Binding kinetics differentiates functional antagonism of orexin‐2 receptor ligands

Orexin receptor antagonism represents a novel approach for the treatment of insomnia that directly targets sleep/wake regulation. Several such compounds have entered into clinical development, including the dual orexin receptor antagonists, suvorexant and almorexant. In this study, we have used equilibrium and kinetic binding studies with the orexin-2 (OX₂) selective antagonist radioligand, [³H]-EMPA, to profile several orexin receptor antagonists. Furthermore, selected compounds were studied in cell-based assays of inositol phosphate accumulation and ERK-1/2 phosphorylation in CHO cells stably expressing the OX2 receptor that employ different agonist incubation times (30 and 5 min, respectively). EMPA, suvorexant, almorexant and TCS-OX-29 all bind to the OX₂ receptor with moderate to high affinity (pk(I) values ≥ 7.5), whereas the primarily OX1 selective antagonists SB-334867 and SB-408124 displayed low affinity (pK(I) values ca. 6). Competition kinetic analysis showed that the compounds displayed a range of dissociation rates from very fast (TCS-OX2-29, k(off) = 0.22 min⁻¹) to very slow (almorexant, k(off) = 0.005 min⁻¹). Notably, there was a clear correlation between association rate and affinity. In the cell-based assays, fast-offset antagonists EMPA and TCS-OX2-29 displayed surmountable antagonism of orexin-A agonist activity. However, both suvorexant and particularly almorexant cause concentration-dependent depression in the maximal orexin-A response, a profile that is more evident with a shorter agonist incubation time. Analysis according to a hemi-equilibrium model suggests that antagonist dissociation is slower in a cellular system than in membrane binding; under these conditions, almorexant effectively acts as a pseudo-irreversible antagonist.