PSAB-OFP, a selective α7 nicotinic receptor agonist, is also a potent agonist of the 5-HT3 receptor
Introduction
5-Hydroxytryptamine 3 (5-HT3) and nicotinic α7 receptors are both members of the superfamily of ligand-gated ion channels. These two receptor subtypes share the greatest similarity within the family displaying ∼30% sequence homology (Maricq et al., 1991). Studies on chimeric receptors composed of α7 receptor amino-terminal sequences linked to carboxy-terminal portions of the 5-HT3 receptor have demonstrated that the agonist recognition site is encoded by the amino terminus (Eiselé et al., 1993). Specific regions within this N-terminal domain have also been identified Changeux et al., 1992, Karlin and Akabas, 1995, Arias, 2000. Homologies, as well as clear differences, have been identified within the ligand-recognition regions for the 5-HT3 and α7 nicotinic acetylcholine receptors Steward et al., 2000, Spier and Lummis, 2000, Yan et al., 1999, Boess et al., 1997 and may help to explain the overlapping, but clearly distinct, pharmacology described for these two receptor subtypes.
Cross-reactivity of various α7 and 5-HT3 receptor ligands has been reported. For example, serotonin is reported to be an antagonist at wild type α7 receptors Palma et al., 1996, Fucile et al., 2002, whilst nicotine and acetylcholine, among other nicotinic acetylcholine receptor agonists, are competitive 5-HT3 receptor antagonists (Gurley and Lanthorn, 1998). Interestingly, 5-HT is an agonist of the L247T mutant α7 receptor Palma et al., 1996, Palma et al., 1997, Fucile et al., 2002, whilst acetylcholine is an agonist at a F107N mutant 5-HT3 receptor (Steward et al., 2000). Moreover, the 5-HT3 receptor antagonist tropisetron (ICS 205-930) has been shown to be a potent, selective partial agonist at α7 nicotinic acetylcholine receptors (Macor et al., 2001), while the α7 receptor selective agonist 3-(2,4)-dimethoxybenzylidene-anabeseine (DMXBA; GTS-21) has been shown to antagonise 5-HT3 receptors Gurley and Lanthorn, 1998, Machu et al., 2001. Interestingly some of the closely related hydroxy-benzylidene metabolites of DMXBA display partial agonism at 5-HT3 receptors (Machu et al., 2001). 3-(2-Hydroxy, 4-methoxybenzylidene)-anabeseine (2-OH-MBA) and 3-(4-hydroxy, 2-methoxybenzylidene)-anabeseine (HMBA; 4OH GTS-21) in particular were identified as partial agonists at both α7 and 5-HT3 receptors Machu et al., 2001, Kem et al., 1996. A further crossover is observed with the ability of 5-hydroxyindole (which lacks the amine group of the 5-HT molecule) to act as a potentiator of both 5-HT3 (Van Hooft et al., 1997) and α7 nicotinic acetylcholine receptors Gurley et al., 2000, Zwart et al., 2002. Once again mutation at L247T in the α7 receptor changes the properties of the receptor such that 5-hydroxyindole displays agonist properties (Gurley and Lanthorn, 2000).
In order to gain more insights into this overlapping pharmacology, we assessed α7 receptor selective agonists for binding to and function on 5-HT3 receptors. Binding was performed on recombinant human 5-HT3 receptors using [3H]GR65630 as a radioligand. Functional activity was assessed using human recombinant 5-HT3 receptors expressed in oocytes, and mouse native 5-HT3 receptors expressed by N1E-115 neuroblastoma cells, using patch clamp and Ca2+ imaging techniques. Using this approach we have identified a selective α7 receptor agonist which displayed high affinity for the 5-HT3 receptor. This compound, (R)-(−)-5′Phenylspiro[1-azabicyclo[2.2.2]octane-3,2′-(3′H)furo[2,3-b]pyridine (PSAB-OFP), was found to be an equipotent, partial agonist at 5-HT3 and human α7 nicotinic acetylcholine receptors.
Section snippets
Radioligand binding assay
Membranes with recombinant human 5-HT3 receptors (RB-HS3M) were from Receptor Biology (Perkin-Elmer Life Sciences, Cambridge, UK). Assays were performed at room temperature in a final volume of 200 μl, containing 20 μl tissue (approx. 3 μg protein), 20 μl [3H]GR65630 (3.4 nM), 140 μl assay buffer (mM): Tris/HCl, 50; EDTA, 1; MgCl2, 5; pH 7.5 at 4 °C) and 20 μl of displacing ligand. These were incubated together for 1 h. Non-specific binding was determined in the presence of 100 μM tropanyl
Results
Using mammalian cell lines stably expressing human α2β4, α3β4, α4β4, α3β2, α4β2 or α7 recombinant nicotinic acetylcholine receptors we confirmed that the reported α7 receptor selective agonist PSAB-OFP (Fig. 1) was selective for α7 over other nicotinic acetylcholine receptor subtypes (Fig. 2). Due to the high homology and reported cross-reactivity between α7 nicotinic and 5-HT3 receptors, PSAB-OFP was also assessed for activity in a binding assay using human recombinant 5-HT3 receptors (see
Discussion
PSAB-OFP has recently been disclosed as a selective α7 nicotinic acetylcholine receptor agonist (Phillips et al., 1999; Astra Arcus USA, patent WO 99/03859). We confirmed the selectivity of this compound for α7 over other nicotinic receptor subtypes, however we found that PSAB-OFP is also a potent agonist of 5-HT3 receptors. PSAB-OFP bound with high affinity to human recombinant 5-HT3 receptors and displayed agonist activity at both human recombinant and native mouse 5-HT3 receptors. In mouse
Acknowledgements
The contributions of Liz Folly, Ella Saunders, James Evans, Annabel Martin, Patty Brown and Ruth Beatty in cell culture are gratefully acknowledged.
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