title: New fluorescent substrate enables quantitative and high-throughput examination of vesicular monoamine transporter 2 (VMAT2).
abstract: Vesicular monoamine transporter 2 (VMAT2) is an essential component of the monoaminergic neurotransmission system in the brain as it transports monoamine neurotransmitters from the neuronal cytosol into the synaptic vesicles and thus contributes to modulation of neurotransmitter release. Considering the continuing interest in VMAT2 as a drug target, as well as a target for the design of imaging probes, we have developed a fluorescent substrate well suited for the study of VMAT2 in cell culture. Herein, we report the synthesis and characterization of a new fluorescent probe, FFN206, as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy, with subcellular localization to VMAT2-expressing acidic compartments without apparent labeling of other organelles. VMAT2 activity can also be measured via microplate reader. The apparent Km of FFN206 at VMAT2 was found to be 1.16 ± 0.10 μM, similar to that of dopamine. We further report the development and validation of a cell-based fluorescence assay amenable to high-throughput screening (HTS) using VMAT2-transfected HEK cells (Z'-factor of 0.7-0.8), enabling rapid identification of VMAT2 inhibitors and measurement of their inhibition constants over a broad range of affinities. FFN206 thus represents a new tool for optical examination of VMAT2 function in cell culture.
authors.affiliation: Department of Chemistry, Columbia University , New York, New York 10027, United States.
authors.forename: Gang
authors.initials: G
authors.lastname: Hu
authors.forename: Adam
authors.initials: A
authors.lastname: Henke
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Karpowicz
authors.forename: Mark S
authors.initials: MS
authors.lastname: Sonders
authors.forename: Frances
authors.initials: F
authors.lastname: Farrimond
authors.forename: Robert
authors.initials: R
authors.lastname: Edwards
authors.forename: David
authors.initials: D
authors.lastname: Sulzer
authors.forename: Dalibor
authors.initials: D
authors.lastname: Sames
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D012680:Sensitivity and Specificity; D054852:Small Molecule Libraries / Q000494:pharmacology; D050493:Vesicular Monoamine Transport Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D054852:Small Molecule Libraries; D050493:Vesicular Monoamine Transport Proteins