A Tale of Two Alpha's: Design of Selective Agonists and Modulators Targeting the a4-a4 Binding Site of (a4)3(b2)2 Nicotinic Acetylcholine Receptors

Abstract

The a4b2 nicotinic acetylcholine receptor (nAChR), belonging to the family of 'Cys-loop' pentameric ligand-gated ion channels, is one of the most abundant subtypes found in the brain. Several researches have concluded its important role in modulating memory, sleep and attention. Thereby, making it an attractive druggable target due to its implication in several neurological, and psychiatric disorders such as Alzheimer's disease, Attention Deficit Hyperactivity Disorder (ADHD) and nicotine addiction. The heteropentameric a4b2 nAChRs are known to arrange in two different stoichiometries; (a4)2(b2)3 and (a4)3(b2)2. Apart from the known and extensively targeted a4-b2 binding sites that are present in both stoichiometries, the distinctive a4-a4 binding site located only in the (a4)3(b2)2 stoichiometry, has become a desirable target to exploit its site-specific functions in formulating treatments for the neurological disorders. The key challenge lies in developing a4-a4 site-selective ligands, as structurally both are closely matched binding sites with only minor differences. Understanding these key differences could pave the way for more potent and selective ligands for the a4-a4 binding site.

NS3920, a pyridine-diazepane compound and NS9283, an oxadiazole analogue has been found to be active at the a4-a4 binding site. Based on the potency of NS3920 and selectivity of NS9283, diazepane substituted pyridyl/phenyl hybrids harbouring common structural features of both ligands were designed, with an aim to improve the biological profile of ligands at the a4-a4 binding site. The synthesis of 5- and 6-monosubstituted as well as 5,6-disubstituted pyridyl/phenyl hybrids was achieved through the Buchwald-Hartwig reaction conditions. The biological evaluation of these hybrids was carried out by employing the TEVC electrophysiology and receptor binding assays on (a4)2(b2)3, (a4)3(b2)2 and mutated (a4)2(b2HQT)3 receptor harbouring three interfaces resembling a4-a4 binding site. The results obtained remained largely inconclusive, thus, leading us to the design of modulators.

Positive allosteric modulators or PAMs are a newfound interest of the scientific community due to their ability to increase the frequency of channel opening without directly activating the receptor itself, thus, reducing the chances of receptor desensitisation, as PAMs bind at a site away from the orthogonal binding site of classical agonists, like ACh. A small pocket in close proximity to the a4-a4 binding site was identified recently by the collaborators at the University of Sydney, which could prove beneficial in unlocking the full potential of receptor. Several co-crystal structures of known agonists and antagonists were analysed that may have a bond vector directed towards the new pocket. Anabasine and nicotine were chosen as the two lead templates to design and synthesise analogues, because of the presence of a bond vector directed straight towards the newly identified pocket. L-proline catalysed Mannich reductive cyclisation principle was employed for the synthesis of several anabasine and nicotine analogues. Side chain substitution was carried out either via CDI-mediated esterification or base-promoted etherification, to install substituents that can directly interact with the site residues of the identified pocket. Preliminary biological studies have shown that four anabasine ester analogues possess positive modulatory activity at a4-a4 binding site which could be due to direct interaction of sidechain substituents with the site residues.