title: Identification and characterization of ML352: a novel, noncompetitive inhibitor of the presynaptic choline transporter.
abstract: The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Ennis
authors.forename: Jane
authors.initials: J
authors.lastname: Wright
authors.forename: Cassandra L
authors.initials: CL
authors.lastname: Retzlaff
authors.affiliation: #Johns Hopkins University Ion Channel Center, Baltimore, Maryland 21205, United States.
authors.forename: Owen B
authors.initials: OB
authors.lastname: McManus
authors.affiliation: #Johns Hopkins University Ion Channel Center, Baltimore, Maryland 21205, United States.
authors.forename: Zhinong
authors.initials: Z
authors.lastname: Lin
authors.affiliation: #Johns Hopkins University Ion Channel Center, Baltimore, Maryland 21205, United States.
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Huang
authors.affiliation: #Johns Hopkins University Ion Channel Center, Baltimore, Maryland 21205, United States.
authors.forename: Meng
authors.initials: M
authors.lastname: Wu
authors.affiliation: #Johns Hopkins University Ion Channel Center, Baltimore, Maryland 21205, United States.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: J Scott
authors.initials: JS
authors.lastname: Daniels
authors.forename: Craig W
authors.initials: CW
authors.lastname: Lindsley
authors.forename: Corey R
authors.initials: CR
authors.lastname: Hopkins
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D000818:Animals; D001549:Benzamides / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D002794:Choline / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D057809:HEK293 Cells; D006426:Hemicholinium 3 / Q000494:pharmacology; D006801:Humans; D007555:Isoxazoles / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009433:Neural Inhibition / Q000187:drug effects; D016548:Prosencephalon / Q000166:cytology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism
chemical_list: D001549:Benzamides; D004791:Enzyme Inhibitors; D007555:Isoxazoles; D026901:Membrane Transport Proteins; C000603315:N-((3-isopropylisoxazol-5-yl)methyl)-4-methoxy-3-((1-methylpiperidin-4-yl)oxy)benzamide; C065637:choline transporter; D006426:Hemicholinium 3; D002794:Choline
keywords: Choline; acetylcholine; drug development; hemicholinium-3; transport