Fret-based assays
AssigneesThe General Hospital Corporation, President and Fellows of Harvard College
InventorsRalph ` Mazitschek, N. Connor Payne, Mark A. Tye
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Abstract
The present application provides compounds and methods for identifying a modulator of a protein of interest using TR-FRET donor attached to the protein of interest and a tracer containing TR-FRET acceptor. Iterations of this compound may also includes attachment of an ATP-binding moiety, an amino acid, and/or a fluorophore.
Claims20
A compound of Formula (I):
- or a pharmaceutically acceptable salt thereof, wherein:
- R3 is an ATP-binding moiety;
- R2 is an amino acid;
- each L1 is independently selected from O, S, S(═O)2, NH, C═O, C═S, and C1-6 alkylene;
- n is an integer from 1 to 12;
- and
- R1 is a fluorophore.
A compound of Formula (II):
- or a pharmaceutically acceptable salt thereof, wherein:
- L2 is C1-3 alkylene;
- or L2 is absent;
- each L1 is independently selected from O, S, S(═O)2, NH, C═O, C═S, and C1-6 alkylene;
- n is an integer from 1 to 12;
- and
- R1 is a fluorophore.
A compound of Formula (IV):
- or a pharmaceutically acceptable salt thereof, wherein:
- each L1 is independently selected from O, S, S(═O)2, NH, C═O, C═S, and C1-6 alkylene;
- n is an integer from 1 to 12;
- and
- R1 is a fluorophore.
A compound of Formula (A):
- or a pharmaceutically acceptable salt thereof, wherein:
- L1 is C1-3 alkylene, or L1 is absent;
- X1 is selected from O and NRN;
- RN is selected from H, C1-3 alkyl, and C1-3 haloalkyl;
- each L2 is independently selected from O, S, S(═O)2, NRN, C═O, C═S, and C1-6 alkylene;
- n is an integer from 0 to 12;
- and
- R1 is selected from H, C1-6 alkyl, and a protecting group.
A method of identifying a compound that modulates a protein of interest, the method comprising:
- (i) providing a sample comprising a protein of interest attached to a FRET donor moiety and a ligand attached to a FRET acceptor moiety, said ligand capable of binding to the protein of interest;
- (ii) exciting the sample with a light of a wavelength capable of being absorbed by the FRET donor moiety, waiting an amount of time sufficient for energy transfer from the FRET donor moiety to the FRET acceptor moiety, and detecting a light of a wavelength emitted by the FRET acceptor moiety by a fluorescence imaging technique;
- (iii) contacting the sample with a test compound;
- (iv) after (iii), detecting a light of a wavelength emitted by the FRET acceptor moiety by a fluorescence imaging technique;
- and
- (v) determining whether the intensity of fluorescence detected from the FRET acceptor moiety in step (iv) is decreased compared to the intensity of fluorescence detected from the FRET acceptor moiety in step (ii),
- wherein said decrease in fluorescence intensity in an indication that the test compound is the modulator the protein of interest.
A compound of Formula (B):
- or a pharmaceutically acceptable salt thereof, wherein:
- L1 is C1-3 alkylene;
- or L1 is absent;
- R1 is selected from H, C1-3 alkyl, C1-3 haloalkyl, —C(═O)RA1, and —C(═O)ORA1;
- and
- each RA1 is selected from H, C1-6 alkyl, and C1-3 haloalkyl.
Dependencies are inferred from the claim text.