Aristotelia Alkaloids as Probes for the Nicotinic Acetylcholine Receptors

ABSTRACT The nicotinic acetylcholine receptors (nAChRs) have been implicated in a variety of central nervous system (CNS) disorders. However, the significant similarity between subtypes has limited the discovery of subtype- selective nAChR antagonists. As a result, the pharmacological utility of the nAChRs depends on the development of improved ligands. Recently, several alkaloids isolated from Aristotelia chilensis were identified as nAChR antagonists that preferentially antagonize the α3β4 subtype over the α4β2 and α7 subtypes. While other α3β4 antagonists exist, they also have poor pharmacokinetic properties and multiple off-target liabilities, making the subtype-selectivity the Aristotelia alkaloids possess particularly unique. In addition, preliminary competitive studies indicate that these alkaloids act through an allosteric mechanism of action, in contrast to other antagonists that are largely orthosteric ligands or channel blockers. Previous studies of the Aristotelia genus have identified >30 unique alkaloids, but few studies have been conducted on their biological activity. My central hypothesis is that these understudied Aristotelia alkaloids are also subtype-selective nAChR antagonists that operate through an allosteric mechanism of action. To investigate this, in Aim 1, I will extract alkaloids from the leaves of A. chilensis through a novel countercurrent chromatography approach, isolating known natural products while also potentially discovering novel alkaloids. In a complementary approach, Aim 2 seeks to access known Aristotelia alkaloids via a series of biomimetic transformations. Armed with a library of alkaloids, Aim 3 will evaluate their activity at multiple nAChRs through a functional assay. Competitive inhibition studies will be done to investigate the potential allosteric mechanism of action, while mutagenetic analysis will identify which residues on the receptors are crucial for activity and reveal differences between subtypes. While making synthetic advancements, this research is both innovative and significant as it will be the most extensive biological evaluation of the Aristotelia alkaloids to date, providing tools to probe the nAChRs as a whole, as the alkaloids establish an entire class of subtype-selective nAChR ligands.