A Novel Approach to Cholinergic Signaling Modulation: Development and Characterization of ML352, a Novel, Noncompetitive Inhibitor of the Presynaptic Choline Transporter

Abstract

The choline transporter (CHT) is a critical determinant of acetylcholine (ACh) synthesis and signaling. Despite the medicinal uses of anticholinergics, CHT remains a largely unexplored target for such endeavors, with only one competitive inhibitor, hemicholinium‐3 (HC‐3), in common use. Thus, we initiated a high‐throughput screen to expand CHT pharmacology, biasing our search toward agents with allosteric modes action. Our efforts identified 5 novel classes of CHT antagonists, with one class subjected to further chemical diversification to improve potency, selectivity and pharmacokinetics. Among these compounds, ML352, was identified as a potent, noncompetitive inhibitor of CHT activity in both transfected cells and nerve terminal preparations. ML352 exhibits little or no inhibitory activity at the ACh homeostatic enzymes or against many other ion channels, transporters and receptors. DMPK studies revealed a high intravenous plasma clearance, a combination of the oral bioavailability and total brain concentrations indicate ML352 to be a suitable probe for investigations of altered in vivo ACh signaling and behavioral effects in rodents. Our efforts reveal ML352 and its derivatives as the first potent and selective CHT inhibitors developed since the identification of HC‐3 more than 50 years ago, providing a novel platform for the treatment of disorders characterized by excess ACh signaling.