Abstract

In the search for improved symptomatic treatment options for neurodegenerative and neuropsychiatric diseases, muscarinic acetylcholine M1 receptors (M1 mAChRs) have received significant attention. Drug development efforts have identified a number of novel ligands, some of which have advanced to the clinic. However, a significant issue for progressing these therapeutics is the lack of robust, translatable and validated biomarkers. One valuable approach to assessing target engagement is to utilize PET tracers. In this study we describe the pharmacological characterization of a selective M1 agonist amenable for in vivo tracer studies. We utilized a novel direct binding assay to identify non-radiolabelled ligands, including LSN3172176, with the favourable characteristics required for a PET tracer. In vitro functional and radioligand binding experiments revealed that LSN3172176 was a potent partial agonist (EC₅₀ 2.4-7.0nM, Emax 43%-73%, displaying binding selectivity for M1 mAChRs (Kd = 1.5nM) which was conserved across species (native tissue Kd=1.02, 2.66, 8, & 1.03 at mouse, rat, monkey and human respectively). Overall selectivity of LSN3172176 appeared to be a product of potency and stabilization of the high-affinity state of the M1 receptor, relative to other mAChR subtypes (M1>M2,M4,M5>M3). In vivo, use of wild-type and mAChR knock-out mice further supported the M1-preferring selectivity profile of LSN3172176 for the M1 receptor (78% reduction in cortical occupancy in M1 KO mice). These findings support the development of LSN3172176 as a potential PET tracer for assessment of M1 mAChR target engagement in the clinic and to further elucidate the function of M1 mAChRs in health and disease.