In vitroactivity: TCS-OX2-29, discovered from high throughput screening (HTS), is a potent and selective OX2 receptor antagonist with IC50 of 40 nM. It displays>250-fold selectivity for OX2 over OX1. 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. TCS-OX2-29 shows selectivity for ion channels, and transporters (<30% inhibition at 10 μM), which includes G-protein coupled receptors associated with food intake including galanin and neuripeptide Y. TCS-OX2-29 Inhibits orexin A induced IP3 accumulation and ERK1/2 phosphorylation in CHO cells transfected with the OX2 receptor.

Kinase Assay: Cell-based inositol phosphate (Cisbio BioAssays, Codolet, France) and ERK1/2 phosphorylation (Surefire, PerkinElmer, Waltham, MA, USA) functional assays were performed in 96-well plates 24 h after seeding with CHO cells stably expressing the human orexin-2 receptor at a density of 25 000 cells/well; full assay details are in the Supporting Information.

Cell Assay: Cell membranes from HEK293 cells transiently expressing the human OX2 receptor were incubated with [3H]-EMPA in Krebs assay buffer (8.5 mM HEPES, 1.3 mM CaCl2, 1.2 mM MgSO4, 118 mM NaCl, 4.7 mM KCl, 4 mM NaHCO3, 1.2 mM KH2PO4, 11 mM glucose, pH 7.4) in a total assay volume of 0.25 mL with a final DMSO concentration of 1%. After 90 min incubation at room temperature, the reaction was terminated by rapid filtration through GF/B 96-well glass fibre plates with 5 × 0.25 mL washes with ddH2O using a Tomtec cell harvester. Bound radioactivity was determined through liquid scintillation using Lablogic SafeScint and detected on a microbeta liquid scintillation counter. Non-specific binding was determined as that remaining in the presence of a 10 μM saturating concentration of the antagonist EMPA. Saturation studies were carried out by incubating membranes (2 μg protein/well) with a range of concentrations of [3H]-EMPA (0.4 nM–15 nM). Radioligand concentrations were determined using SafeScint and a Beckman LS 6000 liquid scintillation counter. Competition binding was performed incubating membranes (2 μg protein/well) with 1.5 nM concentration of [3H]-EMPA and a range of concentrations of the test compound.