Development of a novel high-throughput assay for the investigation of GlyT-1b neurotransmitter transporter function

• Published in: Combinatorial chemistry & high throughput screening Vol. 9; no. 1; p. 9
• Main Authors: Allan, Lynda, Leith, J Lianne, Papakosta, Marianthi, Morrow, John A, Irving, Nicholas G, McFerran, Brian W, Clark, Alan G
• Format: Journal Article
• Language: English
• Published: United Arab Emirates 01.01.2006
• Subjects: Animals; CHO Cells; Cricetinae; Dimethyl Sulfoxide - chemistry; Fluorescent Dyes - chemistry; Glycine - analogs & derivatives; Glycine - antagonists & inhibitors; Glycine - chemistry; Glycine - metabolism; Glycine - pharmacology; Glycine Plasma Membrane Transport Proteins - chemistry; Glycine Plasma Membrane Transport Proteins - drug effects; Glycine Plasma Membrane Transport Proteins - physiology; Humans; Protein Isoforms - chemistry; Protein Isoforms - drug effects; Protein Isoforms - physiology; Sarcosine - analogs & derivatives; Sarcosine - pharmacology; Sensitivity and Specificity; Spectrometry, Fluorescence - instrumentation; Spectrometry, Fluorescence - methods; Structure-Activity Relationship; Time Factors
• ISSN: 1386-2073
• DOI: 10.2174/138620706775213886

The glycine transporter (GlyT-1b) is a Na(+)/Cl(-)-dependent electrogenic transporter which mediates the rapid re-uptake of glycine from the synaptic cleft. Based on its tissue distribution, GlyT-1 has been suggested to co-localise with the NMDA receptor where it may modulate the concentration of glycine at its co-agonist binding site. This data has led to GlyT-1 inhibitors being proposed as targets for disorders such as schizophrenia and cognitive dysfunction. Radiolabelled uptake assays (e.g. [(3)H]glycine) have been traditionally used in compound screening to identify glycine transporter inhibitors. While such an assay format is useful for testing limited numbers of compounds, the identification of novel glycine uptake inhibitors requires a functional assay compatible with high-throughput screening (HTS) of large compound libraries. Here, the authors present the development of a novel homogenous cell-based assay using the FLIPR membrane potential blue dye (Molecular Devices) and FLEXstation. Pharmacological data for the GlyT-1 inhibitors Org 24598 and ALX 5407 obtained using this novel electrogenic assay correlated well with the conventional [(3)H]-glycine uptake assay format. Furthermore, the assay has been successfully miniaturised using FLIPR(3) and therefore has the potential to be used for high-throughput screening.