SB-334867-A: the first selective orexin-1 receptor antagonist

Introduction

Orexin-A and orexin-B are 33 and 28 amino acid peptides respectively, which were recently isolated from the rat hypothalamus and are derived from a 130 amino acid precursor, prepro-orexin (Sakurai et al., 1998). Both peptides bind to two receptors, orexin-1 (OX₁) and orexin-2 (OX₂), although orexin-B displays 10 fold selectivity for OX₂ (Sakurai et al., 1998). In a recombinant system the binding of these ligands to either receptor is associated with an increase in intracellular calcium concentrations ([Ca²⁺]_i) (Smart et al., 1999).

The orexin receptors are located predominantly in the hypothalamus and locus coeruleus (Sakurai et al., 1998; Peyron et al., 1998), but are also found elsewhere in the CNS (Smart, 1999; Van den Pol, 1999). The orexins have been linked to a range of physiological functions (Jerman & Smart, 2001) including the control of feeding and energy metabolism (Sakurai et al., 1998), modulation of neuroendocrine function (Van den Pol, et al., 1998; Smart, 1999), and regulation of the sleep-wake cycle (Piper et al., 2000). However, the study of the role of the orexins in these functions has been hampered by the lack of orexin receptor antagonists (Jerman & Smart, 2001). Therefore, as preliminary studies showed that SB-334867-A (1-(2-Methylbenzoxazol-6-yl)-3-[1,5]napthyridin-4-yl-urea hydrochloride) inhibited an OX₁-mediated calcium response (Smart, 2000), further characterization of this compound's interactions with the orexin receptors has been undertaken. Furthermore, it has recently been reported that neuropeptide Y (NPY), secretin and, to a lesser extent, several other related peptides displace orexin-A binding (Kane et al., 2000). Therefore, these peptides have also been tested as both agonists and antagonists at recombinant human OX₁ and OX₂ receptors expressed in CHO cells using a FLIPR-based functional assay.

Methods

Results

Orexin-A and orexin-B caused a concentration-dependent increase in [Ca²⁺]_i in CHO-OX₁ cells (Figure 1) with pEC₅₀ values of 8.38±0.04 and 7.26±0.05 respectively, (n=12). Similarly, both peptides increased [Ca²⁺]_i in CHO-OX₂ cells (Figure 1), with pEC₅₀ values of 8.20±0.03 and 8.26±0.04 respectively (n=8). However, NPY, secretin, pPYY, [Leu³¹,Pro³⁴]hPYY, hPYY(3-36), NPY free acid, human pancreatic polypeptide and VIP were inactive at both receptors at all concentrations (100 pM – 10 μM) tested (Figure 1 and data not shown). In keeping with this, in the binding studies, orexin-A and orexin-B had pK_i values of 7.79±0.04 and 6.93±0.02 (n=4) respectively at OX₁, whereas NPY and secretin displayed no specific binding to OX₁ receptors at concentrations up to 10 μM.

In CHO-OX₁ cells SB-334867-A (100 pM – 10 μM) inhibited the orexin-A (10 nM) and orexin-B (100 nM)-induced calcium responses in a concentration-dependent manner, with apparent pK_B values of 7.27±0.04 and 7.23±0.03 (n=8), but had no effect on the calcium response elicited by UTP (3 μM), which activated an endogenous purinergic receptor (Figure 2). SB-334867-A also inhibited OX₂-mediated calcium responses, but with lower affinity, causing a 32.7±1.9 and 22.0±4.0% inhibition at 10 μM of the orexin-A (10 nM) and orexin-B (10 nM)-induced responses respectively. SB-334867-A was devoid of agonist properties in either cell-line (data not shown). SB-334867-A also displaced N^6,10-RG-orexin-A binding at human OX₁ receptors, with a pK_i of 7.17±0.04 (n=4). Neither NPY, secretin nor any of the related peptides antagonized the orexin-A (10 nM)-induced calcium responses in either CHO-OX₁ or CHO-OX₂ cells (data not shown).

Discussion

The orexins are a recently discovered family of neuropeptides (Sakurai et al., 1998) which have been linked to a wide range of physiological functions (Smart, 1999), although the elucidation of the mechanisms involved has been hampered by the lack of selective orexin receptor antagonists (Jerman & Smart, 2001). We have demonstrated in a recombinant system that SB-334867-A is an OX₁-selective receptor antagonist. Furthermore, we have shown that NPY, secretin and related peptides were neither agonists nor antagonists at recombinant human OX₁ or OX₂ receptors.

In the present study orexin-A and orexin-B caused a concentration-dependent increase in [Ca²⁺]_i in CHO cells expressing either OX₁ or OX₂ with potencies similar to those reported previously (Smart et al., 1999). Orexin-A was equipotent at OX₁ and OX₂, whilst orexin-B displayed moderate selectivity for OX₂, consistent with the literature (Sakurai et al., 1998; Smart et al., 2000). Furthermore, the potencies at OX₁ were also in keeping with the affinities of orexin-A and orexin-B in the fluorescence-based binding assay.

It has previously been reported that NPY, secretin and related peptides displaced radiolabelled orexin-A binding with moderate to high affinity (Kane et al., 2000). However, in the present study, at equivalent concentrations, none of these peptides displayed any agonist-like activity, nor inhibited the orexin-A induced calcium response, in either CHO-OX₁ or CHO-OX₂ cells. Moreover, NPY and secretin, the two peptides with the highest affinity in the earlier study (Kane et al., 2000), did not display any affinity for OX₁ in the present binding studies. These discrepancies might be explained, at least in part, by the fact that the previous study (Kane et al., 2000) used porcine secretin to define the non-specific binding in crude hypothalamic membranes and thus the specificity of the assay may be questionable. Alternatively, recent evidence suggesting the possibility of a third orexin receptor which couples to adenylate cyclase rather than phospholipase C has been reported (Nanmoko et al., 2000). Therefore, it is possible that NPY and the other peptides were binding to this receptor in the Kane et al. (2000) study, although this putative receptor has only been proposed in PC12 cells to date (Nanmoko et al., 2000).

In the present study SB-334867-A was shown to bind to recombinant human OX₁ receptors with nanomolar affinity, and also to inhibit the OX₁-mediated calcium response at similar concentrations. SB-334867-A also inhibited the OX₂-mediated calcium response, but only at considerably higher concentrations. These inhibitory effects were specific for the orexin receptors as SB-334867-A had no effect on the calcium response elicited by the activation of a purinergic receptor endogenously expressed by CHO cells. Furthermore, SB-334867-A had no appreciable affinity for over 50 G-protein coupled receptors and ion channels in a CEREP screen (Porter, unpublished observations). As agonist-induced receptor desensitization also results in inhibition of orexin receptor-mediated calcium responses (Smart et al., 1999; 2000), it is important to note that SB-334867-A was devoid of agonist-like activity in both CHO-OX₁ and CHO-OX₂ cells. Taken collectively these data demonstrate that SB-334867-A is a selective OX₁ antagonist and thus may be a useful tool for studying the physiological tole of the orexins. Indeed, preliminary reports have recently described how SB-334867-A inhibited natural and orexin-A induced feeding in rats (Arch, 2000) and inhibited orexin-A induced arousal behaviours (Upton, 2000).

In conclusion, we have identified and characterized the first selective OX₁ receptor antagonist, SB-334867-A. Furthermore, we have demonstrated that NPY, secretin and related peptides do not interact with either recombinant human OX₁ or OX₂ receptors.