. Author manuscript; available in PMC: 2024 Jan 1.

Published in final edited form as: Nature. 2022 Nov 30;613(7945):767–774. doi: 10.1038/s41586-022-05588-y

Extended Data Table 1 |.

Summary of binding affinities, cAMP and arrestin recruitment values of fentanyl amino and guano bitopics on μOR

	Binding ^a	cAMP^b			β-arrestin2^b

Ligands	K_i ± SEM (nM)	EC₅₀ (nM)	pEC₅₀ ± SEM	E_max ± SEM (%)	EC₅₀ (nM)	pEC₅₀ ± SEM	E_max ± SEM (%)
C3 amino	> 1000	12300	4.9 ± 0.2	142 ± 20	n.d.	n.d.	8.2 ± 1.1
C3 guano	77 ± 3.0	164	6.8 ± 0.1	107 ± 2.3	n.d.	n.d.	6.9 ± 2.4
C5 amino	1179 ± 13	469	6.3 ± 0.2	90 ± 5.9	n.d.	n.d.	n.d.
C5 guano	4.6 ± 0.7	0.8	9.1 ± 0.1	104 ± 3.1	4710	5.3 ± 0.1	54 ± 3.2
C6 amino	1033 ± 15	844	6.1 ± 0.1	126 ± 4.7	n.d.	n.d.	20 ± 2.3
C6 guano	4.1 ± 0.3	4.7	8.3 ± 0.1	137 ± 2.1	n.d.	n.d.	19 ± 1.4
C7 amino	369 ± 7.0	436	6.4 ± 0.1	109 ± 3.9	n.d.	n.d.	n.d.
C7 guano	41 ± 8.0	62	7.2 ± 0.1	109 ± 2.4	n.d.	n.d.	13 ± 1.4
C9 amino	281 ± 2.2	360	6.4 ± 0.1	106 ± 4.6	66720	4.2 ± 0.2	56 ± 9.4
C9 guano	58 ± 8.0	21.3	7.7 ± 0.1	128 ± 3.2	792	5.1 ± 0.1	120 ± 6.6
C11amino	589 ± 13	293.2	6.5 ± 0.1	105 ± 3.6	n.d.	n.d.	n.d
C11 guano	165 ± 4	1020	6.0 ± 0.1	118 ± 5.2	n.d.	n.d.	n.d.
Fentanyl	9.1 ± 3.1	0.90	9.0 ± 0.1	86 ± 1.8	66.2	7.2 ± 0.1	119 ± 2.7
DAMGO	n.d.	0.40	9.4 ± 0.1	99 ± 4	723	6.1 ± 0.1	100 ± 2.8

K_i binding competition studies were performed with the indicated compound against ¹²⁵I-IBNtxA (0.1 nM) in membranes from CHO cells stably expressing the μOR cloned mouse opioid receptor. Results are presented as K_i ± SEM (nM) from three independent experiments performed in triplicate.

Potency and efficacy data were obtained using agonist induced inhibition measured by cyclic AMP (cAMP) and Tango assay for β-arrestin 2 recruitment, respectively, from three independent experiments performed in triplicate. Efficacy is represented as EC₅₀ (nM) and percent maximal stimulation (E_max) relative to standard agonist DAMGO. “n.d.” Denotes not determined. Efficacy<20%.