On the kinetic features of orexin receptor antagonists at orexin receptors, transduction systems and receptor binding

Orexin receptor antagonists are on the market or under development for the treatment of insomnia and a number of other neuropsychiatric disorders. Currently, suvorexant, lemborexant and daridorexant, three dual orexin receptor antagonists (DORAs) have received market approval by regulatory authorities in the USA, Australia, Europe and/or Japan for the treatment of insomnia. More DORAs and Selective Orexin Receptor Antagonists (SORAs) in addition to orexin receptor agonists are in various stages of preclinical and clinical development: for instance, 1SORAs (selective orexin 1 receptor antagonists) are being developed for the treatment of anxiety, panic, eating disorders, whereas 2SORAs (selective orexin 2 receptor antagonists) are in late clinical stage for the treatment of insomnia and insomnia-related depression. On the other hand, selective orexin 2 receptor agonists are in clinical trials for the treatment of narcolepsy with (NT1) or without cataplexy (NT2) and other aspects of extreme day time sleepiness.

Traditionally, the medium to high throughput screening procedures used to screen / characterize orexin receptor antagonists or agonists (and for that matter ligands acting on a variety of potential drug targets), frequently ignore two aspects of new drugs candidates: possible functional selectivity (biased agonism or antagonism) and intrinsic receptor-ligand kinetic properties (i.e. association and dissociation features at the target level), since most screening protocols are conducted under short incubation time conditions with usually a single functional readout. Here, we report on strategies to characterise orexin receptor ligands (agonists or antagonists) in radioligand binding and calcium mobilization assays (e.g. using the FLIPR ® /Fluorescent Imaging Plate Reader assay) and for a few select DORAs, on ERK activation. We studied clinically effective and/or tool orexin receptor antagonists (almorexant, suvorexant, filorexant, SB-649868, MK1064…), which have been or are being evaluated in clinical trials or are on the market, in these signalling pathways with an emphasis on kinetics. Thus, we investigated calcium mobilization and pERK elevation triggered by orexin A (OXA) in HEK293 cells stably transfected with human OX₁R or OX₂R. We confirmed that the ligands behave as antagonists in either assay. Most ligands do not show significant functional selectivity between the two pathways, except MK-1064, which inhibits calcium mobilization about 35 times more potently than ERK phosphorylation. We also estimated the kinetic properties of the antagonists in radioligand binding, calcium mobilization and pERK assays. The results of radioligand binding and calcium mobilization assays indicate consistently that several of the tested antagonists bind to/dissociate from either or the two orexin receptors very slowly, with equilibrium reached only after several hours. Thus, SB-649868 is a very slow binder at the OX₁R, whereas almorexant is a very slow binder at the OX₂R. By contrast, all tested antagonists (except almorexant at OX₂R), seem to equilibrate with both orexin receptors relatively rapidly in the pERK assay. The different results drawn from the various test systems may relate to experimental design, with different temperature and buffer conditions: some assays were performed with live cells at 37 °C, while others were carried out at room temperature with either intact cells or cell membranes. Our data suggest that orexin receptor ligands may have entirely different effects in different brain cells / nuclei, if biased signalling was to occur. Independent of biased signalling, the kinetic properties of orexin receptor ligands will influence the actual receptor selectivity and duration of action of the compound, as is strongly suggested for clinically relevant antagonists such as almorexant, SB-649868, filorexant or suvorexant. Thus, this paper stresses the importance of studying orexin receptor ‘antagonists’ in different functional assays and to determine their kinetic properties under conditions that are as physiological as feasible.