Characterisation of UB-165 analogues at the alpha3beta4 nicotinic acetylcholine receptor

The α3β4 neuronal nicotinic acetylcholine receptor (nAChR) is implicated in the pre-synaptic modulation of noradrenaline and ACh release in the CNS, making it a valid target for the evaluation of nicotinic drugs. We have exploited a mammalian cell line stably transfected with the rat α3β4 nAChR¹. [³H]Epibatidine bound saturably to a single site (K_d=0.27nM). Immunolocalisation of the α3 subunit revealed strong intracellular labelling with a smaller surface population. Functional surface nAChRs induced rises in fluo-3 calcium fluorescence in response to agonists. Nicotine responses (EC₅₀=13.5µM) were fully antagonised by DHβE, MLA, d-tubocurarine, mecamylamine and αconotoxin AuIB.
A series of analogues of the anatoxin-a/epibatidine hybrid, UB-165² , were examined at the α3β4 nAChR. Removal of the pyridyl chlorine of UB-165, forming DCl-UB, decreased binding affinity and functional potency, 3 and 6 fold respectively. Shifting the 3' pyridyl nitrogen of DCl-UB to the 2' or 4' position produced substantial decreases in binding affinities and loss of functional potency. Addition of an extra nitrogen to the pyridyl ring of DCl-UB decreased binding affinities (10 fold) and functional potencies (2 fold) for the pyrimidinyl and pyridazinyl compounds. Greater decreases were observed for the pyrazinyl member. At the α3β4 nAChR pharmacophore the 3' pyridyl nitrogen confers maximal potency; this is diminished by the introduction of an additional nitrogen.