Comparison Of Novel Azetidine/Pyrrolidine-Containing Compounds With The Nicotinic Acetylcholine Receptor Partial Agonist Sazetidine-A On Intracellular Calcium Increases In SH-SY5Y Cells.

Jack Brown1, Sarah Foale1, Antonio Fuela2, John Fossey2, Susan Wonnacott1. 1Department of Biology and Biochemistry, University of Bath, Bath, Bath and North East Somerset BA2 7AY, UK, 2School of Chemistry, University of Birmingham, Birmingham B15 2TT, UK

Nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels with high relative calcium permeability (Dajas-Bailador et al. 2002). Sazetidine-A has been reported to act as a partial agonist and a desensitizing agent at α4β2 nAChRs (Xiao et al. 2006; Zwart et al. 2008), and to reduce nicotine self-administration in rats (Levin et al. 2010), indicating potential as a smoking cessation aid. In this study, the effects on nAChRs of five novel compounds containing an azetidine and/or pyrrolidine group (Compounds A-E) have been compared with sazetidine-A in radioligand binding assays in rat brain membranes and intracellular calcium assays in SH-SY5Y cells.

To label nAChRs, rat brain membranes were prepared by differential centrifugation, incubated with [3H]epibatidine (1 nM) with or without drugs for 1.5 h and vacuum filtered. Bound [3H]epibatidine was then measured by liquid scintillation counting. Both nicotine and sazetidine-A competed with [3H]epibatidine for binding, with IC50 values of 33 nM and 87 nM respectively. Compounds A-E (100 μM) did not significantly affect [3H]epibatidine binding, indicating that they do not interact with the agonist binding site of nAChRs.

SH-SY5Y cells were cultured in 96-well plates, pre-loaded with fluo-3 AM and stimulated with drugs in the presence or absence of antagonists or test drugs, n≥3. Changes in fluorescence (F538) were measured for 20 s. Nicotine (100 μM) and the selective agonist PNU-282987 (3 μM; in the presence of the selective potentiator PNU-120596, 10 μM) were used to activate α3* and α7 nAChRs respectively. Both elicited rapid increases in fluorescence of 32.8 ± 6.01 % and 41.2 ± 9.33 % respectively. Sazetidine-A (100 μM) also provoked an increase in fluorescence of 32.2 ± 8.14 % that was prevented by the nicotinic antagonist mecamylamine (100 μM). Interestingly, this response was potentiated in the presence of PNU-120596 in a methyllycaconitine-sensitive manner by 154 ± 28.6 %, consistent with α7 nAChR agonism. In contrast, compounds A-E (100 μM) did not elicit a response, supporting the absence of nicotinic agonist activity.

Sazetidine-A and compounds A-E were also examined for their ability to block nicotinic responses in SH-SY5Y cells. Sazetidine-A blocked both α3*- (IC50 = 228 nM) and α7-nAChR (IC50 = 174 nM) mediated increases in fluorescence in contrast to its purported α4β2-selectivity. Compounds A-E (100 μM) also blocked α3* responses, whereas α7 responses were significantly inhibited by compounds C and D. To examine the nicotinic specificity of compounds A-E, they were evaluated for inhibition of calcium increases evoked by KCl (100 mM). KCl induced increases in fluorescence were unaffected by sazetidine-A and compounds A and B, partially inhibited by compound C, and fully inhibited by compounds D and E. These data reveal distinct pharmacological profiles for compounds A-E. Compounds A and B were more selective in inhibiting α3* nAChR-induced responses. Furthermore, hitherto unreported interactions of sazetidine-A with α7 nAChRs have been demonstrated.

Dajas-Bailador F.A. et al., (2002) J Neurochem 81,606-614.

Levin, E.D. et al., (2010) JPET, 332,933-999.

Xiao, Y. et al., (2006) Mol pharm, 70,1454-1460.

Zwart, R. et al., (2008). Mol pharm, 73,1838-1843.