Abstract
Two promising lead structures of small molecular orexin receptor agonist have been reported, but without detailed analyses of the pharmacological properties. One of them, 1-(3,4-dichlorophenyl)-2-[2-imino-3-(4-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl]ethan-1-ol (Yan 7874), is commercially available, and we set out to analyze its properties. As test system we utilized human OX1 and OX2 orexin receptor-expressing Chinese hamster ovary (CHO) K1 cells as well as control CHO-K1 and neuro-2a neuroblastoma cells. Gq-coupling was assessed by measurement of intracellular Ca2+ and phospholipase C activity, and the coupling to Gi and Gs by adenylyl cyclase inhibition and stimulation, respectively. At concentrations above 1 μM, strong Ca2+ and low phospholipase C responses to Yan 7874 were observed in both OX1- and OX2-expressing cells. However, a major fraction of the response was not mediated by orexin receptors, as determined utilizing the non-selective orexin receptor antagonist N-biphenyl-2-yl-1-{[(1-methyl-1H-benzimidazol-2-yl)sulfanyl]acetyl}-L-prolinamide (TCS 1102) as well as control CHO-K1 cells. Yan 7874 did not produce any specific adenylyl cyclase response. Some experiments suggested an effect on cell viability by Yan 7874, and we thus analyzed this. Within a few hours of exposure, Yan 7874 markedly changed cell morphology (shrunken, rich in vacuoles), reduced growth, promoted cell detachment, and induced necrotic cell death. The effect was equal in cells expressing orexin receptors or not. Thus, Yan 7874 is a weak partial agonist of orexin receptors. It also displays strong off-target effects in the same concentration range, culminating in necrotic cell demise. This makes Yan 7874 unsuitable as orexin receptor agonist.
Highlights
Orexin receptor stimulation is expected to have important pharmacological uses, most obviously in symptomatic treatment of narcolepsy ([1,2,3]; reviewed in [4,5,6,7]), and maybe other sleep and vigilance disorders
The results indicate that Yan 7874 is a weak partial orexin receptor agonist
The results indicate a necrotic mechanism for the cell death, and the idea is supported by the rapid induction of cell death and the apparent decrease in the ATP level
Summary
Orexin receptor stimulation is expected to have important pharmacological uses, most obviously in symptomatic treatment of narcolepsy ([1,2,3]; reviewed in [4,5,6,7]), and maybe other sleep and vigilance disorders. The first series has so far only been published through a patent [15], while the second series was published in a peer-reviewed journal [16] Common for these studies is that very few details of the pharmacological properties of even the lead structures have been published, and for what is published, the assay details etc. The original publications [15, 16] remain as sole sources of information; lack of a commercial source for these compounds is the likely explanation for the lack of other, independent studies. These compounds are of high interest, for the drug use and for use as research tools. We set out to examine this ligand for a set of orexin receptor responses representing different G-protein outputs, utilizing Chinese hamster ovary K1 (CHO-K1) cells expressing recombinant human orexin receptors as the model
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