Kinase experiment:

Saturation, association, and dissociation binding studies are performed for [3H]Vilanterol to determine receptor binding kinetics at the β2-AR (equilibrium dissociation constant (KD), total number of receptors (Bmax), association rate (kon), and dissociation rate (koff) are calculated). For saturation binding, membranes (in a volume of 1.4 mL to avoid ligand depletion) are incubated with increasing concentrations of [3H]Vilanterol (~0.01-1.3 nM) for 5 h before filtration. For association binding, membranes are incubated with different concentrations of [3H]Vilanterol (~0.1-1.9 nM) for varying incubation times up to 1 h before filtration. For dissociation binding, membranes are preincubated for 1 h with a fixed concentration of [3H]Vilanterol (~1.1 nM) before dissociation is initiated by a 1:20 dilution in binding buffer (containing 10 μM cold Vilanterol) and then incubated for varying times up to 8 h before filtration. Saturation binding is also completed for [3H]CGP12177 (increasing concentrations of ~0.01-2.8 nM) in the same format as described above for [3H]Vilanterol. To determine the affinity of β2-AR agonists and antagonists, competition binding displacement studies are completed in which membranes are incubated with a fixed concentration of [3H]Vilanterol (~0.2 nM) and increasing concentrations of unlabeled agonist/antagonist for 5 h before filtration. All competition binding displacement studies are completed in the presence of 100 μM Gpp(NH)p to ensure that binding curves are monophasic[1].

Vilanterol trifenatate is a novel and selective agonist of β2-AR with a PEC50 value of 10.37±0.05 [1].

Vilanterol trifenatate is a novel long-acting β2-AR agonist (LABA) with 24h activity in development for inhaled once daily treatment. In the radioligand binding studies, Vilanterol trifenatate has shown the binding affinity in the one-affinity site model with pKD values of 9.44±0.07 and 10.82±0.12 in the presence Gpp(NH)p and absence Gpp(NH)p, respectively. In dissociation studies, Vilanterol trifenatate has been reported to bind from the β2-AR with a dissociation t1/2 value of 3.5 min in the presence of Gpp(NH)p. Vilanterol trifenatate has been found to have a good selectivity for β2-AR over the other β-AR receptor subtypes(β1and β3) with pEC50 values of 10.37±0.05, 6.98±0.03 and 7.36±0.03, respectively. Vilanterol trifenatate has exhibited at least 1000-fold selectivity over both β1-and β3-AR subtypes [1].

References:
[1] Slack RJ1, Barrett VJ, Morrison VS, Sturton RG, Emmons AJ, Ford AJ, Knowles RG.In vitro pharmacological characterization of vilanterol, a novel long-acting β2-adrenoceptor agonist with 24-hour duration of action.J Pharmacol Exp Ther. 2013 Jan; 344(1):218-30.