Identification and characterization of ml352: A novel, noncompetitive inhibitor of the presynaptic choline transporter

Elizabeth A. Ennis, Jane Wright, Cassandra L. Retzlaff, Owen B. McManus, Zhinong Lin, Xiaofang Huang, Meng Wu, Min Li, J. Scott Daniels, Craig W. Lindsley, Corey R. Hopkins, Randy D. Blakely

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)417-427
Number of pages11
JournalACS Chemical Neuroscience
Volume6
Issue number3
DOIs
StatePublished - Mar 18 2015

Fingerprint

Choline
Cholinergic Agents
Hemicholinium 3
Norepinephrine Plasma Membrane Transport Proteins
Serotonin Plasma Membrane Transport Proteins
Functional analysis
Dopamine Plasma Membrane Transport Proteins
Pharmacokinetics
Acetylcholinesterase
choline transporter
Synaptosomes
Ion Channels
Metabolism
Acetylcholine
Assays
Binding Sites
Throughput
Membranes
Imaging techniques
Pharmacology

Keywords

  • acetylcholine
  • Choline
  • drug development
  • hemicholinium-3
  • transport

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Physiology
  • Cognitive Neuroscience

Cite this

Ennis, E. A., Wright, J., Retzlaff, C. L., McManus, O. B., Lin, Z., Huang, X., ... Blakely, R. D. (2015). Identification and characterization of ml352: A novel, noncompetitive inhibitor of the presynaptic choline transporter. ACS Chemical Neuroscience, 6(3), 417-427. https://doi.org/10.1021/cn5001809

Identification and characterization of ml352 : A novel, noncompetitive inhibitor of the presynaptic choline transporter. / Ennis, Elizabeth A.; Wright, Jane; Retzlaff, Cassandra L.; McManus, Owen B.; Lin, Zhinong; Huang, Xiaofang; Wu, Meng; Li, Min; Daniels, J. Scott; Lindsley, Craig W.; Hopkins, Corey R.; Blakely, Randy D.

In: ACS Chemical Neuroscience, Vol. 6, No. 3, 18.03.2015, p. 417-427.

Research output: Contribution to journalArticle

Ennis, EA, Wright, J, Retzlaff, CL, McManus, OB, Lin, Z, Huang, X, Wu, M, Li, M, Daniels, JS, Lindsley, CW, Hopkins, CR & Blakely, RD 2015, 'Identification and characterization of ml352: A novel, noncompetitive inhibitor of the presynaptic choline transporter', ACS Chemical Neuroscience, vol. 6, no. 3, pp. 417-427. https://doi.org/10.1021/cn5001809
Ennis, Elizabeth A. ; Wright, Jane ; Retzlaff, Cassandra L. ; McManus, Owen B. ; Lin, Zhinong ; Huang, Xiaofang ; Wu, Meng ; Li, Min ; Daniels, J. Scott ; Lindsley, Craig W. ; Hopkins, Corey R. ; Blakely, Randy D. / Identification and characterization of ml352 : A novel, noncompetitive inhibitor of the presynaptic choline transporter. In: ACS Chemical Neuroscience. 2015 ; Vol. 6, No. 3. pp. 417-427.
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