Mapping of the binding sites for the OX1 orexin receptor antagonist, SB-334867, using orexin/hypocretin receptor chimaeras.

The binding sites for agonists and antagonist of orexin receptors are not know, hampering progressive drug design approaches. In the current study, we utilized chimaeric orexin receptor approach to map the receptor areas contributing to the selectivity of the classical antagonist, SB-334867, for OX(1) receptors. Altogether ten chimaeras between OX(1) and OX(2) orexin receptors were utilized. The receptors were transiently expressed in HEK-293 cells. The ability (K(B)) of SB-334867 to inhibit orexin-A-induced inositol phosphate release (phospholipase C activity) was measured. The results, in synthesis, suggest that there are several possible interactions contributing to the high affinity binding, all of which are not required simultaneously. This is indicated by the fact that most of the chimaeras display affinity (at least somewhat) higher than OX(2). As previously shown for the agonist distinction, the second quarter of the receptor, from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 seems to be most central also for SB-334867 binding, but also the third quarter, from the transmembrane helix 4 to the transmembrane helix 6 is able to contribute (and compensate for loss of other sites). A previous study has suggested that amino acids conserved between OX(1) and OX(2) receptors would somehow confer selectivity for subtype-selective antagonists. In contrast to previous findings, our results indicate that the amino acids distinct between the receptor subtypes are in key position.