Broad spectrum GPCR binding agents

Abstract

To provide broad-spectrum G-Protein coupled receptor (GPCR) binding agents, detectable / isolatable compounds comprising such binding agents (e.g., broad-spectrum GPCR binding agents linked to a functional element and / or solid surface), and methods of use thereof for the detection / isolation of GPCRs.SOLUTION: The present invention provides a composition comprising a broad-spectrum G-protein coupled receptor (GPCR) binding agent represented by formula (CLZP1), which is attached to a functional element or a solid surface, and a method of detecting or quantifying GPCRs in a sample, the method comprising contacting the sample with the composition and detecting or quantifying a functional element of a signal produced thereby.SELECTED DRAWING: None

Description

[Technical field]

[0001] Provided herein are broad-spectrum G protein-coupled receptor (GPCR) binding agents, detectable / isolatable compounds comprising such binding agents (e.g., broad-spectrum GPCR binding agents linked to functional elements and / or solid surfaces), and methods of their use for the detection / isolation of GPCRs. [Background technology]

[0002] G protein-coupled receptors (GPCRs) are an important class of transmembrane proteins. They are involved in multiple diseases, making them targets of many modern medicines and are also intensively studied for the development of new drugs. Therefore, tools are needed that allow interrogating GPCR-ligand interactions in living cells. Summary of the Invention

[0003] Provided herein are broad-spectrum G protein-coupled receptor (GPCR) binding agents, detectable / isolatable compounds comprising such binding agents (e.g., broad-spectrum GPCR binding agents linked to functional elements and / or solid surfaces), and methods of their use for the detection / isolation of GPCRs.

[0004] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, wherein the broad-spectrum GPCR binding agent is [ka] wherein [ka] is the point of attachment to a functional element of the broad-spectrum GPCR-binding agent, a solid surface, or a linker between the broad-spectrum GPCR-binding agent and a functional element or solid surface.

[0005] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder conjugated to a functional element or a solid surface, wherein the broad-spectrum GPCR binder

Chemical formula

Chemical formula

[0006] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder conjugated to a functional element or a solid surface, wherein the broad-spectrum GPCR binder

Chemical formula

Chemical formula

[0007] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder conjugated to a functional element or a solid surface, wherein the broad-spectrum GPCR binder

Chemical formula

Chemical formula

[0008] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder

Chemical formula

Chemical formula

[0009] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder

Chemical formula

Chemical formula

[0010] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder

Chemical formula

[0011] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder [Chemical formula] comprises, wherein in the formula [Chemical formula] is a binding site to a functional element of a broad-spectrum GPCR binder, a solid surface, or a linker between the broad-spectrum GPCR binder and the functional element or the solid surface, and a double bond may be present as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[0012] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder [Chemical formula] comprises, wherein in the formula [Chemical formula] is a binding site to a functional element of a broad-spectrum GPCR binder, a solid surface, or a linker between the broad-spectrum GPCR binder and the functional element or the solid surface, and a double bond may be present as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[0013] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is

Chemical formula

Chemical formula

[0014] In some embodiments, provided herein is a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is

Chemical formula

Chemical formula

[0015] In some embodiments, provided herein is a composition described herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, NTRP2, etc.) comprising a solid surface selected from deposited particles, a film, glass, a tube, a well, a self-assembled monolayer, a surface plasmon resonance chip, or a solid support having an electronically conductive surface. In some embodiments, the deposited particles are magnetic particles.

[0016] In some embodiments, provided herein is a composition described herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, NTRP2, etc.) comprising a functional element selected from a detectable element, an affinity element, and a capture element. In some embodiments, the detectable element comprises a fluorescent dye molecule, a chromophore, a radionuclide, an electron-opaque molecule, an MRI contrast agent, a SPECT contrast agent, or a mass tag.

[0017] In some embodiments, a broad-spectrum GPCR binder of a composition described herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, NTRP2, etc.) is directly bound to a functional element or a solid surface. In some embodiments, a broad-spectrum GPCR binder of a composition described herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, NTRP2, etc.) is bound to a functional element or a solid surface via a linker. In some embodiments, the linker comprises [(CH2)2O] nを wherein n is from 1 to 20. In some embodiments, the linker is bound to the broad-spectrum GPCR binder and / or the functional element by an amide bond.

[0018] In some embodiments, provided herein is [Chemical formula] A composition comprising the structure of, where n ranges from 0 to 8 and X is a functional element or a solid surface.

[0019] In some embodiments, provided herein is [Chemical formula] A composition comprising the structure of, where n ranges from 0 to 8, m ranges from 0 to 8, and X is a functional element or a solid surface.

[0020] In some embodiments, provided herein is [Chemical formula] A composition comprising the structure of, where n ranges from 0 to 8, m ranges from 0 to 8, and X is a functional element or a solid surface.

[0021] In some embodiments, provided herein is [Chemical formula] A composition comprising the structure of, where n ranges from 0 to 8, m ranges from 0 to 8, and X is a functional element or a solid surface.

[0022] In some embodiments, provided herein is [Chemical formula] A composition comprising the structure of, where n ranges from 0 to 8 and X is a functional element or a solid surface.

[0023] In some embodiments, provided herein is [Chemical formula] A composition comprising the structure, wherein n is from 0 to 8, m is from 0 to 8, and X is a functional element or a solid surface.

[0024] In some embodiments, provided herein is

Chemical formula

[0025] In some embodiments, provided herein is

Chemical formula

[0026] In some embodiments, provided herein is

Chemical formula

[0027] In some embodiments, provided herein is

Chemical formula

[0028] In some embodiments, provided herein is

Chemical formula

[0029] In some embodiments, provided herein is a composition comprising a functional element (X) that is a fluorescent dye molecule.

[0030] In some embodiments, provided herein is a composition comprising an amitriptyline-based structure as shown in FIG. 15, or a nortriptyline-based form of one of the structures shown in FIG. 15. In some embodiments, the amitriptyline-based structure as shown in FIG. 15 or its nortriptyline-based form is provided with an alternative linker, a fluorescent dye molecule (or other X group), or a point of attachment on the ring system of amitriptyline or nortriptyline. In some embodiments, alternative linkers and X groups are provided herein, and alternative points of attachment are provided, for example, by AMTRP1, AMTRP2, NTRP1, NTRP2, and NTRP2.

[0031] In some embodiments, the compositions herein include non-natural abundance ratios of one or more stable heavy isotopes.

[0032] In some embodiments, provided herein is a method for detecting or quantifying a GPCR in a sample, comprising contacting the sample with a composition described herein (e.g., a composition comprising a linked GPCR binder and a functional group or solid surface), and detecting or quantifying a functional element of the signal thereby generated. In some embodiments, the functional element of the signal thereby generated is detected or quantified by fluorescence, mass spectrometry, optical imaging, magnetic resonance imaging (MRI), and energy transfer.

[0033] In some embodiments, provided herein is a method for isolating a GPCR from a sample, comprising contacting the sample with a composition described herein (e.g., a composition comprising a linked GPCR binder and a functional group or a solid surface), and separating the functionally bound GPCR, as well as the functional element or solid surface, from the unbound portion of the sample. In some embodiments, characterizing the identity of a GPCR in a sample comprises isolating the GPCR from the sample and analyzing the isolated GPCR by mass spectrometry.

[0034] In some embodiments, provided herein is a method for monitoring the interaction between a GPCR and an unmodified biomolecule, comprising contacting the sample with a composition described herein (e.g., a composition comprising a linked GPCR binder and a functional group or a solid surface).

[0035] In some embodiments, any of the methods described herein are performed using a sample selected from a cell, cell lysate, body fluid, tissue, biological sample, in vitro sample, and environmental sample.

[0036] In some embodiments, provided herein is a system comprising a composition described herein (e.g., a composition comprising a linked GPCR binder and a functional group), wherein the functional element is a fluorescent dye molecule; (b) a fusion of a GPCR with a peptide component of a bioluminescent protein or bioluminescent complex, wherein the emission spectrum of the bioluminescent protein or bioluminescent complex overlaps with the excitation spectrum of the fluorescent dye molecule. In some embodiments, the system comprises a kit, cell, cell lysate, or reaction mixture. In some embodiments, the fusion comprises a GPCR and a peptide component of a bioluminescent complex, wherein the system further comprises one or more additional components of the bioluminescent complex (e.g., a polypeptide component of the bioluminescent complex) and a substrate for the bioluminescent complex.

[0037] In some embodiments, provided herein is a method comprising: (a) contacting a fusion of a GPCR and a bioluminescent protein with (i) a composition described herein (e.g., a composition comprising a linked GPCR binder and a functional group) in which the functional element is a fluorescent dye molecule and the emission spectrum of the bioluminescent protein overlaps the excitation spectrum of the fluorescent dye molecule, and (ii) a substrate of the bioluminescent protein; and (b) detecting the wavelength of light within the range of the excitation spectrum of the fluorescent dye molecule resulting from bioluminescence resonance energy transfer from the bioluminescent protein to the fluorescent dye molecule when a broad-spectrum GPCR binder binds to the GPCR.

[0038] In some embodiments, provided herein is a method comprising: (a) contacting a fusion of a GPCR and a peptide component of a bioluminescence complex with (i) a composition described herein (e.g., a composition comprising a linked GPCR binder and a functional group) in which the functional element is a fluorescent dye molecule and the emission spectrum of the bioluminescent protein overlaps the excitation spectrum of the fluorescent dye molecule, (ii) a polypeptide component of the bioluminescence complex, and (iii) a substrate of the bioluminescent protein; and (b) detecting the wavelength of light within the range of the excitation spectrum of the fluorescent dye molecule resulting from bioluminescence resonance energy transfer from the bioluminescence complex to the fluorescent dye molecule when a broad-spectrum GPCR binder binds to the GPCR.

Brief Description of the Drawings

[0039]

Figure 1

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Figure 16

[0040] Definitions Methods and materials similar or equivalent to those described herein can be used in the practice and testing of the embodiments described herein, but some preferred methods, compositions, devices, and materials are described herein. However, before explaining the materials and methods of the present invention, it should be understood that the specific molecules, compositions, methodologies, or protocols described herein may vary according to routine experimentation and optimization and that the present invention is not limited thereto. Also, it should be understood that the terms used in the description are for the purpose of merely describing a particular type or embodiment and are not intended to limit the scope of the embodiments described herein.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Thus, in the context of the embodiments described herein, the following definitions apply.

[0042] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "GPCR" are references to one or more GPCRs and their equivalents known to those of skill in the art.

[0043] As used herein, the term "and / or" includes any and all combinations of the listed items, including each of the listed items individually. For example, "A, B, and / or C" includes A, B, C, AB, AC, BC, and ABC, each of which is considered to be individually described by the description "A, B, and / or C".

[0044] As used herein, the term "comprise" and its linguistic variants mean the presence of the described features (s), elements (s), method steps (s), etc., without excluding the presence of additional features (s), elements (s), method steps (s), etc. Conversely, the term "consisting of" and its linguistic variants mean the presence of the described features (s), elements (s), method steps (s), etc., and exclude features (s), elements (s), method steps (s), etc. not described, except for impurities normally associated therewith. The expression "consisting essentially of" means the described features (s), elements (s), method steps (s), etc., and additional features (s), elements (s), method steps (s), etc. that do not substantially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open-ended language "comprising". Such embodiments include a plurality of closed embodiments of "consisting of" and / or "consisting essentially of", which may alternatively be claimed or described using such language.

[0045] As used herein, the term "isomer" refers to compounds having the same composition and molecular weight but different physical and / or chemical properties. This structural difference may be in the configuration or the ability to rotate the plane of polarization.

[0046] As used herein, the terms "stereoisomer" or "geometric isomer" refer to a set of compounds that have the same number and types of atoms and share the same bond connectivity between those atoms, but have different three-dimensional structures. The terms "stereoisomer" or "geometric isomer" refer to any member of this series of compounds.

[0047] As used herein, the term "clozapine" refers to a compound having the following structure.

Chem.

[0048] As used herein, the term "loxapine" refers to a compound having the following structure.

Chem.

[0049] As used herein, the term "quetiapine" refers to a compound having the following structure.

Chem.

[0050] As used herein, the term "risperidone" refers to a compound having the following structure.

Chem.

[0051] As used herein, the term "olanzapine" refers to a compound having the following structure. [Chemical formula] The moiety or substituent of olanzapine of the molecular entity includes an olanzapine structure connected at any suitable bonding point to another molecular entity (e.g., a solid surface, a functional element, etc.).

[0052] As used herein, the term "amitriptyline" refers to a compound having the following structure. [Chemical formula] The moiety or substituent of amitriptyline of the molecular entity includes an amitriptyline structure connected at any suitable bonding point to another molecular entity (e.g., a solid surface, a functional element, etc.). Amitriptyline and the amitriptyline moiety contain C=C. Amitriptyline is symmetric, but substitution that breaks the symmetry gives rise to two geometric isomers of the double bond. Thus, the amitriptyline moiety may exist as the cis isomer (Z), the trans isomer (E), or a mixture of the two.

[0053] As used herein, the term "nortriptyline" refers to a compound having the following structure. [Chemical formula] The moiety or substituent of the molecular entity nortriptyline comprises a nortriptyline structure linked at any suitable point of attachment to another molecular entity (e.g., a solid surface, a functional element, etc.). Nortriptyline and nortriptyline moieties contain C=C. Nortriptyline is symmetric, but substitutions that break the symmetry give rise to two geometric isomers of the double bond. Thus, the nortriptyline moiety may exist as the cis isomer (Z), the trans isomer (E), or a mixture of the two.

[0054] As used herein, the term "tracer" refers to a compound or agent of interest that binds to an analyte of interest (e.g., a protein of interest (e.g., a GPCR), etc.) and exhibits a quantifiable or detectable property (e.g., detected or quantified by a suitable biochemical or biophysical technique (e.g., optically, magnetically, electrically, by resonance imaging, by mass, by radiation, etc.)). The tracer may include a compound or agent of interest that binds to the analyte of interest and is linked (e.g., directly or via a suitable linker) to a fluorescent dye molecule, a radionuclide, a mass tag, a magnetic resonance imaging (MRI) contrast agent, a planar scintigraphy (PS) agent, a positron emission tomography (PET) agent, a single photon emission computed tomography (SPECT) agent, and a computed tomography (CT) contrast agent (e.g., a metal ion chelator, an isotope, or a radionuclide by a bound metal ion).

[0055] As used herein, the term "sample" is used in its broadest sense. In another sense, it means a specimen or culture obtained from any source, as well as biological and environmental samples. A biological sample is obtained from an animal (including a human) and may include fluids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. A sample may also refer to a cell lysate or a purified form of an enzyme, peptide, and / or polypeptide described herein. A cell lysate may include cells lysed with a lysing agent or a lysate such as rabbit reticulocyte lysate or wheat germ lysate. A sample may also include a cell-free expression system. Environmental samples include environmental substances such as, for example, surface materials, soil, water, crystals, and industrial samples. However, these examples should not be construed as limiting the types of samples applicable to the present invention.

[0056] As used herein, the term "linear connecting atom" refers to the backbone atoms of a chain or polymer, excluding pendant, side-chain, or H atoms that do not form the main chain or backbone.

[0057] As used herein, the term "functional element" refers to a detectable, reactive, affinity, or otherwise bioactive agent or moiety that is attached (e.g., directly or via a suitable linker) to a compound or moiety described herein. Other additional functional elements that may be used in the embodiments described herein include "localization elements", "detection elements", and the like.

[0058] As used herein, the term "capture element" refers to a molecular entity that forms a covalent interaction with a corresponding "capture agent".

[0059] As used herein, the term "affinity element" refers to a molecular entity that forms a stable non-covalent interaction with a corresponding "affinity agent".

[0060] As used herein, the term "solid support" is used with respect to any solid or stationary material to which reagents such as substrates, mutant proteins, drug-like molecules, and other test components can bind or may bind. Examples of solid supports include microscope slides, wells of microtiter plates, cover slips, beads, particles, resins, cell culture flasks, and many other suitable ones as well. The beads, particles, or resins can be magnetic or paramagnetic.

[0061] When used in a chemical structure, the representation

Chem.

[0062] Detailed Description Provided herein are broad-spectrum G protein-coupled receptor (GPCR) binders, detectable / separable compounds comprising such binders (e.g., functional elements and / or broad-spectrum GPCR binders linked to a solid surface), and methods of using the same for the detection / separation of GPCRs.

[0063] In some embodiments, provided herein are labeled GPCR ligands. Experiments were conducted during the development of the embodiments herein to demonstrate the selection of binding point(s) on a GPCR binder that generates a set of promiscuous tracers that retain the binding profile of the parent drug molecule but include additional functionality such as linked functional elements, solid surfaces, etc. The labeled GPCR ligands described herein are used in any suitable assay.

[0064] In some embodiments, provided herein are compounds that bind to broad-spectrum GPCRs (e.g., are specific for GPCRs but not specific between GPCRs). In some embodiments, provided herein are

Chem.

[0065] In some embodiments, provided herein are analogs or derivatives of CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, and NTRP2. In some embodiments, CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, or analogs or derivatives thereof are directly (via a single covalent bond) attached to a functional element or a solid surface. In some embodiments, CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, or analogs or derivatives thereof are indirectly (via a linker) attached to a functional element or a solid surface.

[0066] In some embodiments, provided herein are the compounds of this specification (including, for example, CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, analogs or derivatives thereof, etc.), wherein

Chemical formula

[0067] In some embodiments, the linker provides sufficient distance between moieties in the compounds or compositions herein (e.g., between a broad-spectrum GPCR binder and a detectable element, a solid surface, etc.) such that each can function without being hindered (or being hindered as little as possible) by binding to others. For example, the linker provides sufficient distance such that the GPCR binder can bind to the GPCR and the detectable moiety is detectable (e.g., without interference between the two, or with minimal interference). In some embodiments, the linker is from 5 angstroms to 1000 angstroms in length (including both ends) to separate a GPCR binder herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, analogs or derivatives thereof, etc.) and a functional element (e.g., a detectable element, a solid surface, etc.). Suitable linkers separate the compounds herein and the functional element by 5 Å, 10 Å, 20 Å, 50 Å, 100 Å, 150 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, 1000 Å, and any suitable range therein (e.g., 5 - 100 Å, 50 - 500 Å, 150 - 700 Å, etc.). In some embodiments, the linker separates the compounds and the functional element herein by 1 to 200 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any suitable range therein (e.g., 2 - 20, 10 - 50, etc.)).

[0068] In some embodiments, the linker comprises one or more (e.g., 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range therebetween)-(CH2)2O-(oxyethylene) groups (e.g., -(CH2)2O-(CH2)2O-(CH2)2O-(CH2)2O-, -(CH2)2O-(CH2)2O-(CH2)2O-(CH2)2O-CH2)2O-, -(CH2)2O-(CH2)2O-(CH2)2O-(CH2)2O-CH2)2O-(CH2)2O-, etc.). In some embodiments, the linker is -(CH2)2O-(CH2)2O-(CH2)2O-(CH2)2O-.

[0069] In some embodiments, the linker comprises two or more "linker moieties" (L 1 , L 2 , etc.). In some embodiments, the linker comprises zero, one, or two of a cleavable (e.g., enzymatically cleavable, chemically cleavable, etc.) moiety (Y) and additional "linker moieties" (L 1 , L 2 , etc.). In some embodiments, the linker moiety is a straight or branched chain comprising any combination of an alkyl chain, an alkenyl chain, or an alkynyl chain, and backbone heteroatoms (e.g., O, S, N, P, etc.). In some embodiments, the linker moiety comprises one or more backbone groups selected from -O-, -S-, -CH=CH-, =C=, carbon-carbon triple bond, C=O, NH, SH, OH, CN, etc. In some embodiments, the linker moiety comprises one or more substituents, pendants, side chains, etc. comprising any suitable organic functional group (e.g., OH, NH2, CN, =O, SH, halogen (e.g., Cl, Br, F, I), COOH, CH3, etc.).

[0070] In certain embodiments, the linker moiety is an alkyl carbamate group (e.g., (CH2) n OCONH, (CH2) nIt includes, such as -NHCOO. In some embodiments, the alkyl carbamate is oriented such that the -NH terminus is directed towards the GPCR binder and the COO terminus is directed towards the functional element or the solid surface. In some embodiments, the alkyl carbamate is oriented such that the -COO terminus is directed towards the GPCR binder and the -NH terminus is directed towards the functional element or the solid surface. In some embodiments, the linker or linker moiety comprises a single alkyl carbamate group. In some embodiments, the linker or linker moiety comprises two or more alkyl carbamate groups (e.g., 2, 3, 4, 5, 6, 7, 8, etc.).

[0071] In some embodiments, the linker moiety comprises C, S, N, and / or O atoms linearly connected in excess of 1. In some embodiments, the linker moiety comprises one or more alkyl carbamate groups. In some embodiments, the linker moiety comprises one or more alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.). In some embodiments, the linker moiety comprises from 1 to 200 linearly connected atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any suitable range therein (e.g., 2 - 20, 10 - 50, 6 - 18)). In some embodiments, the linker moiety is a length of from 1 to 200 linearly connected atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any suitable range therein (e.g., 2 - 20, 10 - 50, 6 - 18)).

[0072] Exemplary linkers for connecting a "drug" (e.g., including the GPCR binders of the present specification such as CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, analogs or derivatives thereof, etc.) and a functional element (e.g., a detectable element, a solid surface, etc.) are shown in FIG. 14. Such exemplary linkers are used with any suitable GPCR binder and functional element described herein.

[0073] In some embodiments, the compositions described herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, analogs or derivatives thereof, etc.) are biocompatible (e.g., cell-compatible) and / or cell-permeable. Thus, in some embodiments, suitable functional elements (e.g., detectable elements, capture elements) are those that are cell-compatible and / or cell-permeable in the context of such compositions. In some embodiments, a composition containing additional elements can enter cells across the cell membrane (e.g., via diffusion, endocytosis, active transport, passive transport, etc.) when added extracellularly. In some embodiments, suitable functional elements and linkers are selected based on cell compatibility and / or cell permeability in addition to their specific functions.

[0074] In certain embodiments, the functional element has a detectable property that enables the detection of a compound herein (including, for example, CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) or an analyte (such as a GPCR) bound thereto. Detectable functional elements include those having characteristic electromagnetic spectrum properties such as luminescence or absorption, magnetism, electron spin resonance, capacitance, dielectric constant, or conductivity, as well as ferromagnetic, paramagnetic, diamagnetic, luminescent, electrochemiluminescent, fluorescent, phosphorescent, chromogenic, antigenic, or having a unique mass functional groups. Functional elements include, but are not limited to, nucleic acid molecules (such as DNA or RNA (such as oligonucleotides or nucleotides)), proteins (such as luminescent proteins, peptides, contrast agents (such as MRI contrast agents), radionuclides, affinity tags (such as biotin or streptavidin), haptens, amino acids, lipids, lipid bilayers, solid supports, fluorescent dye molecules, chromophores, reporter molecules, radionuclides, electron-opaque molecules, MRI contrast agents (such as manganese, gadolinium(III), or iron oxide particles), or their coordinators, etc.). Methods for detecting specific functional elements or methods for isolating compositions containing specific functional elements and those bound thereto are understood.

[0075] In some embodiments, the functional group is or includes a solid support. Suitable solid supports include deposited particles such as magnetic particles, sepharose, or cellulose beads; membranes; glass, such as slide glass; cellulose, alginate, plastic, or other synthetically prepared polymers (such as the wells of Eppendorf tubes or multiwell plates); self-assembled monolayers; surface plasmon resonance chips; or solid supports with an electronically conductive surface; and the like.

[0076] Exemplary detectable functional elements include haptens (e.g., molecules useful for enhancing immunogenicity such as hemocyanin from keyhole limpet), cleavable labels (e.g., photocleavable biotin), and fluorescent labels (e.g., coumarin modified with N-hydroxysuccinimide (NHS) and succinimide or sulfonosuccinimide-modified BODIPY (detectable by UV and / or visible excitation fluorescence detection), rhodamine (R110, rhodol, CRG6, Texas methyl red (TAMRA), Rox5, FAM, or fluorescein), coumarin derivatives (e.g., 7-aminocoumarin, and 7-hydroxycoumarin, 2-amino-4-methoxynaphthalene, 1-hydroxypyrene, resorufin, phenalenone or benzophenalenone (U.S. Patent No. 4,812,409)), acridinone (U.S. Patent No. 4,810,636), anthracene, and derivatives of alpha and beta-naphthol, fluorinated xanthene derivatives including fluorinated fluorescein and rhodol (e.g., U.S. Patent No. 6,162,931), and bioluminescent molecules (e.g., luciferase (e.g., opophorus derivative luciferase (e.g., U.S. Patent Application No. 12 / 773,002; U.S. Patent Application No. 13 / 287,986; incorporated herein by reference in its entirety) or GFP or GFP derivatives)). Fluorescent (or bioluminescent) functional elements can be used to sense changes in systems such as phosphorylation in real time. Fluorescent molecules such as chemical sensors for metal ions may be employed to label proteins that bind the composition. Bioluminescent or fluorescent functional groups such as BODIPY, rhodamine green, GFP, or infrared dyes are used as functional elements and may be employed, for example, in interaction studies (e.g., using BRET, FRET, LRET, or electrophoresis).

[0077] Functional elements of another class include molecules detectable using electromagnetic radiation, including, but not limited to, xanthene fluorophore molecules, dansyl fluorophore molecules, coumarin and coumarin derivatives, fluorescent acridinium moieties, benzo[a]pyrene-based fluorophore molecules, as well as 7-nitrobenz-2-oxa-1,3-diazole, and 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diamino-propionic acid. Preferably, the fluorescent molecule has a high quantum yield of fluorescence at a wavelength different from that of natural amino acids, and more preferably, has a high quantum yield of fluorescence that can be excited in the visible portion of the spectrum, or both the UV and visible portions. When excited at a preselected wavelength, the molecule is detectable at low concentrations visually or using conventional fluorescence detection methods. Electrochemiluminescent molecules such as ruthenium chelates and their derivatives, or nitroxide amino acids and their derivatives, are detectable below the femtomolar range.

[0078] In some embodiments, the functional element is a fluorescent dye molecule. Suitable fluorescent dye molecules for linking to the compounds herein (e.g., for forming a fluorescent tracer) include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile Red, Nile Blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphyrin, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA FLuoR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate, etc.), quantum dot nanocrystals, and the like, but are not limited thereto.In some embodiments, the fluorescent dye molecule is a rhodamine analog (e.g., a carboxyrhodamine analog), such as those described in U.S. Patent Application No. 13 / 682,589, which is hereby incorporated by reference in its entirety.

[0079] In addition to fluorescent molecules, various molecules having physical properties based on the interaction and response of the molecule to electromagnetic fields and radiation are used in the compositions and methods described herein. These properties include absorption in the UV, visible, and infrared regions of the electromagnetic spectrum, Raman activity, and the presence of chromophores that can be further enhanced by resonance Raman spectroscopy, electron spin resonance activity, and nuclear magnetic resonance and molecular weight via, for example, a mass spectrometer.

[0080] In some embodiments, the functional element is a capture element. In some embodiments, the capture element is a substrate of a protein (e.g., an enzyme), and the capture agent is the protein. In some embodiments, the capture element is a "covalent substrate", or one that forms a covalent bond with the protein or enzyme to which it reacts. The substrate may include a reactive group (e.g., a modified substrate) that forms a covalent bond with the enzyme upon interaction with the enzyme, or the enzyme may be a mutant version that cannot reconcile the covalently bound intermediate with the substrate. In some embodiments, the substrate is recognized by a mutant protein (e.g., a mutant dehalogenase) that forms a covalent bond with it. In such embodiments, the interaction of the substrate with the wild-type version of the protein (e.g., dehalogenase) results in the product and regeneration of the wild-type protein, while the interaction of the substrate (e.g., haloalkane) with the mutant version of the protein (e.g., dehalogenase) results in the formation of a stable bond (e.g., covalent bond formation) between the protein and the substrate. The substrate may be any substrate suitable for any mutant protein that is modified to form an ultra-stable or covalent bond with a substrate that is normally only temporarily bound by a protein. In some embodiments, the protein is a mutant hydrolase or dehalogenase. In some embodiments, the protein is a mutant dehalogenase and the substrate is a haloalkane. In some embodiments, the haloalkane is an alkane (e.g., C2-C 20 ) capped by a terminal halogen (e.g., Cl, Br, F, I, etc.). In some embodiments, the haloalkane is of the formula A-X, where X is a halogen (e.g., Cl, Br, F, I, etc.) and A is an alkane containing 2 to 20 carbons. In certain embodiments, A includes a straight-chain segment of 2 to 12 carbons. In certain embodiments, A is a straight-chain segment of 2 to 12 carbons. In some embodiments, the haloalkane may include any additional pendant or substitution that does not interfere with its interaction with the mutant dehalogenase.

[0081] In some embodiments, the capture agent is a SNAP tag and the capture element is benzylguanine (see, e.g., Crivat G, Taraska JW, (January, 2012). Trends in Biotechnology 30(1):8-16; which is incorporated herein by reference in its entirety). In some embodiments, the capture agent is a CLIP tag and the capture element is benzylcytosine (see, e.g., Gautier, et al. Chem Biol. 2008 Feb;15(2):128-36.; which is incorporated herein by reference in its entirety).

[0082] In some embodiments, the functional element is an affinity element (e.g., something that binds to an affinity agent). Examples of such pairs include an antibody as the affinity agent and an antigen as the affinity element; a His tag as the affinity element and a nickel column as the affinity agent; and a protein and a small molecule (e.g., streptavidin and biotin) that have high affinity as the affinity agent and the affinity element, respectively. Examples of affinity molecules include, for example, immunogenic molecules (e.g., epitopes of proteins, peptides, carbohydrates, or lipids) (e.g., any molecule useful for preparing an antibody specific to that molecule)); biotin, avidin, streptavidin, and their derivatives; metal-binding molecules; and molecules such as fragments and combinations of these molecules. Exemplary affinity molecules include His5 (HHHHH) (SEQ ID NO: 15), HisX6 (HHHHHH) (SEQ ID NO: 16), C-myc (EQKLISEEDL) (SEQ ID NO: 17), Flag (DYKDDDDK) (SEQ ID NO: 18), SteptTag (WSHPQFEK) (SEQ ID NO: 19), HA tag (YPYDVPDYA) (SEQ ID NO: 20), thioredoxin, cellulose-binding domain, chitin-binding domain, S-peptide, T7 peptide, calmodulin-binding peptide, C-terminal RNA tag, metal-binding domain, metal-binding reactive group, amino acid reactive group, intein, biotin, streptavidin, and maltose-binding protein. Another example of an affinity molecule is dansyl lysine. Antibodies that interact with the dansyl ring are commercially available (Sigma Chemical; St. Louis, Mo.) or can be prepared using known protocols as described in Antibodies: A Laboratory Manual (Harlow and Lane, 1988).

[0083] In some embodiments, provided herein are methods for detecting, isolating, analyzing, characterizing, etc., GPCRs in a system (e.g., a cell, cell lysate, sample, biochemical solution or mixture, tissue, organism, etc.) using the compounds provided herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) alone or in combination with a functional element (e.g., directly or via a suitable linker).

[0084] In some embodiments, provided herein is a method for detecting one or more GPCRs in a sample, the method comprising contacting the sample with a compound provided herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.). In some embodiments, provided herein is a method for isolating one or more GPCRs from a sample.

[0085] In some embodiments, provided is a method for characterizing a sample by analyzing the presence, amount, or population of GPCRs (e.g., which GPCRs are present and / or in what amounts) in the sample by contacting the sample with a compound provided herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.).

[0086] In some embodiments, provided herein is a method for diagnosing a disease state, comprising detecting the presence or amount of one or more GPCRs in a sample from a subject by contacting the sample with a compound provided herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.), wherein the presence or amount of one or more GPCRs in the sample indicates the disease, state, or predisposition thereto.

[0087] In some embodiments, provided herein is a method of monitoring a subject's response to a therapeutic treatment, comprising: (a) detecting the presence or amount of one or more GPCRs in a sample from the subject by contacting the sample with a compound herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, analogs or derivatives thereof, etc.) prior to administration of the therapeutic treatment; and (b) detecting the presence or amount of one or more GPCRs in a sample from the subject by contacting the sample with a compound herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, analogs or derivatives thereof) subsequent to administration of the therapeutic treatment, wherein a change in the presence or amount of one or more GPCRs indicates the subject's response to the therapeutic treatment.

[0088] In some embodiments, the GPCRs bound by the compounds herein are detected, quantified, and / or isolated by any means that utilizes the unique properties of the compounds and / or functional elements bound thereto, including electrophoresis, gel filtration, high-pressure or high-speed liquid chromatography, mass spectrometry, affinity chromatography, ion-exchange chromatography, chemical extraction, magnetic bead separation, precipitation, hydrophobic interaction chromatography (HIC), or any combination thereof. The isolated GPCR(s) may be employed in structural and functional studies for diagnostic use, preparation of biological or pharmaceutical reagents, as a tool for drug development, and for isolation and characterization of protein complexes, and the like, for the study of protein interactions.

[0089] In some embodiments, methods are provided for detecting and / or quantifying a compound (e.g., including CC CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) and / or an analyte (e.g., GPCR) bound thereto in a sample. In some embodiments, techniques for the detection and / or quantification of a compound (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, its analogs or derivatives, etc.) and / or an analyte (e.g., GPCR) bound thereto in the present specification rely on the uniqueness of functional elements (e.g., capture elements, affinity elements, detectable elements (e.g., fluorescent dye molecules, luciferase, chelated radionuclides, chelated contrast agents, etc.)) bound to the compound and / or specific modifications to the compound (e.g., mass tags (e.g., heavy isotopes (e.g., 13 C, 15 N, 2 H, etc.)). For example, if a compound (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) in the present specification is linked to a fluorescent dye molecule or other luminescent functional element, a system, device, and / or apparatus provided for detecting, quantifying, or monitoring the amount or change in the emitted light (e.g., fluorescence) may be used to detect / quantify the compound and / or an analyte (e.g., GPCR) bound thereto in a sample. In some embodiments, the detection, quantification, and / or monitoring is provided by a device, system, or apparatus including one or more of a spectrophotometer, fluorometer, luminometer, photomultiplier tube, photodiode, nephelometer, photon counter, electrode, ammeter, voltmeter, capacitance sensor, flow cytometer, CCD, etc.

[0090] In addition to fluorescent functional elements, various functional elements having physical properties based on the interaction and response of functional elements to electromagnetic fields and radiation can be used to detect the compounds (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, analogs or derivatives thereof, etc.) and / or the bound GPCRs of the present specification. These properties include absorption in the UV, visible, and infrared regions of the electromagnetic spectrum, Raman activity, the presence of chromophores that can be further enhanced by resonance Raman spectroscopy, electron spin resonance activity, nuclear magnetic resonance, and molecular weight via, for example, a mass spectrometer.

[0091] In some embodiments, the compounds herein bind to a broad spectrum of GPCRs including Class A, Class B, Class C, Frizzled class, adhesion class protein GPCRs, and other seven transmembrane proteins. In some embodiments, the binders herein are, for example, 5-hydroxytryptamine receptor, acetylcholine receptor (muscarinic), adenosine receptor, adrenergic receptor, angiotensin receptor, apelin receptor, bile acid receptor, bombesin receptor, bradykinin receptor, cannabinoid receptor, chemokine receptor, cholecystokinin receptor, Class A orphan, complement peptide receptor, dopamine receptor, endothelin receptor, formyl peptide receptor, free fatty acid receptor, galanin receptor, ghrelin receptor, glycoprotein hormone receptor, gonadotropin releasing hormone receptor, histamine receptor, hydroxycarboxylic acid receptor, leukotriene receptor, lysophospholipid (LPA) receptor, lysophospholipid (S1P) receptor, melanin concentrating hormone receptor, melanocortin receptor, melatonin receptor, neuromedin U receptor, neuropeptide FF / neuropeptide AF receptor, neuropeptide W / neuropeptide B receptor, neuropeptide Y receptor, neurotensin receptor, opioid receptor, opsin receptor, orexin receptor, P2Y receptor, prokineticin receptor, prolactin releasing peptide receptor, prostanoid receptor, protease activated receptor, QRFP receptor, relaxin family peptide receptor, somatostatin receptor, succinate receptor, tachykinin receptor, thyrotropin releasing hormone receptor, trace amine receptor, urotensin receptor, vasopressin and oxytocin receptor, calcitonin receptor, corticotropin releasing factor receptor, glucagon receptor family, parathyroid hormone receptor, VIP and PACAP receptor, calcium sensing receptor, Class C orphan, GABA BBinds to multiple different GPCRs such as receptors, metabotropic glutamate receptors, taste 1 receptors, class Frizzled GPCRs, adhesion class GPCRs, and / or GPCRs of multiple GPCR families. In some embodiments, the compounds herein bind to GPCRs of any suitable organism. In some embodiments, the compounds herein bind to human GPCRs and / or homologs and analogs from other organisms.

[0092] In some embodiments, the binding agents herein are broad-spectrum GPCR binding agents. Thus, the binding agents herein may bind to GPCRs of multiple (e.g., 2, 3, 4, 5, 10, 20, 30, 40, or more) GPCR classes or GPCR families. In some embodiments, the binding agents herein bind to multiple (e.g., 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, or more) distinct GPCRs.

[0093] In some embodiments, the GPCR binding agents and tracers described herein are used in systems and methods using such systems that further comprise a bioluminescent protein (or bioluminescent complex) to generate bioluminescence resonance energy transfer (BRET) for the detection, characterization, and monitoring of GPCRs. Accordingly, the disclosure includes bioluminescent polypeptides, bioluminescent complexes and their components, and materials and methods related to bioluminescence resonance energy transfer (BRET).

[0094] In some embodiments, provided herein are assays, devices, methods, and systems that incorporate a bioluminescent polypeptide and / or a bioluminescent complex (of peptide(s) and / or polypeptide component(s)) based on, for example, structurally, functionally, etc., Oplophorus gracilirostris luciferase, NanoLuc luciferase (Promega Corporation; U.S. Patent No. 8,557,970; U.S. Patent No. 8,669,103; which are hereby incorporated by reference in their entireties), and / or NanoBiT (US9,797,889; which is hereby incorporated by reference in its entirety), or NanoTrip (U.S. Provisional Patent Application No. 62 / 684,014). As described below, in some embodiments, the assays, devices, methods, and systems herein incorporate commercially available NanoLuc-based technologies (e.g., NanoLuc luciferase, NanoBRET, NanoBiT, NanoTrip, NanoGlo, etc.), while in other embodiments, various combinations, modifications, or derivatives from commercially available NanoLuc-based technologies are employed.

[0095] PCT Application No. PCT / US2010 / 033449, U.S. Patent No. 8,557,970, PCT Application No. PCT / 2011 / 059018, and U.S. Patent No. 8,669,103 (each of which is hereby incorporated by reference in its entirety and for all purposes) describe compositions and methods containing bioluminescent polypeptides. Such polypeptides are used in the embodiments herein and can be used in conjunction with the assays and methods described herein.

[0096] In some embodiments, the assays, methods, devices, and systems described herein include the bioluminescent polypeptide of SEQ ID NO: 5, or a bioluminescent polypeptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity to SEQ ID NO: 5. In some embodiments, the bioluminescent polypeptide is fused to a GPCR or otherwise linked to a component of the assays, methods, devices, and / or systems described herein.

[0097] U.S. Patent Application No. PCT / US14 / 26354 and U.S. Patent No. 9,797,889 (each of which is hereby incorporated by reference in its entirety and for all purposes) describe compositions and methods for the organization of bioluminescent complexes. Such complexes, as well as their peptide and polypeptide components, are used in the embodiments herein and can be used in conjunction with the assays, methods, devices, and / or systems described herein. In some embodiments, provided herein is a polypeptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 9, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6. In some embodiments, provided herein is a peptide having at least 60% (e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 10, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8. In some embodiments, provided herein is a peptide having at least 60% (e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 11, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8.In some embodiments, any of the aforementioned NanoBiT-based peptides or polypeptides is fused to a GPCR or otherwise linked to (e.g., fused to, chemically linked to, etc.) components of the assays, methods, devices, and / or systems described herein.

[0098] U.S. Provisional Patent Application No. 62 / 684,014 (which is hereby incorporated by reference in its entirety for all purposes) describes compositions and methods for the organization of bioluminescent complexes. Such complexes, as well as their peptide and polypeptide components, are used in the embodiments herein and can be used in conjunction with the assays and methods described herein. In some embodiments, provided herein is a polypeptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 12, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 9. In some embodiments, provided herein is a peptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 11, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8. In some embodiments, provided herein is a peptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 13, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7.In some embodiments, provided herein is a peptide having at least 60% (e.g., 66%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 14, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8. In some embodiments, any of the aforementioned NanoTrip-based peptides or polypeptides is fused to a GPCR or otherwise linked (e.g., fused, chemically linked, etc.) to a component of an assay, method, device, and / or system described herein.

[0099] PCT Application No. PCT / US13 / 74765 and U.S. Patent Application No. 15 / 263,416 (which are hereby incorporated by reference in their entireties for all purposes) describe bioluminescence resonance energy transfer (BRET) systems and methods (e.g., incorporating NanoLuc-based technology). Such systems and methods, as well as bioluminescent polypeptides and their fluorescent dye molecule binding components, are used in the embodiments herein and can be used in conjunction with the assays, methods, devices, and systems described herein.

[0100] In some embodiments, any NanoLuc-based, NanoBiT-based, and / or NanoTrip-based peptides, polypeptides, complexes, fusions, etc. may be used in BRET-based applications with the assays, methods, devices, and systems described herein.

[0101] As used herein, the term "energy acceptor" refers to any small molecule (e.g., a chromophore), macromolecule (e.g., a self-fluorescent protein, a phycobiliprotein, a nanoparticle, a surface, etc.), or molecular complex that generates a readily detectable signal (e.g., via resonance energy transfer) in response to energy absorption. In certain embodiments, the energy acceptor is a fluorescent dye molecule or other detectable chromophore (e.g., any fluorescent dye molecule or other detectable chromophore described herein or understood in the art).Suitable fluorescent dye molecules include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile Red, Nile Blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphyrin, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA FLuoR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate, etc.), quantum dot nanocrystals, etc., but are not limited thereto. In some embodiments, the fluorescent dye molecule is a rhodamine analog (e.g., a carboxyrhodamine analog) such as those described in U.S. Patent Application No. 13 / 682,589, which is hereby incorporated by reference in its entirety.

[0102] In some embodiments, a system is provided that includes (a) a fusion of a GPCR and a bioluminescent protein (or a component of a bioluminescent complex); and (b) a broad-spectrum GPCR-binding moiety of the present specification linked to a fluorescent dye molecule, wherein the emission spectrum of the bioluminescent protein overlaps the excitation spectrum of the fluorescent dye molecule such that BRET is detectable between the bioluminescent protein and the fluorescent dye molecule when the broad-spectrum GPCR-binding moiety binds to the GPCR. Similar BRET systems (e.g., utilizing NANOLUC luciferase) are described, for example, in International Patent Application No. PCT / US13 / 74765, which is hereby incorporated by reference in its entirety, and embodiments thereof will be used in the systems and methods of the present specification.

[0103] U.S. Patent No. 10,107,800; U.S. Patent No. 9,869,670; and U.S. Patent No. 9,797,890 (each incorporated herein by reference in its entirety) describe a two-component system for organizing a bioluminescent complex from a peptide component and a polypeptide component. U.S. Provisional Patent Application No. 62 / 684,014 (incorporated herein by reference in its entirety) describes a three-component system for organizing a bioluminescent complex from three peptide components and a polypeptide component. In some embodiments, such systems (and related methods) are used in the embodiments herein. For example, the peptide component of the bioluminescent complex is provided as a fusion with one or more GPCRs. When such a fusion is contacted with the broad-spectrum GPCR fluorescent tracer described herein and the polypeptide component of the bioluminescent complex, a BRET signal is detectable. However, non-target GPCRs that are not fused to the peptide component of the bioluminescent complex will not generate a BRET signal, even though they are bound to the broad-spectrum GPCR fluorescent tracer. In some embodiments, the peptide tag (e.g., fused to a GPCR) and other components of the bioluminescent complex system (e.g., polypeptide component, substrate, etc.) are described in the above patents / patent applications and / or are commercially available as NanoBiT and / or NanoTrip technology (Promega Corp., Madison, WI). In some embodiments, the peptide tag fused to a GPCR for BRET applications exhibits high affinity for the polypeptide component (e.g., and / or additional peptide components) of the bioluminescent complex, such that upon introduction of the appropriate components, the bioluminescent complex is formed without facilitation.

[0104] In some embodiments, the BRET application of the technology described herein relies on a GPCR protein structure that is minimally destabilized by genetic fusion to the peptide component of the bioluminescent complex. In some embodiments, the peptide exhibits high affinity for the polypeptide component of the bioluminescent complex and / or other peptide components (e.g., HiBiT). In some embodiments, the fusion occurs N-terminally, C-terminally, and internally within the GPCR. In some embodiments, the small size of the peptide tag enables minimal genetic manipulation of the protein. Experiments conducted during the development of the embodiments herein demonstrated that peptide tags (e.g., components of bioluminescent complexes (e.g., HiBiT)) can be readily inserted into the GPCR of interest via CRISPR-Cas, obviating the need to overexpress the GPCR and / or enabling interrogation of GPCR-ligand interactions without using membrane preparations. In some embodiments, the polypeptide component of the bioluminescent complex (e.g., LgBiT) is not cell permeable, so the signal is detected only on the cell surface. This feature of the detection method enables monitoring of both the cell surface expression level and internalization of the GPCR.

[0105] Figure 1 shows an exemplary BRET embodiment of the compositions and methods herein. The fluorescence signal is generated via energy transfer between the HiBiT-tagged GPCR protein (via formation of the bioluminescent HiBiT-LgBiT complex) and the fluorescent ligand, which enables real-time monitoring of GPCR-fluorescent ligand interactions. Substituting the fluorescent ligand with an unlabeled ligand results in loss of the BRET signal, enabling determination of both the kinetic and binding data of the unlabeled compound. The advantage of this approach is the detection of only the interaction between the fluorescent ligand and the HiBiT-GPCR fusion. Since other interactions of the fluorescent ligand are not detected, the background is significantly reduced and the sensitivity of BRET signal detection is improved (Figure 2).

[0106] In some embodiments, provided in certain embodiments herein are, for example, U.S. Patent No. 7,238,842; U.S. Patent No. 7,425,436; U.S. Patent No. 7,429,472; U.S. Patent No. 7,867,726, each of which is incorporated herein by reference in its entirety, systems comprising mutant proteins (e.g., mutant hydrolases (e.g., mutant dehalogenases)) that covalently bind their substrates (e.g., haloalkane substrates). Such proteins may be provided as fusions with GPCRs. In other embodiments, such proteins are used to capture GPCRs bound to the agents described herein (e.g., when the functional group is a substrate of the mutant protein).

Example

[0107]

Chemical formula

[0108]

Chemical formula

[0109] [Chemical formula] A 10 mL microwave vial equipped with a stir bar was charged with SL-1202 (56 mg, 0.21 mmol), tert-butyl (3-(piperazin-1-yl)propyl)carbamate (104 mg, 126 μmol), K2CO3 (74 mg, 0.53 mmol), and dioxane (4 mL). The vial was placed in a microwave reactor and heated to 120 °C for 1 hour. Consumption of the starting material was confirmed by HPLC analysis, and then the solution was filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0→20% MeOH / DCM) to afford 72 mg (73% yield) of amidine SL-1236 as a yellow solid. 1 1H-NMR (400 MHz, DMSO-d6) δ 7.33 (td, J = 7.8, 1.5 Hz, 1H), 7.26 - 7.10 (m, 2H), 7.09 - 6.92 (m, 2H), 6.87 - 6.71 (m, 3H), 2.94 (app.q, J = 6.6 Hz, 2H), 2.42 (s, 3H, partially overlapping with DMSO-d5), 2.30 (t, J = 7.3 Hz, 2H, partially overlapping with DMSO-d5 - 12 C and partially overlapping), 1.66 - 1.46 (m, 2H), 1.37 (s, 9H); HRMS (ESI) C 25 H 33 ClN5O2 [M + H] + Calculated for: 470.2300; Found: 470.2300.

[0110] [Chemical formula] A 50 mL flask equipped with a stir bar was charged with amidine SL-1236 (72 mg, 0.15 mmol) and cleavage cocktail (10 mL, 85:15:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22 °C for 20 h, at which point HPLC indicated complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to afford 72 mg (97% yield) of primary amine SL-1239 as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ 7.81 (s, 2H), 7.50 - 7.36 (m, 2H), 7.32 (dd, J = 7.8, 1.6 Hz, 1H), 7.14 - 6.99 (m, 2H), 6.99 - 6.76 (m, 3H), 3.96 (br.s, 1H), 3.54 (br.s.1H), 3.31 (br.s, 5H), 2.53 - 2.51 (m, 2H, overlapping with DMSO-d5) 2.88 (m, 2H), 2.00 - 1.87 (m, 2H); HRMS (ESI) C 20 H 25 ClN5[M+H] + Calculated for: 370.1798; Found: 370.1790.

[0111] [Chemical formula] A 25 mL flask equipped with a stir bar was charged with SL-1239 (12 mg, 25 μmol), 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azacosan-20-oic acid (14 mg, 37 μmol), HATU (12 mg, 31 μmol), NEt3 (24 μL, 0.17 mmol), and DMF (6 mL). The resulting pale yellow solution was stirred at 22 °C for 1 h, at which point HPLC indicated complete consumption of the starting material, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to afford 18 mg (quantitative yield) of amide SL-1448 as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.58 - 7.51 (m, 1H), 7.47 (dd, J = 7.8, 1.6 Hz, 1H), 7.227.20 (m 1H), 7.20 - 7.08 (m, 3H), 6.97 (d, J = 8.5 Hz, 1H), 3.94 (br.s, 4H), 3.76 (t, J = 5.8 Hz, 2H), 3.61 (m, 12H), 3.49 (m, 6H), 3.38 (t, J = 6.3 Hz, 2H), 3.28 - 3.03 (m, 4H), 2.50 (t, J = 5.8 Hz, 2H), 2.11 - 1.86 (m, 2H), 1.43 (s, 9H). MS (ESI) C 36 H 54 ClN6O7 [M + H] + Calculated value for: 717.37; Measured value: 717.58.

[0112]

Chemical formula

[0113]

Chemical formula

[0114]

Chemical formula

[0115]

Chemical Structure

[0116]

Chem.

[0117]

Chemical Structure

[0118]

Chem.

[0119]

Chem.

[0120]

Chem.

[0121]

Chemical Structure

[0122]

Chemical formula

[0123]

Chemical formula

[0124] [Chemical formula] A 25 mL flask equipped with a stir bar was charged with SL-1432 (4.8 mg, 11 μmol), BODIPY576 / 589SE (4.8 mg, 11 μmol), DIPEA (14 μL, 79 μmol), and DMF (10 mL). The resulting deep purple solution was stirred at 22 °C for 3 h, at which point HPLC indicated complete consumption of the starting material, and the reaction mixture was purified by preparative HPLC (C18, 5→95% MeCN / H2O, 0.05% TFA) to afford 4.3 mg (52% yield) of the amide SL-1434 as a deep purple film. 1H-NMR (400 MHz, MeOD) δ 7.42 (ddd, J = 8.3, 7.3, 1.6 Hz, 1H), 7.34 (dd, J = 7.7, 1.6 Hz, 1H), 7.28 - 7.16 (m, 4H), 7.16 - 7.10 (m, 3H), 7.10 - 7.01 (m, 2H), 6.98 (d, J = 8.7 Hz, 1H), 6.88 (d, J = 4.0 Hz, 1H), 6.37 (dd, J = 3.9, 2.5 Hz, 1H), 6.30 (d, J = 4.0 Hz, 1H), 4.23 (s, 2H), 4.09 - 3.55 (br.s, 4H), 3.55 - 3.40 (br.s, 4H), 3.40 - 3.32 (m, 2H), 3.27 (t, J = 7.2 Hz, 1H), 3.20 (t, J = 7.2 Hz, 1H), 3.17 - 3.08 (m, 2H), 2.93 (s, 3H), 2.59 (t, J = 7.4 Hz, 2H); HRMS (ESI) C 38 H 40 Calculated for BClF2N9O2[M + H]+: 738.3055; Found: 738.3055.

[0125]

Chem.

[0126]

Chem.

[0127]

Chem.

[0128]

Chem.

[0129]

Chemical Structure

[0130]

Chem.

[0131]

Chem.

[0132]

Chem.

[0133]

Chem.

[0134]

Chemical Structure

[0135]

Chemical Structure

[0136]

Chem.

[0137]

Chem.

[0138]

Chemical Structure

[0139]

Chemical Structure

[0140]

Chemical Structure

[0141]

Chem.

[0142]

Chem.

[0143]

Chemical formula

[0144]

Chemical formula

[0145]

Chemical formula

[0146]

Chemical Structure

[0147]

Chemical Structure

[0148]

Chemical formula

[0149]

Chemical formula

[0150] [Chemical formula] A 25 mL flask equipped with a stir bar was charged with SL-1545 (6 mg, 10 μmol), BODIPY576 / 589SE (3.5 mg, 8 μmol), DIPEA (14 μL, 82 μmol), and DMF (8 mL). The resulting dark purple solution was stirred at 22 °C for 3 h, at which point HPLC indicated complete consumption of the starting material, and the reaction mixture was purified by preparative HPLC (C18, 5→95% MeCN / H2O, 0.05% TFA) to afford 2 mg (27% yield) of the amide SL-1516 as a dark purple film. 1H-NMR (400 MHz, MeOD) δ 7.28 (ddd, J = 8.0, 7.2, 1.7 Hz, 1H), 7.25 (s, 1H), 7.24 - 7.18 (m, 4H), 7.15 (ddd, J = 8.0, 7.2, 1.3 Hz, 1H), 7.01 (d, J = 4.6 Hz, 1H), 6.96 - 6.85 (m, 2H), 6.55 (q, J = 1.2 Hz, 1H), 6.34 (td, J = 4.2, 1.8 Hz, 2H), 4.03 (s, 4H), 3.74 (t, J = 5.8 Hz, 2H), 3.59 (d, J = 3.7 Hz, 12H), 3.53 (t, J = 5.6 Hz, 2H), 3.46 (br.s, 4H), 3.41 - 3.34 (m, 4H), 3.27 (d, J = 7.6 Hz, 2H), 3.18 (t, J = 7.0 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 2.48 (t, J = 5.7 Hz, 2H), 2.34 (d, J = 1.2 Hz, 3H), 1.95 (p, J = 6.8 Hz, 2H); MS (ESI) C 46 H 59 BF2N9O6S [M + H] + Calculated value for: 914.44; Measured value: 914.26.

[0151]

Chemical Structure

[0152]

Chem.

[0153]

Chem.

[0154]

Chem.

[0155]

Chem.

[0156]

Chem.

[0157]

Chem.

[0158]

Chem.

[0159]

Chem.

[0160]

Chem.

[0161] Synthesis of Amitriptyline Fluorescent Tracer:

Chemical Structure

[0162]

Chemical formula

[0163]

Chemical Structure

[0164] A 25 mL round-bottom flask equipped with a stir bar was charged with SL-1822 (90 mg, 0.25 mmol) and a 2 M solution of diethylamine in THF (12 mL, 25 mmol). The reaction mixture was stirred at 50 °C for 20 hours, at which point HPLC indicated complete consumption of the starting material, and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 100% EtOAc / heptane) to afford 26 mg (35% yield) of the amine SL-1823 as a clear oil. 1 1H-NMR (400 MHz, CDCl3) δ 7.37 (d, J = 2.3 Hz, 1H), 7.28 (dd, J = 8.0, 6.6 Hz, 2H), 7.23 - 7.16 (m, 3H), 7.14 (dd, J = 8.2, 2.3 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 3.07 - 2.95 (m, 2H), 2.95 - 2.81 (m, 2H), 2.64 (s, 4H), 2.31 (s, 6H); 13 13C NMR (100 MHz, CDCl3) δ 13C-NMR (101 MHz, CDCl3) δ 142.0, 141.6, 131.9, 131.3, 130.0, 128.4, 128.3, 127.9, 126.0, 124.8, 78.8, 77.3, 77.0, 76.7, 58.3, 45.1, 36.7, 36.2, 18.5; HRMS (ESI) C 20 H 23 CLN [M + H] + Calculated for: 312.1519; Found: 312.1516.

[0165]

Chemical Structure

[0166] A 50 mL round-bottom flask equipped with a stir bar and a septum was charged with SL-1824 (25 mg, 80 μmol), K2CO3 (33 mg, 0.24 mmol), Pd(OAc)2 (1.8 mg, 8.0 μmol), and [dcpp2BF4] (9.8 mg, 16 μmol). The flask was evacuated and filled back with argon (repeated 3 times). Degassed DMSO (2 mL) and H2O (0.2 mL) were added, the reaction vessel was evacuated and filled back with carbon monoxide (repeated 3 times). CO was bubbled through the solution for 5 minutes. The resulting yellow suspension was heated to 110 °C for 18 hours under a CO balloon, and at that point HPLC analysis showed complete consumption of the starting material. The reaction mixture was diluted with MeOH (3 mL), passed through a syringe filter, and purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to give 25 mg (97% yield) of a mixture of E / Z of carboxylic acid SL-1825 as a clear oil. 1H-NMR (400 MHz, reported for a mixture of E / Z isomers in MeOD) δ 8.06 - 7.67 (m, 2H), 7.49 - 6.92 (m, 5H), 5.89 (m, 1H), 3.49 - 3.34 (m, 2H), 3.25 (m, 2H), 3.09 - 2.90 (m, 1H), 2.81 (d, J = 12.1 Hz, 7H), 2.66 - 2.40 (m, 2H); MS (ESI) C 21 H 24 NO2[M + H] + Calculated for: 322.18; Found: 322.15.

[0167]

Chem.

[0168]

Chem.

[0169]

Chemical Structure

[0170]

Chem.

[0171]

Chem.

[0172]

Chemical Structure

[0173]

Chemical Structure

[0174]

Chemical Structure

[0175]

Chemical formula

[0176]

Chemical formula

[0177] A 50 mL round-bottom flask equipped with a stir bar was charged with SL-1828 (270 mg, 0.74 mmol) and a 2 M solution of diethylamine in THF (37 mL, 74 mmol). The reaction mixture was stirred at 50 °C for 17 h, at which point HPLC indicated complete consumption of the starting material and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 20% MeOH / DCM) to afford 125 mg (54% yield) of the amine SL-1829 as a clear oil. 1 1H-NMR (400 MHz, CDCl3) δ 7.39 - 7.27 (m, 3H), 7.24 - 7.18 (m, 3H), 7.16 - 7.03 (m, 2H), 3.08 - 2.95 (m, 2H), 2.95 - 2.83 (m, 2H), 2.63 (s, 4H), 2.31 (s, 6H); 13 13C-NMR (100 MHz, CDCl3) δ 145.4, 141.6, 133.4, 128.8, 128.4, 126.1, 126.0, 121.7, 58.4, 45.1, 36.7, 36.7, 18.5; HRMS (ESI) C 20 H 23 ClN [M + H] +Calculation for: 312.1519; Measured value: 312.1516.

[0178]

Chem.

[0179] [Chem.] A 50 mL round-bottom flask equipped with a stir bar and a septum was charged with SL-1832 (100 mg, 0.32 mmol), K2CO3 (130 mg, 0.96 mmol), Pd(OAc)2 (7.2 mg, 32 μmol), and [dcpp2BF4] (39 mg, 64 μmol). The flask was evacuated and filled with argon and then backfilled (repeated 3 times). Degassed DMSO (8 mL) and H2O (0.8 mL) were added, the reaction vessel was evacuated, and filled with carbon monoxide and then backfilled (repeated 3 times). CO was bubbled through the solution for 5 minutes. The resulting yellow suspension was heated at 110 °C for 22 hours under a CO balloon, at which point HPLC analysis showed complete consumption of the starting material. The reaction mixture was diluted with MeOH (8 mL), filtered through a syringe filter, and purified by preparative HPLC (C18, 5→95% MeCN / H2O, 0.05% TFA) to give 54 mg (51% yield) of SL-1834-E as a clear oil and 51 mg (48% yield) of SL-1834-Z as a clear oil. The E isomer has a shorter retention time than the Z isomer. Characteristic data for SL-1834-E: 1 1H-NMR (400 MHz, MeOD) δ 7.82 (dd, J = 8.0, 1.8 Hz, 1H), 7.78 (d, J = 1.8 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.36 - 7.24 (m, 3H), 7.23 - 7.16 (m, 1H), 5.93 (t, J = 7.3 Hz, 1H), 3.39 (t, J = 9.0 Hz, 2H), 3.26 (q, J = 7.4 Hz, 2H), 3.02 (d, J = 14.9 Hz, 1H), 2.82 (d, J = 8.1 Hz, 7H), 2.60 (dd, J = 16.9, 8.3 Hz, 2H); 13 13C NMR (100 MHz, MeOD) δ 169.6, 147.7, 146.1, 140.6, 139.7, 138.7, 132.8, 131.1, 129.7, 129.6, 129.6, 129.1, 128.5, 127.4, 126.5, 58.0, 34.8, 32.7, 26.2; HRMS (ESI) C 21 H 24 NO2 [M + H] +Calculated value for: 322.1807; Measured value: 322.1807. Characteristic data for SL-1834-Z: 1 H-NMR (400 MHz, MeOD) δ 7.97 (d, J = 1.7 Hz, 1H), 7.92 (dd, J = 7.8, 1.8 Hz, 1H), 7.37 - 7.25 (m, 2H), 7.25 - 7.13 (m, 2H), 7.10 (dd, J = 7.2, 1.9 Hz, 1H), 5.89 (t, J = 7.3 Hz, 1H), 3.55 - 3.36 (m, 2H), 3.26 (q, J = 8.6 Hz, 2H), 2.97 (d, J = 25.6 Hz, 2H), 2.82 (d, J = 6.3 Hz, 6H), 2.68 - 2.45 (m, 2H); 13 C NMR (100 MHz, MeOD) δ 169.5, 147.6, 145.4, 141.2, 140.8, 138.2, 131.7, 131.3, 130.7, 129.5, 129.1, 129.0, 128.8, 127.4, 125.9, 58.0, 34.5, 32.8, 26.2; HRMS (ESI) C 21 H 24 NO2 [M + H] + Calculated value for: 322.1807; Measured value: 322.1807.

[0180]

Chemical formula

[0181] A 10 mL flask equipped with a stir bar was charged with SL-1839 (20 mg, 35 μmol) and the cleavage cocktail (4 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22 °C for 75 minutes, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure. The crude residue was used in the next step without further purification. MS(ESI) C 23 H 30 N3O3 + [M+H] + Calculated value for: 364.2; Measured value: 364.3. Single peak in HPLC at 254 nm.

[0182] [Chemical formula] A 25 mL flask equipped with a stir bar was charged with SL-1834-Z TFA salt (26 mg, 81 μmol), tert-butyl (2-aminoethyl)carbamate (39 mg, 0.10 mmol), HATU (16 mg, 0.10 mmol), EtN(iPr)2 (72 μL, 0.40 mmol), and DMF (8 mL). The resulting pale yellow solution was stirred at 22 °C for 17 hours, at which point HPLC analysis showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5→95% MeCN / H2O, 0.05% TFA) to give 33 mg (89% yield) of amide SL-1840 as a clear oil. MS(ESI) C 28 H 38 NO3[M+H] + Calculated value for: 464.3; Measured value: 464.4; Single peak in HPLC at 254 nm.

[0183] A 10 mL flask equipped with a stir bar was charged with SL-1840 (15 mg, 35 μmol) and cleavage cocktail (4 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22 °C for 85 minutes, at which point HPLC indicated complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure. The crude residue was used in the next step without further purification. MS(ESI)C 23 H 30 N3O3 + [M+H] + Calculated for: 364.2; Found: 364.4; Single peak by HPLC at 254 nm.

[0184]

Chemical Structure

[0185]

Chemical Structure

[0186] A 10 mL flask equipped with a stir bar was charged with SL-1846 (3.8 mg, 4.6 μmol) and cleavage cocktail (4 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22 °C for 60 min, at which point HPLC indicated complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH and the solvent was removed under reduced pressure. The crude residue was used in the next step without further purification. MS (ESI) C 34 H 52 N4O6 2+ [M + H] 2+Calculated value for / 2: 306.2; Measured value: 306.4; Single peak in HPLC at 254 nm.

[0187] To a solution of SL-1848 (4 mg, 5 μmol) in DMF (6 mL) was added DIPEA (6.0 μL, 33 μmol), followed by the addition of NanoBRET590 SE (2.0 mg, 4.7 μmol, Promega). The resulting solution was reacted at 22 °C for 23 h, at which point HPLC analysis showed complete consumption of the starting material. The solvent was removed under vacuum, and the crude residue was purified by preparative RP HPLC (5→95% MeCN / H2O buffered with 0.5% TFA) to afford 3.0 mg (70% yield) of SL-1850 as a purple thin film. HPLC: 99% purity at 254 nm. 1 1H-NMR (400 MHz, MeOD) δ 7.60 (dd, J = 8.1, 1.9 Hz, 1H), 7.55 (d, J = 1.9 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.28 (td, J = 4.5, 4.1, 2.7 Hz, 2H), 7.26 - 7.17 (m, 5H), 7.17 - 7.10 (m, 1H), 7.01 (d, J = 4.6 Hz, 1H), 6.91 (d, J = 4.0 Hz, 1H), 6.34 (t, J = 3.1 Hz, 1H), 6.31 (d, J = 4.0 Hz, 1H), 5.86 (t, J = 7.3 Hz, 1H), 3.65 (t, J = 6.0 Hz, 2H), 3.56 - 3.43 (m, 16H), 3.41 (d, J = 5.6 Hz, 2H), 3.35 (m, 4H), 3.27 (d, J = 7.7 Hz, 2H), 3.19 (s, 2H), 2.97 (s, 1H), 2.77 (d, J = 13.9 Hz, 7H), 2.63 (t, J = 7.7 Hz, 2H), 2.55 (t, J = 8.8 Hz, 2H), 2.40 (t, J = 6.0 Hz, 2H); HRMS (SI) C 50 H 63 BF2N7O7 + Calculated value for [M + H]+: 922.4850; Measured value: 922.4847.

[0188]

Chemical Structure

[0189]

Chemical Structure

[0190] Sequence WT OgLuc (SEQ ID NO: 1) JPEG2025093913000131.jpg20164

[0191] WT OgLuc Lg (SEQ ID NO: 2) JPEG2025093913000132.jpg20164

[0192] WT OgLuc β9 (SEQ ID NO: 3) JPEG2025093913000133.jpg1236

[0193] WT OgLuc β10 (SEQ ID NO: 4) JPEG2025093913000134.jpg745

[0194] NanoLuc (SEQ ID NO: 5) JPEG2025093913000135.jpg19164

[0195] NanoLuc Lg (SEQ ID NO: 6) JPEG2025093913000136.jpg18164

[0196] NanoLuc β9 (SEQ ID NO: 7) JPEG2025093913000137.jpg741

[0197] NanoLuc β10 (SEQ ID NO: 8) JPEG2025093913000138.jpg746

[0198] LgBiT (SEQ ID NO: 9) JPEG2025093913000139.jpg18164

[0199] SmBiT (SEQ ID NO: 10) JPEG2025093913000140.jpg837

[0200] HiBiT (SEQ ID NO: 11) JPEG2025093913000141.jpg937

[0201] LgTrip(3546) (SEQ ID NO: 12) JPEG2025093913000142.jpg18164

[0202] SmTrip9 (SEQ ID NO: 13) JPEG2025093913000143.jpg837

[0203] β9 / β10 dipeptide (SEQ ID NO: 14) JPEG2025093913000144.jpg968

[0204] His5 (SEQ ID NO: 15) JPEG2025093913000145.jpg620

[0205] HisX6 (SEQ ID NO: 16) JPEG2025093913000146.jpg723

[0206] C-myc (SEQ ID NO: 17) JPEG2025093913000147.jpg731

[0207] Flag (SEQ ID NO: 18) JPEG2025093913000148.jpg732

[0208] SteptTag (SEQ ID NO: 19) JPEG2025093913000149.jpg630

[0209] HA tag (SEQ ID NO: 20) JPEG2025093913000150.jpg733

Claims

1. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 1】 wherein 【Chemistry 2】 is the point of attachment of the broad-spectrum GPCR-binding agent to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR-binding agent and the functional element or solid surface.

2. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 3】 wherein 【Chemistry 4】 is the point of attachment of the broad-spectrum GPCR-binding agent to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR-binding agent and the functional element or solid surface.

3. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 5】 wherein 【Chemistry 6】 is the point of attachment of the broad-spectrum GPCR-binding agent to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR-binding agent and the functional element or solid surface.

4. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 7】 wherein 【Chemistry 8】 is the point of attachment of the broad-spectrum GPCR-binding agent to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR-binding agent and the functional element or solid surface.

5. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 9】 wherein 【Chemistry 10】 is the point of attachment of the broad-spectrum GPCR-binding agent to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR-binding agent and the functional element or solid surface.

6. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 11】 wherein 【Chemistry 12】 is the point of attachment of the broad-spectrum GPCR-binding agent to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR-binding agent and the functional element or solid surface.

7. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 13】 wherein 【Chemistry 14】 is the point of attachment of the broad-spectrum GPCR-binding agent to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR-binding agent and the functional element or solid surface.

8. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 15】 wherein 【Chemistry 16】 is the point of attachment of said broad-spectrum GPCR-binding agent to said functional element, to a solid surface, or to a linker between said broad-spectrum GPCR-binding agent and said functional element or solid surface, and said broad-spectrum GPCR-binding agent may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

9. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 17】 wherein 【Chemistry 18】 is the point of attachment of said broad-spectrum GPCR-binding agent to said functional element, to a solid surface, or to a linker between said broad-spectrum GPCR-binding agent and said functional element or solid surface, and said broad-spectrum GPCR-binding agent may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

10. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 19】 wherein 【Chemistry 20】 is the point of attachment of said broad-spectrum GPCR-binding agent to said functional element, to a solid surface, or to a linker between said broad-spectrum GPCR-binding agent and said functional element or solid surface, and said broad-spectrum GPCR-binding agent may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

11. 1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binding agent bound to a functional element or solid surface, said broad-spectrum GPCR binding agent comprising: 【Chemistry 21】 wherein 【Chemical 22】 is the point of attachment of said broad-spectrum GPCR-binding agent to said functional element, to a solid surface, or to a linker between said broad-spectrum GPCR-binding agent and said functional element or solid surface, and said broad-spectrum GPCR-binding agent may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

12. The composition according to any one of claims 1 to 11, wherein the solid surface is selected from a deposited particle, a membrane, glass, a tube, a well, a self-assembled monolayer, a surface plasmon resonance chip, or a solid support having an electronically conductive surface.

13. The composition of claim 12 , wherein the deposition particles are magnetic particles.

14. The composition of any one of claims 1 to 11, wherein the functional element is selected from a detectable element, an affinity element, and a capture element.

15. 15. The composition of claim 14, wherein the detectable entity comprises a fluorophore, a chromophore, a radionuclide, an electron opaque molecule, an MRI contrast agent, a SPECT contrast agent, or a mass tag.

16. The composition of any one of claims 1 to 11, wherein the broad-spectrum GPCR-binding agent is directly bound to the functional element or solid surface.

17. The composition of any one of claims 1 to 11, wherein the broad-spectrum GPCR-binding agent is attached to the functional element or solid surface via a linker.

18. The linker is [(CH 2 ) 2 O] n 18. The composition of claim 17, comprising:

19. 18. The composition of claim 17, wherein the linker is attached to the broad-spectrum GPCR-binding agent and / or the functional element by an amide bond.

20. The structure: 【Chemistry 23】 2. The composition of claim 1, comprising:

21. The structure: 【Chemistry 24】 3. The composition of claim 2, comprising:

22. The structure: 【Chemistry 25】 4. The composition of claim 3, comprising:

23. The structure: 【Chemistry 26】 5. The composition of claim 4, comprising: wherein n is 0-8, m is 0-8, and X is a functional entity or a solid surface.

24. The structure: 【Chemical 27】 6. The composition of claim 5, comprising:

25. The structure: 【Chemistry 28】 7. The composition of claim 6, comprising:

26. The structure: 【Chemical 29】 8. The composition of claim 7, comprising: wherein n is 0-8, m is 0-8, and X is a functional entity or a solid surface.

27. The structure: 【Chemistry 30】 10. The composition of claim 8 or 9, comprising:

28. The structure: 【Chemistry 31】 12. The composition of claim 10 or 11, comprising:

29. The structure: 【Chemistry 32】 12. The composition of claim 10 or 11, comprising: wherein n is 0 to 8 and X is a functional element or a solid surface.

30. The structure: 【Chemical Formula 33】 10. The composition of claim 8 or 9, comprising:

31. The composition of any one of claims 15 to 30, wherein X is a fluorescent dye molecule.

32. 32. The composition of any one of claims 1 to 31, comprising a non-natural abundance of one or more heavy stable isotopes.

33. A method for detecting or quantifying a GPCR in a sample, the method comprising contacting the sample with a composition according to any one of claims 1 to 32, and detecting or quantifying a functional component of a signal generated thereby.

34. 34. The method of claim 33, wherein the functional components of the signal generated thereby are detected or quantified by fluorescence, mass spectrometry, optical imaging, magnetic resonance imaging (MRI), and energy transfer.

35. A method for isolating a GPCR from a sample, the method comprising contacting the sample with a composition according to any one of claims 1 to 32, and separating the bound GPCR as well as the functional element or solid surface from unbound portions of the sample.

36. A method for characterizing the identity of a GPCR in a sample, comprising isolating the GPCR from a sample by the method of claim 35 and analyzing the isolated GPCR by mass spectrometry.

37. A method for monitoring an interaction between a GPCR and an unmodified biomolecule, the method comprising contacting the sample with a composition according to any one of claims 1 to 32.

38. The method of any one of claims 33 to 37, wherein the sample is selected from a cell, a cell lysate, a body fluid, a tissue, a biological sample, an in vitro sample, and an environmental sample.

39. 1. A system comprising: (a) a composition according to any one of claims 1 to 32, wherein the functional entity is a fluorescent dye molecule; (b) a fusion of a GPCR with a peptide component of a bioluminescent protein or bioluminescent complex, wherein the emission spectrum of the bioluminescent protein or bioluminescent complex overlaps with the excitation spectrum of the fluorescent dye molecule.

40. 40. The system of claim 39, comprising a kit, a cell, a cell lysate, or a reaction mixture.

41. The system of claim 39 , wherein the fusion comprises a GPCR and a peptide component of a bioluminescent complex, and the system further comprises one or more additional components of the bioluminescent complex and a substrate for the bioluminescent complex.

42. 1. A method comprising: (a) a fusion of a GPCR with a bioluminescent protein, (i) a composition according to any one of claims 1 to 32, wherein the functional element is a fluorophore and the emission spectrum of the bioluminescent protein overlaps with the excitation spectrum of the fluorophore; and (ii) contacting with a substrate for the bioluminescent protein; (b) detecting wavelengths of light within the excitation spectrum of the fluorescent dye molecule that result from bioluminescence resonance energy transfer from the bioluminescent protein to the fluorescent dye molecule when the broad-spectrum GPCR binding agent binds to the GPCR.

43. 1. A method comprising: (a) a fusion of a GPCR with a peptide component of a bioluminescent complex; (i) the composition according to any one of claims 1 to 32, wherein the functional element is a fluorophore and the emission spectrum of the bioluminescent protein overlaps with the excitation spectrum of the fluorophore; (ii) a polypeptide component of the bioluminescent complex, and (iii) contacting the bioluminescent protein with a substrate; (b) detecting wavelengths of light within the excitation spectrum of the fluorescent dye molecule that result from bioluminescence resonance energy transfer from the bioluminescent complex to the fluorescent dye molecule when the broad spectrum GPCR binding agent binds to the GPCR.

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