Searching for structural determinants of agonist selectivity in nicotinic acetylcholine receptors
The α4β2 and α7 receptors are the most abundant nicotinic acetylcholine receptors (nAChRs) in the brain, where they contribute to cognition, reward, nociception and mood. They are also implicated in depression, schizophrenia, cognitive deficit and in addiction to nicotine (Nic). Activation of these receptors by partial agonists is considered as a valid strategy to intervene therapeutically in the aforementioned dysfunctions. For clinical scenarios such as Nic addiction receptor subtype-specificity may increase clinical efficacy by decreasing off-target effects. Designing subtype-specific agonists is however problematic, mainly because of the highly conserved nature of the agonist binding site in the nAChR family. This thesis focuses on identifying elements in nAChR subtypes to aid the design of novel smoking cessation drugs. Functional and radioligand binding assays showed that novel C(10) cytisine (Cyt) derivatives maintain the potency and efficacy of the smoking cessation drug Cyt at α4β2 nAChR but had no significant activity at α7 subtype. Molecular docking in combination with functional assays showed that differences in residues in the complementary side of the agonist site do not account for the selectivity of the C(10) Cyt derivatives. However, molecular dynamics simulations revealed a conserved arginine residue (R101) in β3-strand that impairs agonist binding in α7 nAChR. In α4β2, the arginine residue (R106) establishes an inter-subunit electrostatic interaction with an aspartate residue (D185) in loop B, an interaction that is needed for functional expression. In α7, the aspartate residue is exchanged for glycine (G174), which makes the side chain of R101 highly mobile, allowing it to orientate towards the agonist binding site, which ultimately weakens agonist binding. In accord, stabilising R101 by introducing mutation G174D increases the potency of Cyt and its C(10) derivatives at α7 nAChR. Functional assays of R101 mutants suggested that this arginine affects agonist-activation and the onset of decay of agonist responses. R101 also couples to a channel mutation (L9’T) that influences gating. The mobility of the side chain of R101 sets the electrostatic landscape of the regions reached by the side chain. This effect may underlie the effects of R101 on α7 nAChR and raise the possibility that R101 may be a good target to increase receptor subtype-specificity.
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