	US 11,834,489 B2
	Methods for quantifying ligand efficacy in G-protein coupled receptors using single-molecule fluorescence energy transfer
Scott C. Blanchard, Ithaca, NY (US); Gabriel Glenn Gregorio, Ithaca, NY (US); and Brian Kobilka, Ithaca, NY (US)
Assigned to CORNELL UNIVERSITY, Ithaca, NY (US); and THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY, Palo Alto, CA (US)
Appl. No. 16/642,181
Filed by Cornell University, Ithaca, NY (US)
PCT Filed Aug. 31, 2018, PCT No. PCT/US2018/049275 § 371(c)(1), (2) Date Feb. 26, 2020, PCT Pub. No. WO2019/046826, PCT Pub. Date Mar. 7, 2019.
Claims priority of provisional application 62/553,708, filed on Sep. 1, 2017.
Prior Publication US 2021/0072155 A1, Mar. 11, 2021
Int. Cl. G01N 21/64 (2006.01); C12Q 1/6818 (2018.01); C07K 14/705 (2006.01); G01N 33/58 (2006.01); G01N 33/94 (2006.01); G01N 33/542 (2006.01)

CPC C07K 14/70571 (2013.01) [G01N 21/6408 (2013.01); G01N 33/542 (2013.01); G01N 33/582 (2013.01); G01N 33/9433 (2013.01); G01N 2021/6421 (2013.01); G01N 2333/4719 (2013.01)]

16 Claims

1. A method for determining a molecular efficacy of a ligand, the method comprising:

obtaining a labelled beta-2 adrenergic receptor (β₂AR) and contacting the labelled β₂AR with the ligand;

detecting an effective rate of generating G_s(GTP) from G_s(GDP) provided by the ligand (“ligand effective rate”); and

detecting an apparent EC₅₀for GDP binding of the ligand (“ligand EC₅₀”);

wherein

η₁is the ligand effective rate normalized to an effective rate of adrenaline in generating G_s(GTP) from G_s(GDP);

η₂is the ligand EC₅₀normalized to an apparent EC₅₀for GDP binding of adrenaline; and

a value greater than 1.0 for the product of η₁and η₂indicates the ligand is more efficacious than adrenaline.