compounds of formula (I). These compounds are typically obtained by a methodology based on the use of reactive groups to conjugate two organic molecules. Said methods are within the general knowledge of the person skilled in the art and are described, for example, in Bioconjugate Techniques, GT Hermanson, Academic Press, Second Edition, 2008, pp. 169-211. To obtain the GTP-gamma-O compound, GTP is first reacted with a compound of formula G2-L-G1, and the intermediate compound thus formed is conjugated with a lanthanide complex. In the above formula G2-L-G1, L is a divalent linking group as defined above; G1 is a reactive electrophilic group capable of reacting with the OH functional group of the phosphate at the gamma position of GTP; G2 is the lanthanide complex Ln 3+ is a reactive group capable of reacting with the reactive group (G3) possessed by

[0026] The conjugation reaction between the intermediate compound (bearing reactive group G2) and the lanthanide complex (bearing reactive group G3) results in the formation of a covalent bond involving one or more atoms of the reactive group.

[0027] In one embodiment, the electrophilic group G1 is an OH group activated by a reactant which forms as an intermediate a reactive species capable of reacting with the hydroxyl function of the phosphoric acid in the gamma position, or Leaving group type reactive groups, such as aliphatic chlorides, aliphatic bromides, aliphatic iodides, aliphatic mesylates, aliphatic tosylates, and aliphatic triflates.

[0028] In one embodiment, the reactive groups G2 and G3 are independently selected from one of the following groups: acrylamide, an amine which may be activated (e.g. cadaverine or ethylenediamine), an activated ester, an aldehyde, an alkyl halide, an anhydride, an aniline, an azide, an aziridine, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine such as monochlorotriazine or dichlorotriazine, a hydrazine (including a hydrazide), an imidoester, an isocyanate, an isothiocyanate, a maleimide, a sulfonyl halide, a thiol, a ketone, an acid halide, a succinimidyl ester, a hydroxysuccinimidyl ester, a hydroxysulfosuccinimidyl ester, an azidonitrophenyl, an azidophenyl, a 3-(2-pyridyldithio)propionamide, a glyoxal, a triazine, an acetylene group, in particular a group of the formula: [ka] wherein w varies from 0 to 8, v is equal to 0 or 1, and Ar is a saturated or unsaturated 5- or 6-membered heterocycle containing 1 to 3 heteroatoms, optionally substituted with halogen atoms.

[0029] G2 and G3 may be derived from those forms protected with suitable protecting groups.

[0030] The reactive groups G2 and G3 are preferably selected independently from amines (optionally protected in the form of -NHBoc), succinimidyl esters, hydroxysuccinimidyl esters, haloacetamides, hydrazines, halotriazines, isothiocyanates, maleimide groups or carboxylic acids (optionally protected in the form of -CO2Me or -CO2tBu groups), in the latter case requiring activation of the acid in the form of an ester in order to be able to react with nucleophiles.

[0031] To obtain the GTP-gamma-N compound, GTP can be reacted directly with a lanthanide complex if the lanthanide complex has an NH2 group. GTP can also be reacted with a lanthanide complex of the formula 2HN-(CH2) n -NH2 (wherein n is as defined above and one of the amino groups may be protected by a protecting group), and the resulting intermediate compound can then be coupled with a lanthanide complex functionalized with a reactive group G3 as defined above.

[0032] The compounds of the present invention can bind to G proteins. This property is confirmed by an immunoassay based on the FRET principle, by incubating a membrane preparation containing GPCR and Gα protein in the presence of a pair of FRET partners consisting of an anti-Gα protein antibody labeled with a receptor fluorophore and the compounds of the present invention. The incubation is carried out in the presence or absence of a non-hydrolyzable or slowly hydrolyzable GTP analog such as GTPγS. When each partner of the FRET pair binds to the same Gα protein, a FRET signal appears, thereby confirming that the compounds of the present invention have bound to Gα protein. Thus, the compounds of the present invention can be advantageously used to identify molecules capable of altering the activation of G protein-coupled receptors by FRET methods.

[0033] (synthesis) The synthesis of GTP linked at the gamma position to a lanthanide complex is illustrated in Schemes 1-19.

[0034] (Synthesis of GTP-gamma-O (GTPγO) family compounds) Compound 2, which is the precursor of the compound of the invention (lanthanide complex GTP), can be synthesized according to protocols known to those skilled in the art. Starting from commercially available GTP, compound 1 is obtained by introducing a linking group "L1" at the gamma position of GTP by nucleophilic displacement of GTP with a linking group having a leaving group (I, Br, mesyl, tosyl) at the alpha position and a protected amino group at its omega position. When P=CBz or COCF3, analogous examples of coupling can be used (see WO 2009 / 105077 or WO 2009 / 091847). The protecting group is removed using deprotection conditions corresponding to the protecting group (see WO 2009 / 014612). Compound 2 is then covalently attached to the lanthanide complex via an amide bond (Scheme 1) using conventional methods known to those skilled in the art. [ka]

[0035] Another option for attaching the lanthanide complex at the gamma position of GTP is to use "click chemistry". To do so, it is first necessary to introduce either an azide group (Scheme 2) or an acetylene group (Scheme 3). As mentioned above, these groups are introduced via a nucleophilic substitution reaction of the linking group L2 or L3 with GTP (Schemes 2 and 3). Examples of attachment of nucleotides to linking groups are described, for example, by Hacker et al. (The Journal of Organic Chemistry, 2012, 77(22), 17450). [ka] [ka]

[0036] Conversely, an acetylene group can be introduced into GTP in a first step, giving compounds of the series 6. Examples of coupling to other nucleotides are available in the literature (Journal of the American Chemical Society, 2003, 125, 9588). Cycloaddition reactions can then be carried out with lanthanide complexes functionalized in the azide form to give CLn 3+ Compounds 7a to 7d can be obtained.

[0037] (Synthesis of GTP-gamma-N family compounds) As in the case of the GTP-gamma-O family, the GTP-gamma-N compounds can be prepared following a similar strategy: NH2-functionalized lanthanide complexes are directly condensed to the GTP molecule without the insertion of a linker (Scheme 4). [ka]

[0038] Lanthanide complexes can be attached using GTP-gamma-N molecules 9a-e that already contain a pre-introduced terminal NH2 linking group. [ka]

[0039] To avoid the use of some equivalents of the diamino linking group, one of the functional groups is protected, for example with CBz or trifluoroacetate (COCF3) groups. Subsequent removal of the protecting group (under conditions that avoid chemical hydrolysis of GTP) gives the same compounds 9a-e. Lanthanide complexes are attached to these compounds using conventional methods known to those skilled in the art (Scheme 6). [ka]

[0040] (Synthesis of GTP-gamma-C family compounds) The GTP-gamma-C system is synthesized by condensation of guanosine diphosphate (GDP) with phosphonic acids whose terminal position can be substituted in various ways. When the terminal position is a protected (trifluoroacetamide 15a-f or CBz 16a-f) amine function, these compounds are prepared using methods described in the literature (EP 0 959 077 (TFA), WO 2012 / 150866, and The Journal of Organic Chemistry, 1984, 49, 1158 (CBz)). In a first step, the amine function is deprotected by condensation of N-phthalimidoalkyl bromides (12a-f) with triethyl phosphite, followed by treatment with hydrazine. Hydrolysis of the ethyl phosphonate in the presence of hydrobromic acid gives compounds 14a-f. To prevent self-condensation reactions during the coupling reaction with GDP, the primary amine function is protected with either CBz or trifluoroacetamide. This reaction sequence can provide intermediate compounds 15a-f and 16a-f. [ka]

[0041] Instead of introducing a masked primary amine function, it is also possible to use GTP-gamma-C, which terminates in an acetylene unit at the end of the linker. This function allows the "click chemistry" reaction of acetylenic GTP-gamma-C with lanthanide complexes that have an azide function available for conjugation reactions. Scheme 8 briefly describes the various intermediates that were prepared according to protocols available in the literature (Bioorganic Medicinal Chemistry, 2018, 26, 191 and Angewandte Chemie International Edition, 2011, 50, 10699). Condensation of commercially available acetylenic alkyl bromide derivatives with trimethylsilyl phosphite, followed by hydrolysis, gives 18a-f. [ka]

[0042] Another option is to introduce functional groups allowing bioconjugation reactions into the linker of the GTP-gamma-C compound. In this approach, an azide group is introduced into the phosphonic acid using the sequence described in Scheme 9. Protocols are described, for example, in Chemistry, A European Journal, 2010, 16, 12718, Langmuir, 2010, 26, 10725, or The Journal of Organic Chemistry, 2012, 77, 10450. The dibrominated derivatives are reacted with triethyl phosphite to give compounds 20a-f. The azide function is then introduced by a simple nucleophilic substitution with sodium azide. Subsequent hydrolysis of the diester in the presence of TMS-Br gives derivatives 22a-f. [ka]

[0043] The coupling of compounds 15a-f, 16a-f, 18a-f, or 22a-f with lanthanide complexes is carried out using conventional methods known to those skilled in the art (Schemes 10-13). [ka] [ka] [ka] [ka]

[0044] As in the case of GTP, the same synthetic strategy can be adopted to couple GPPNHP analogues (commercially available, CAS:64564-03-0) with lanthanide complexes at gamma position. For derivatives substituted at gamma O, the synthesis is described, for example, in scheme 14. For derivatives substituted at gamma N, the synthesis is described in schemes 15 and 16. These couplings are also exemplified in WO2009 / 068751, where ATP analogues are used. [ka] [ka] [ka]

[0045] As in the case of GTP, the same synthetic strategy can be adopted to combine GPPCH2P analogues (commercially available, CAS: 13912-93-1 or 10470-57-2, Na salt form) with lanthanide complexes at gamma position.For derivatives substituted with gamma O, the synthesis is described, for example, in scheme 17.For derivatives substituted with gamma N, the synthesis is described in schemes 18 and 19.These bonds are also exemplified in WO2009 / 068751, where ATP analogues are used. [ka] [ka] [ka] EXAMPLES

[0046] The invention is illustrated by the following examples given by way of illustration.

[0047] (Abbreviations used) BRET: Bioluminescence Resonance Energy Transfer BSA: bovine serum albumin DMSO: Dimethyl sulfoxide EDC: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide eq: equivalent ESI+: positive electrospray ionization mode FRET: Förster Resonance Energy Transfer GDP: guanosine diphosphate GPCR: G protein-coupled receptor GTP: guanosine triphosphate GTPγS: guanosine 5'-[γ-thio]triphosphate h: time HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC: High-performance liquid chromatography LC-MS: High performance liquid chromatography coupled with mass spectrometry LRMS: Low resolution mass spectrometry MES: 2-(N-morpholino)ethanesulfonic acid MOPS: 3-morpholino-1-propanesulfonic acid NCS: Isothiocyanates NHS: N-hydroxysuccinimide Py: Pyridine GPCR: G protein-coupled receptor AT:Ambient temperature TEA: Triethylamine TRIS: Trishydroxymethylaminomethane UPLC: Ultra-high performance liquid chromatography UPLC-MS: Ultra-high performance liquid chromatography coupled with mass spectrometry UV: Ultraviolet light

[0048] (Chromatography) Analytical and preparative high performance liquid chromatography (HPLC) procedures were performed on two instruments: Analytical HPLC: ThermoScientific, P4000 quaternary pump, UV1000 detector with deuterium lamp (190-350 nm), Waters XBridge C 18 Analytical column (3.5 μm, 4.6 × 100 mm) Preparative HPLC: Shimadzu, 2 LC-8A pumps, Varian ProStar diode array UV detector, Waters XBridge prep C 18 Preparative column (5μm: 19×100mm or 50×150mm)

[0049] Analytical ultra-performance liquid chromatography (UPLC) procedures were performed on a Waters Acquity HClass instrument equipped with either a PDA diode array UV detector or an SQD2 single quadrupole mass detector as the detector. The probe used was electrospray in positive mode with a capillary voltage of 3.2 kV and a cone voltage of 30 V.

[0050] Gradient A: Waters Xbridge C 18 Column (5 μm, 10 × 100 mm), A / water 25 mM triethylammonium acetate, pH 7, B / acetonitrile, t = 0–5 min: 1% B, t = 10 min: 10% B, 20 ml.min -1 (260 nm)

[0051] Gradient B Waters Xbridge C 18 Column (5 μm, 4.6 × 100 mm), A / water 25 mM triethylammonium acetate, pH 7, B / acetonitrile, t = 0–2 min: 2% B, t = 19 min: 40% B, 1 ml.min -1

[0052] Gradient C Waters Xbridge C 18 Column (3.5 μm, 4.6 × 100 mm), A / water 5 mM ammonium acetate, pH 5, B / acetonitrile, t = 0 min: 2% B, t = 1 min: 2% B, t = 15 min: 40% B, 1 ml.min -1

[0053] Gradient D Waters Xbridge C 18 Column (5 μm, 19 × 100 mm), A / water 5 mM ammonium acetate, pH 6.6, B / acetonitrile, t = 0 min: 2% B, t = 2.5 min: 2% B, t = 27 min: 40% B, 12 ml.min -1 (280 and 320 nm)

[0054] Gradient E Waters Xbridge C 18 Column (5 μm, 19 × 100 mm), A / water 25 mM triethylammonium acetate, pH 7, B / acetonitrile, t = 0 min: 2% B, t = 2 min: 2% B, t = 20 min: 50% B, 20 ml.min -1 (280 and 320 nm)

[0055] Gradient F Waters Acquity C 18 Column (300 Å, 1.7 μm, 2.1 × 50 mm), A / water 5 mM ammonium acetate, pH 5, B / acetonitrile, t = 0 min: 2% B, t = 0.2 min: 2% B, t = 5 min: 40% B, 0.6 ml.min -1

[0056] Gradient G Waters Xbridge C 18 Column (300 Å, 5 μm, 10 × 100 mm), A / water 25 mM triethylammonium acetate, B / acetonitrile, t = 0 min: 2% B, t = 19 min: 20% B, 5 ml.min -1

[0057] Gradient H Waters Xbridge C 18 Column (5 μm, 4.6 × 100 mm), A / water 25 mM triethylammonium acetate, pH 7, B / acetonitrile, t = 0–2 min: 2% B, t = 19 min: 50% B, 1 ml.min -1

[0058] Gradient I Waters Xbridge C 18 Column (5 μm, 10 × 100 mm), A / water 25 mM triethylammonium acetate, pH 7, B / acetonitrile, t = 0 min: 2% B, t = 2 min: 2% B, t = 27 min: 50% B, 3.5 ml.min -1 (280 nm)

[0059] Gradient J Waters Xbridge C 18 Column (5 μm, 19 × 100 mm), A / water 25 mM triethylammonium acetate, B / acetonitrile, t = 0 min: 5% B, t = 17 min: 45% B, 20 ml.min -1

[0060] Gradient K Waters Xbridge C 18 Column (300 Å, 5 μm, 10 × 100 mm), water 25 mM triethylammonium acetate, B / acetonitrile, t = 0 min: 2% B, t = 19 min: 40% B, 5 ml.min -1

[0061] (Preparation of Compounds) Preparation Example 1: GTP gamma-N-pentyl-NH2 (9c) [ka] EDC (40 mg; 210 μmol, 11 eq) and cadaverine (58 mg, 573 μmol, 30 eq) in MOPS (pH 8) were added to a round-bottom flask containing a solution of GTP (10 mg; 19.1 μmol, 1 eq) in water (500 μl). The reaction mixture was stirred overnight at AT and then purified by preparative HPLC (gradient A). A white solid was obtained, corresponding to compound 9c (5 mg). (ESI+):calculated C 15 H 28 N7O 13 P3[M+H] + ,m / z=608.10,found 608.55.

[0062] Preparation Example 2: GTP gamma-O-hexyl-C2 [ka] A solution of complex C2 (473 μg, 700 nmol) functionalized in NCS (isothiocyanate) form in DMSO (200 μl) was added all at once to a solution of compound 2c (0.454 mg, 700 nmol) in 100 mM carbonate buffer (pH 9, 500 μl). The mixture was stirred overnight at AT. The progress of the reaction was monitored by HPLC (gradient B) and UPLC-MS (gradient C). After this period the reaction was complete. The reaction mixture was directly purified by semi-preparative HPLC (gradient D) to give compound GTP-gamma-O-hexyl-C2 (125 μg, 96 nmol, 14%) as a white powder. LRMS (ESI+): calculated for C 40 H 47 EuN 10 O 22 P3S - [M+3H] 2+ ,m / z=650.06,found 650.32.

[0063] Preparation Example 3: GTP gamma-O-hexyl-C3 [ka] A solution of complex C3 (0.647 mg, 708 nmol) functionalized in dichlorotriazine form in 100 mM carbonate buffer (pH 9, 200 μl) was added all at once to a solution of compound 2c (0.442 mg, 710 nmol) in 100 mM carbonate buffer (pH 9, 200 μl). The mixture was stirred overnight at 4° C. The progress of the reaction was monitored by UPLC-MS (gradient C). After this period, the reaction was complete. The reaction mixture was directly purified by preparative HPLC (gradient E) to give compound GTP-gamma-O-hexyl-C3 (0.11 mg, 70 nmol, 10%) as a white powder. LRMS (ESI+): calculated for C 50 H 53 ClEuN 15 O 22 P3 - [M+3H] 2+,m / z=749.59,found 750.29.

[0064] Preparation Example 4: GTP-gamma-N-C2 [ka] Complex C2 (0.366 mg, 2 μmol) was dissolved in 500 mM MES buffer (pH 5.5, 150 μl) to give a yellow solution. To the reaction mixture, GTP (1.32 mg, 2.4 μmol) was added, followed by EDC (1.92 mg, 10 μmol) all at once. The mixture was stirred at AT for 40 h. The progress of the reaction was monitored by UPLC-MS (gradient F). After this period, the reaction was partial and did not proceed further. The reaction mixture was directly purified by preparative HPLC (gradient G) to give GTP-gamma-N-C2 (60 nmol, 3%) as a colorless oil. LRMS (ESI+): calculated for C 33 H 34 N9O 21 P3Eu - [M] - ,m / z=1137.6,found 1138.2.

[0065] Preparation Example 5: GTP-gamma-N-C3 [ka] Complex C3 (0.366 mg, 480 nmol) was dissolved in 500 mM MES buffer (pH 5.5, 150 μl) to give a yellow solution. To the reaction mixture, GTP (0.317 mg, 576 nmol) was added, followed by EDC (0.460 mg, 2.4 μmol) all at once. The mixture was stirred at AT overnight. The progress of the reaction was monitored by UPLC-MS (gradient F). After this period, the reaction was partial and did not proceed further. The reaction mixture was directly purified by preparative HPLC (gradient G) to give GTP-gamma-N-C3 (45.6 nmol, 9.5%) as a colorless oil. LRMS (ESI+): calculated for C 41 H 40 N 11 O 21 P3Eu- [M+2H] + ,m / z=1269.7,found 1269.

[0066] Preparation Example 6: GTP-gamma-N-octyl-C2 [ka] Compound 9e (50.0 μl, 500 nmol) was dissolved in water (100 μl). Pyridine (300 μl) and triethylamine (6 μl) were added to the reaction mixture. The reaction mixture was added all at once to a test tube containing complex C2 (0.438 mg, 650 nmol) functionalized in NCS (isothiocyanate) form. The mixture was stirred at 24° C. for 12 h. The progress of the reaction was monitored by UPLC-MS (gradient F). After this period, the reaction was partial. The reaction mixture was directly purified by preparative HPLC (gradient G) to give GTP-gamma-N-octyl-C2 (16.9 nmol, 3.4%) as a colorless oil. LRMS (ESI+): calculated for C 42 H 52 N 11 O 21 P3SEu - [M+3H] 2+ ,m / z=663.5,found 663.9.

[0067] Preparation 7: GTP-gamma-N-octyl-C3 [ka] A solution of complex C3 (400 μg, 438 nmol) functionalized in dichlorotriazine form in water (200 μl) was added all at once to a solution of compound 9e (0.545 mg, 840 nmol) in 100 mM carbonate buffer (pH 9, 500 μl). The mixture was stirred overnight at 20° C. The progress of the reaction was monitored by HPLC (gradient H) and UPLC-MS (gradient C). After this period the reaction was complete. The reaction mixture was directly purified by semi-preparative HPLC (gradient I) to give compound GTP-gamma-N-octyl-C3 (7.6 μg, 5 nmol, 1%) as a white powder. LRMS (ESI+): calculated for C 52 H 58 ClEuN 16 O 21 P3 - [M+3H] 2+ ,m / z=763.11,found 763.29.

[0068] Preparation 8: GTP-gamma-N-hexyl-C11 [ka] A solution of complex C11 (1.47 mg, 1 μmol) functionalized in NHS ester form in anhydrous DMSO (122 μl) was added all at once to a solution of compound 9d (0.62 mg, 1 μmol) in 50 mM HEPES buffer (pH 8, 900 μl). The mixture was stirred at 24° C. for 1 h. The progress of the reaction was monitored by UPLC-MS (gradient F). After this period, the reaction was complete. The reaction mixture was directly purified by preparative HPLC (gradient J) to give compound GTP-gamma-N-hexyl-C11 (0.52 mg, 258 nmol, 26%) as a white powder. LRMS (ESI+): calculated for C 80 H 109 N 20 O 27 P3Tb 3+ [MH] 2+ ,m / z=1018.9,found 1019.6,[M+2Na + -3H] 2+ ,m / z=1040.8,found 1041.6.

[0069] Preparation 9: GTP-gamma-N-octyl-C11 [ka] A solution of complex C11 (0.76 mg, 500 nmol) functionalized in NHS ester form in anhydrous DMSO (102 μl) was added all at once to a solution of compound 9e (0.32 mg, 500 nmol) in 50 mM HEPES buffer (pH 8, 300 μl). The mixture was stirred at 24° C. for 1 h. The progress of the reaction was monitored by UPLC-MS (gradient F). After this period, the reaction was complete. The reaction mixture was directly purified by preparative HPLC (gradient K) to give compound GTP-gamma-N-octyl-C11 (0.22 mg, 105 nmol, 21%) as a white powder. LRMS (ESI+): calculated for C 82 H 117 N 20 O 27 P3Tb 3+ [M-2H] + ,m / z=2064.8,found 2064.1.

[0070] Preparation 10: GTP-gamma-N-octyl-thiosuccinimidyl-C2 [ka] A solution of compound 9e (0.649 mg, 1 μmol) in water (100 μl) was diluted with 50 mM HEPES buffer (pH 8, 757 μl) to obtain a colorless solution. To the reaction mixture, a solution of complex C2 (750 μl, 1 μmol) functionalized in NHS (N-hydroxysuccinimide ester) form in anhydrous DMSO (143 μl) was added in one portion. The reaction was stirred at 25° C. for 1 h. The progress of the reaction was monitored by UPLC-MS (gradient B). After this period, the reaction was not complete. However, the reaction mixture was purified twice by preparative HPLC (gradient E). The fractions corresponding to the expected product were collected and then concentrated under reduced pressure to obtain compound GTP-gamma-N-octyl-thiosuccinimidyl-C2 (0.241 μmol, 24%) as a colorless solution. LRMS (ESI+): calculated for C 51 H 68 EuN 13 O 24 P3S2 + [M+H] + ,m / z=1555.8,found 1555.8.

[0071] (Binding test) (material) Membrane preparations of cells expressing GPCRs and G-alpha-i proteins (Gαi) were purchased from Perkin Elmer or Euroscreen. The table below summarizes the cell backgrounds and reference numbers of the different samples used. [Table 1] The DSV36S antibody was developed by Cisbio Bioassays and is available from Cisbio Bioassays upon request (number DSV36S). The antibody was labeled with the fluorescent probe d2 (red receptor) compatible with TR-FRET detection. The DSV36S antibody binds to switch II of the Gαi protein. ·GTP and GTPγS nucleotides were purchased from Sigma Aldrich (catalog numbers G8877 and G8634, respectively). · White-bottom 384-well low volume white plates were purchased from Greiner Bio One (cat. no. 784075). Non-hydrolyzable / slowly hydrolyzable GTP analogs labeled with lanthanide-complexed donor fluorophores were synthesized at Cisbio Bioassays.

[0072] (method) (Preparation of Reagents) Unless otherwise stated, all reagents were diluted in 50 mM Tris-HCl buffer (pH 7.4), 10 mM MgCl2, 0.1% BSA, 10 mM NaCl. Membranes were prepared at 4X and dispensed at 1 μg or 10 μg per well (amounts specified in each figure legend). GTPγS nucleotides (non-specific signal conditions) were prepared at 6.67X to a final concentration in the well of 100 μM. Anti-Gαi antibody DSV36S-d2 used for detection was prepared at 4X to target a final concentration in the well of 10 nM. Non-hydrolyzable / slowly hydrolyzable GTP analogs labeled with fluorescent donor probes (lanthanide complexes) were prepared at 4X to target the final concentrations in the wells stated in each figure legend.

[0073] (Reagent distribution in 384-well plates) Membranes expressing GPCR and G protein: 5 μl GTPγS buffer or nucleotides (for non-specific signal conditions): 3 μl Non-hydrolyzable / slowly hydrolyzable GTP analogue-donor: 5μl ·Anti-Gαi antibody-receptor (DSV36S-d2): 5μl Buffer: 2μl

[0074] Non-specific signals (background fluorescence noise) were measured in wells containing an excess of GTPγS (10 or 100 μM).

[0075] (HTRF signal reading) After incubating the plates at 21° C. for 20 h, the HTRF signal was measured using a PHERAstar reader (BMG Labtech) with the following configuration: Module: HTRF (excitation 337nm, emission 665nm and 620nm) Excitation: 40 laser flashes or 100 lamp flashes Read window: Delay 60μs, Integration 400μs

[0076] (Signal Processing) The HTRF ratio was calculated from the raw signals at 665 nm and 620 nm according to the following formula: HTRF ratio = [(signal at 665 nm) / (signal at 620 nm)] x 10000

[0077] (Exam Format) Figure 2 represents the test format used to detect the binding of compounds of formula (I) to G proteins. A membrane preparation containing a GPCR and a Gαi protein was incubated in the presence or absence of a non-hydrolyzable or slowly hydrolyzable GTP analog (typically GTPγS), in the presence of a test compound and an anti-Gα protein antibody labeled with a receptor fluorophore. If the two FRET partners (test compound and anti-Gα protein antibody) bind to the same Gα protein, a FRET signal appears (left side of the figure: "total signal"), which is distinguished from a non-specific signal (right side of the figure: background noise corresponding to "non-specific signal") that contains a large excess of unlabeled GTPγS (substituting the fluorescent GTP analog).

[0078] Example 1: GTPgN-C2 analog binding assay The ability of the GTPgN-C2 (Preparation Example 2) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G proteins was confirmed using membrane preparations (10 μg per well) of CHO-K1 cells expressing delta opioid GPCR and Gαi protein. GTPgN-C2 was used at a final concentration of 6 nM in the well. The membrane was incubated in the absence or presence of a large excess of GTPγS (100 μM). The difference in the TR-FRET signal observed between these two conditions (HTRF ratio, total signal / nonspecific signal = 16.6) indicates that the GTPgN-C2 analog can bind to Gαi protein and generate a TR-FRET signal together with the anti-Gαi antibody-receptor (Figure 3).

[0079] (Example 2: GTPgN-C3 analog binding assay) The ability of the GTPgN-C3 (Preparation Example 5) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G proteins was confirmed using membrane preparations (1 μg per well) of HEK293 cells expressing delta opioid GPCR and Gαi protein. GTPgN-C3 was used at a final concentration of 1 nM in the well. The membrane was incubated in the absence or presence of a large excess of GTPγS (100 μM). The difference in the TR-FRET signal observed between these two conditions (HTRF ratio, total signal / nonspecific signal = 1.7) indicates that the GTPgN-C3 analog can bind to Gαi protein and generate a TR-FRET signal together with the anti-Gα antibody-receptor (Figure 4).

[0080] Example 3: GTPgN-Octyl-C2 Analog Binding Assay Using membrane preparations (10 μg per well) of CHO-K1 cells expressing delta opioid GPCR and Gαi protein, the ability of the GTPgN-octyl-C2 (Preparation Example 6) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G protein was confirmed. GTPgN-octyl-C2 was used at a final concentration of 6 nM in the well. The membrane was incubated in the absence or presence of a large excess of GTPγS (100 μM). The difference in the TR-FRET signal observed between these two conditions (HTRF ratio, total signal / nonspecific signal = 12.4) indicates that the GTPgN-octyl-C2 analog can bind to Gαi protein and generate a TR-FRET signal together with the anti-Gαi antibody-receptor (Figure 5).

[0081] Example 4: GTPgN-octyl-C11 analog binding assay The ability of the GTPgN-octyl-C11 (Preparation Example 9) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G proteins was confirmed using membrane preparations (10 μg per well) of CHO-K1 cells expressing delta opioid GPCR and Gαi protein. GTPgN-octyl-C11 was used at a final concentration of 6 nM in the well. The membrane was incubated in the absence or presence of a large excess of GTPγS (100 μM). The difference in the TR-FRET signal observed between these two conditions (HTRF ratio, total signal / nonspecific signal = 7.6) indicates that the GTPgN-octyl-C11 analog can bind to Gαi protein and generate a TR-FRET signal together with the anti-Gαi antibody-receptor (Figure 6).

[0082] Example 5: GTPgN-Octyl-C3 Analog Binding Assay The ability of the GTPgN-octyl-C3 (Preparation Example 7) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G proteins was confirmed using membrane preparations (1 μg per well) of HEK293 cells expressing delta opioid GPCR and Gαi protein. GTPgN-octyl-C3 was used at a final concentration of 1 nM in the well. The membrane was incubated in the absence or presence of a large excess of GTPγS (100 μM). The difference in the TR-FRET signal observed between these two conditions (HTRF ratio, total signal / nonspecific signal = 1.4) indicates that the GTPgN-octyl-C3 analog can bind to Gαi protein and generate a TR-FRET signal together with the anti-Gαi antibody-receptor (Figure 7).

[0083] Example 6: GTPgO-Hexyl-C2 Analog Binding Assay Using membrane preparations (10 μg per well) of CHO-K1 cells expressing delta opioid GPCR and Gαi protein, the ability of the GTPgO-hexyl-C2 (Preparation Example 2) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G protein was confirmed. GTPgO-hexyl-C2 was used at a final concentration of 6 nM in the well. The membrane was incubated in the absence or presence of a large excess of GTPγS (100 μM). The difference in the TR-FRET signal observed between these two conditions (HTRF ratio, total signal / nonspecific signal = 7.1) indicates that the GTPgO-hexyl-C2 analog can bind to Gαi protein and generate a TR-FRET signal together with the anti-Gαi antibody-receptor (Figure 8).

[0084] Example 7: GTPgO-Hexyl-C3 Analog Binding Assay Using membrane preparations (1 μg per well) of HEK293 cells expressing delta opioid GPCR and Gαi protein, the ability of the GTPgO-hexyl-C3 (Preparation Example 3) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G protein was confirmed. GTPgO-hexyl-C3 was used at a final concentration of 1 nM in the well. The membrane was incubated in the absence or presence of a large excess of GTPγS (100 μM). The difference in the TR-FRET signal observed between these two conditions (HTRF ratio, total signal / nonspecific signal = 1.3) indicates that the GTPgO-hexyl-C3 analog can bind to Gαi protein and generate a TR-FRET signal together with the anti-Gαi antibody-receptor (Figure 9).

[0085] Example 8: GTPgN-Octyl-C2 Analog Binding Assay Using membrane preparations (10 μg per well) of CHO-K1 cells expressing dopamine D2S GPCR and Gαi protein, the ability of the GTPgN-octyl-C2 (Preparation Example 6) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G protein was confirmed. GTPgN-octyl-C2 was used at a final concentration of 6 nM in the well. The membrane was incubated in the absence or presence of a large excess of GTPγS (100 μM). The difference in the TR-FRET signal observed between these two conditions (HTRF ratio, total signal / nonspecific signal = 3.8) indicates that the GTPgN-octyl-C2 analog can bind to Gαi protein and generate a TR-FRET signal together with the anti-Gαi antibody-receptor (Figure 10).

[0086] Example 9: Effect of membrane concentration and GTP analog-lanthanide concentration on GTPgN-octyl-C2 analog binding assay The ability of the GTPgN-octyl-C2 (Preparation Example 6) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G proteins was confirmed using membrane preparations (1 or 10 μg / well) of CHO-K1 cells expressing delta opioid GPCR and Gαi protein. GTPgN-octyl-C2 was used at a final concentration of 2 or 6 nM in the well. The membrane was incubated in the absence or presence of a large excess of GTPγS (100 μM). The difference in the TR-FRET signal observed between these two conditions (HTRF ratio, total signal (S) / nonspecific signal (N)) indicates that the GTPgN-octyl-C2 analog can bind to Gαi protein and generate a TR-FRET signal together with the anti-Gαi antibody-receptor (Figures 11A and 11B). Also, Figure 11A clearly shows that increasing the amount of membrane from 1 μg to 10 μg per well increases the signal amplitude (S / N), and Figure 11B clearly shows that increasing the concentration of GTPgN-octyl-C2 from 2 nM to 6 nM increases the signal amplitude (S / N).

[0087] Example 10: GTPg N-octyl-thiosuccinimidyl-C2 analog binding assay Using membrane preparations (10 μg per well) of CHO-K1 cells expressing delta opioid GPCR and Gαi protein, the ability of the GTPgN-octyl-thiosuccinimidyl-C2 (Preparation Example 10) / anti-Gαi antibody DSV36S-d2 pair to generate a specific TR-FRET signal by binding to G protein was confirmed. The reagent was diluted in 50 mM Tris-HCl buffer (pH 7.4), 60 mM MgCl2, 0.1% BSA, and 150 mM NaCl. GTPgN-octyl-thiosuccinimidyl-C2 was used at a final concentration of 7.5 nM in the well. The fluorescent conjugate was incubated in the absence or presence of the membrane preparation. The difference in TR-FRET signal observed between these two conditions (HTRF ratio, total signal / nonspecific signal = 2.8) indicates that the GTPgN-octyl-thiosuccinimidyl-C2 analogue can bind to Gαi proteins and generate a TR-FRET signal together with the anti-Gαi antibody-receptor (Figure 12).

Claims

1. The following formula: 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 The compound is selected from one of:

2. The following formula: 【Chemistry 10】 2. The compound of claim 1 ,

3. 1. A method for identifying a molecule capable of altering activation of a G protein-coupled receptor (GPCR), comprising: a) incubating a membrane preparation comprising a GPCR and a Gα protein in the presence of a pair of FRET partners composed of (i) a compound according to claim 1 or 2 and (ii) an anti-Gα protein antibody labeled with a receptor fluorophore, and a test molecule; b) determining whether a FRET signal appears A method comprising:

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