Broad spectrum gpcr binding agents

Patent metadata

Field	Value
Publication number	US-2020200765-A1
Application number	US-201916703534-A
Country	US
Kind code	A1
Filing date	Dec 4, 2019
Priority date	Dec 4, 2018
Publication date	Jun 25, 2020
Grant date	—

Abstract

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 a functional element and/or solid surface), and methods of use thereof for the detection/isolation of GPCRs.

First claim

1 . A composition comprising a broad-spectrum G-protein coupled receptor (GPCR) binding agent attached to a functional element or solid surface, wherein the broad-spectrum GPCR binding agent comprises:

wherein is the point of attachment of the broad-spectrum GPCR binding agent to the functional element, solid surface, or a linker between the broad-spectrum GPCR binding agent and the functional element or solid surface, and wherein the broad-spectrum GPCR binding agent may exist as the cis isomer (Z), trans isomer (E), or a mixture of the two.

2 - 11 . (canceled)

12 . The composition of claim 1 , wherein the solid surface is selected from a sedimental particle, a membrane, glass, a tube, a well, a self-assembled monolayer, a surface plasmon resonance chip, or a solid support with an electron conducting surface.

13 . The composition of claim 12 , wherein the sedimental particle is a magnetic particle.

14 . The composition of claim 1 , wherein the functional element is selected from a detectable element, an affinity element, and a capture element.

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

16 . The composition of claim 1 , wherein the broad-spectrum GPCR binding agent is attached to the functional element or solid surface directly.

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

18 . The composition of claim 17 , wherein the linker comprises [(CH 2 ) 2 O] n , wherein n is 1-20.

19 . 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 composition of claim 1 , comprising a structure of:

wherein n is 0-8, and wherein X is a functional element or solid surface

wherein n is 0-8, wherein m is 0-8, and wherein X is a functional element or solid surface;

wherein n is 0-8, wherein m is 0-8, and wherein X is a functional element or solid surface;

wherein n is 0-8, wherein m is 0-8, and wherein X is a functional element or solid surface;

wherein n is 0-8, and wherein X is a functional element or solid surface;

wherein n is 0-8, wherein m is 0-8, and wherein X is a functional element or solid surface;

wherein n is 0-8, wherein m is 0-8, and wherein X is a functional element or solid surface;

wherein n is 0-8 and wherein X is a functional element or solid surface;

wherein n is 0-8 and wherein X is a functional element or solid surface;

wherein n is 0-8 and wherein X is a functional element or solid surface; or

wherein n is 0-8 and wherein X is a functional element or solid surface.

21 - 30 . (canceled)

31 . The composition of claim 20 , wherein X is a fluorophore.

32 . The composition of claim 1 , comprising a non-natural abundance of one or more stable heavy isotopes.

33 . A method of detecting or quantifying GPCRs in a sample, comprising contacting the sample with a composition of claim 1 and detecting or quantifying the functional element of a signal produced thereby.

34 . The method of claim 33 , wherein the functional element of a signal produced thereby is detected or quantified by fluorescence, mass spectrometry, optical imaging, magnetic resonance imaging (MM), and energy transfer.

35 . A method of isolating GPCRs from a sample, comprising contacting the sample with a composition of claim 1 and separating the functional element or the solid surface, as well as the bound GPCRs, from the unbound portion of the sample.

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

37 . A method of monitoring interactions between GPCRs and unmodified biomolecules comprising contacting the sample with a composition of any claim 1 .

38 . The method of claim 33 , wherein the sample is selected from a cell, cell lysate, body fluid, tissue, biological sample, in vitro sample, and environmental sample.

39 . A system comprising:
(a) composition of claim 1 , wherein the functional element is a fluorophore; and (b) a fusion of a GPCR and a bioluminescent protein or a peptide component of a bioluminescent complex, wherein the emission spectrum of the bioluminescent protein or the bioluminescent complex overlaps the excitation spectrum of the fluorophore.

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

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

42 . A method comprising:
(a) contacting a fusion of a GPCR and a bioluminescent protein, with
(i) a composition of claim 1 wherein the functional element is a fluorophore and wherein the emission spectrum of the bioluminescent protein overlaps the excitation spectrum of the fluorophore, and
(ii) a substrate for the bioluminescent protein; and
(b) detecting a wavelength of light within the excitation spectrum of the fluorophore resulting from bioluminescence resonance energy transfer from the bioluminescent protein to the fluorophore when the broad-spectrum GPCR binding agent is bound to the GPCR.

43 . A method comprising:
(a) contacting a fusion of a GPCR and a peptide component of a bioluminescent complex, with
(i) a composition of claim 1 wherein the functional element is a fluorophore and wherein the emission spectrum of the bioluminescent protein overlaps the excitation spectrum of the fluorophore,
(ii) a polypeptide component of the bioluminescent complex. and
(iii) a substrate for the bioluminescent protein; and
(b) detecting a wavelength of light within the excitation s

Assignees

• Promega Corp

Inventors

• Levin Sergiy
• Boursier Michelle
• Ohana Rachel Friedman
• Kirkland Thomas
• Wood Keith

Classifications

• G01N33/6848 Primary

  Methods of protein analysis involving mass spectrometry · CPC title

• C07C233/83

  of an acyclic saturated carbon skeleton · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N24/088

  Assessment or manipulation of a chemical or biochemical reaction, e.g. verification whether a chemical reaction occurred or whether a ligand binds to a receptor in drug screening or assessing reaction kinetics · CPC title

• G01N33/582

  with fluorescent label · CPC title