Validation of a High-Throughput Calcium Mobilization Assay for the Human Trace Amine-Associated Receptor 1

The human trace amine-associated receptor 1 (hTAAR1) is a G protein-coupled receptor (GPCR) that is widely expressed in monoaminergic nuclei in the central nervous system and has therapeutic potential for multiple diseases, including drug addiction and schizophrenia. Thus, identification of novel hTAAR1 ligands is critical to advancing our knowledge of hTAAR1 function and to the development of therapeutics for a wide range of diseases. Herein we describe the development of a robust, 3-addition high-throughput screening (HTS) calcium mobilization assay using stable CHO-Gα_q16-hTAAR1 cells, which functionally couple hTAAR1 to the promiscuous Gα_q16 protein and thus allow signal transduction to occur through mobilization of internal calcium. Our previously established 96-well hTAAR1 assay was first miniaturized to the 384-well format and optimized to provide an assay with a Z' factor of 0.84, which is indicative of a robust HTS assay. Using the 3-addition protocol, 22,000 compounds were screened and yielded a ~1% agonist hit rate and a ~0.2% antagonist hit rate. Of the antagonist hits, two confirmed hits are the most potent hTAAR1 antagonists identified to date (IC₅₀ = 206 and 281 nM). While scientists have been studying hTAAR1 for years, the lack of suitable hTAAR1 antagonists has been a major roadblock for studying the basic pharmacology of hTAAR1. Thus, these new ligands will serve as valuable tools to study hTAAR1-mediated signaling mechanisms, therapeutic potential, and in vivo functions.