Biochemical and behavioural characterization of EMPA,a novel high-affinity,selective antagonist for the OX2 receptor |
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Authors: | P Malherbe E Borroni L Gobbi H Knust M Nettekoven E Pinard O Roche M Rogers-Evans JG Wettstein J-L Moreau |
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Affiliation: | Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland |
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Abstract: | Background and purpose:The OX2 receptor is a G-protein-coupled receptor that is abundantly found in the tuberomammillary nucleus, an important site for the regulation of the sleep-wake state. Herein, we describe the in vitro and in vivo properties of a selective OX2 receptor antagonist, N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA).Experimental approach:The affinity of [3H]EMPA was assessed in membranes from HEK293-hOX2-cells using saturation and binding kinetics. The antagonist properties of EMPA were determined by Schild analysis using the orexin-A-or orexin-B-induced accumulation of [3H]inositol phosphates (IP). Quantitative autoradiography was used to determine the distribution and abundance of OX2 receptors in rat brain. The in vivo activity of EMPA was assessed by reversal of [Ala11,D-Leu15]orexin-B-induced hyperlocomotion during the resting phase in mice and the reduction of spontaneous locomotor activity (LMA) during the active phase in rats.Key results:[3H]EMPA bound to human and rat OX2-HEK293 membranes with KD values of 1.1 and 1.4 nmol·L−1 respectively. EMPA competitively antagonized orexin-A-and orexin-B-evoked accumulation of [3H]IP at hOX2 receptors with pA2 values of 8.6 and 8.8 respectively. Autoradiography of rat brain confirmed the selectivity of [3H]EMPA for OX2 receptors. EMPA significantly reversed [Ala11,D-Leu15]orexin-B-induced hyperlocomotion dose-dependently during the resting phase in mice. EMPA, injected i.p. in rats during the active phase, reduced LMA dose-dependently. EMPA did not impair performance of rats in the rotarod procedure.Conclusions and implications:EMPA is a high-affinity, reversible and selective OX2 receptor antagonist, active in vivo, which should prove useful for analysis of OX2 receptor function. |
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Keywords: | EMPA orexin OX2 antagonist binding kinetics inositol phosphate accumulation Schild analysis autoradiography rat brain distribution orexin-B-induced hyperlocomotion ex vivo receptor occupancy |
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