Characterisation of the kinetics of binding of almorexant and suvorexant at the orexin-2 receptor

Richard Mould, Jason Brown, Christopher J. Langmead. Heptares Therapeutics, Welwyn Garden City, Hertfordshire, UK

Orexin signalling involving the OX1 and OX2 receptors have been shown to promote wakefulness and arousal responses. For these reasons interest has developed around using orexin antagonists as a novel treatment for insomnia. Of the existing orexin receptor antagonists in clinical development little has been published about their receptor kinetics. In this study we have used equilibrium and kinetic radioligand binding studies at the OX2 receptor to understand the profile of a range of antagonists, including the clinical development compounds, almorexant and suvorexant.

Antagonist affinities for the OX2 WT receptor were determined using [3H]-EMPA inhibition assays in transient HEK-293-hOX2 cell membranes (2 μg/well) in Krebs’ buffer containing 1.5 nM radioligand. Total and non specific counts were determined by 1% DMSO vehicle and 10μM cold EMPA, respectively. Plates were then incubated for 1.5 hours at room temperature and filtered through GF/B filter plates soaked in 0.5% PEI with 5 x 0.5ml washes in ddH20. Bound radioligand on filter plate was counted on a microbeta counter. IC50 values were determined by fitting to a four parameter sigmoidal dose response and converted to pKI values using the KD value determined using saturation binding.

To measure association kinetics of [3H]-EMPA, membranes were incubated in the presence of 1.5nM radioligand at varying time points up to three hours. Dissociation kinetics were measured by adding a saturating concentration of cold EMPA (100 μM) to membranes pre-incubated for three hours in the presence of 1.5nM [3H] EMPA. Kinetics of non-labelled compounds were determined by performing association kinetics assays in the presence of increasing concentrations of cold compound (Dowling & Charlton, 2006).

[3H]-EMPA bound to the OX2 receptor with high affinity (KD = 1.39 ± 0.19 nM; n = 3) and in a monophasic manner with a maximal binding capacity 2050cpm (16.51 ± 0.28 pmol. mg protein-1).

Association and dissociation of the radioligand were relatively rapid, with equilibrium being reached within 25 min. Association and dissociation rate constants for a range of test antagonists are shown in Table 1; both constants varied for different antagonists, most notably in their dissociation rate, where suvorexant, SB-334867 and almorexant displayed slower kinetics than either EMPA or TCS-OX2-29. Kinetic pKD values agree well with those generated by equilibrium competition binding.

Table 1. Binding parameters for five orexin receptor antagonists at the OX2 receptor. Kinetic pKD values are calculated from the ratio of the association and dissociation rate constants. All values are derived from n≥3 with standard deviation quoted below.

These data indicate that both association and dissociation rate constants can vary widely for antagonists of the same receptor. The kinetics of competitive binding may prove a useful method for assessing novel orexin receptor antagonists in drug discovery.

Dowling, Charlton (2006) Quantifying the association and dissociation rates of unlabelled antagonists at the muscarinic M3 receptor. Br J Pharmacol.148(7): 927–937.