PSAB-OFP, a selective α7 nicotinic receptor agonist, is also a potent agonist of the 5-HT3 receptor

https://doi.org/10.1016/S0014-2999(02)02273-2Get rights and content

Abstract

5-Hydroxytryptamine 3 (5-HT3) and α7 nicotinic receptors share high sequence homology and pharmacological cross-reactivity. An assessment of the potential role of α7 receptors in many neurophysiological processes, and hence their therapeutic value, requires the development of selective α7 receptor agonists. We used a recently reported selective α7 receptor agonist, (R)-(−)-5′Phenylspiro[1-azabicyclo[2.2.2] octane-3,2′-(3′H)furo[2,3-b]pyridine (PSAB-OFP) and confirmed its activity on human recombinant α7 receptors. However, PSAB-OFP also displayed high affinity binding to 5-HT3 receptors. To assess the functional activity of PSAB-OFP on 5-HT3 receptors we studied recombinant human 5-HT3 receptors expressed in Xenopus oocytes, as well as native mouse 5-HT3 receptors expressed in N1E-115 neuroblastoma cells, using whole-cell patch clamp and Ca2+ imaging. Our results show that PSAB-OFP is an equipotent, partial agonist of both α7 and 5-HT3 receptors. We conclude that it will be necessary to identify the determinant of this overlapping pharmacology in order to develop more selective α7 receptor ligands.

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.

References (39)

Cited by (35)

  • Design of α7 nicotinic acetylcholine receptor ligands using the (het)Aryl-1,2,3-triazole core: Synthesis, in vitro evaluation and SAR studies

    2016, European Journal of Medicinal Chemistry
    Citation Excerpt :

    A combination of sigma and spiranic bonds or fusion with the tertiary amine or the (het)Ar fractions are often observed to link the three key parts together. Numerous representative drugs which bind to the α7 nAchR receptor have been with a high affinity answer this general description [21–30]. It seems that quinuclidine represents a chosen structure in the obtaining of drugs having potential beneficial effects in CNS disorders such as AD [31,32].

  • Neuronal nicotinic receptors as analgesic targets: It's a winding road

    2013, Biochemical Pharmacology
    Citation Excerpt :

    Thus, peripheral α7 nAChRs could contribute to α7-mediated analgesia. A subset of α7 nAChR agonists also antagonize spinal 5-HT3 receptor activity [37,38]. After inflammation or injury, 5-HT3 receptor activation contributes to nociceptive facilitation via supraspinal descending pathways [39,40].

  • Discovery of novel α7 nicotinic receptor antagonists

    2010, Bioorganic and Medicinal Chemistry Letters
  • High affinity binding of epibatidine to serotonin type 3 receptors

    2008, Journal of Biological Chemistry
    Citation Excerpt :

    Similar actions are observed for the nAChR channel blockers, chlorpromazine and QX222 (48). Other studies also show that tropisetron, which is a selective 5-HT3R antagonist, binds with high affinity and is a potent partial agonist at the α7 nAChR (66), whereas the α7 agonist, PSAB-OFP, acts as a potent agonist at 5-HT3Rs (67). Relevant to our results, the nAChR agonists, anabaseine and epibatidine, are shown to block serotonin-activated currents in oocytes expressing 5-HT3ARs with IC50 values of 14 and 8 μm, respectively (51, 52).

View all citing articles on Scopus
View full text