Identification and Development of NPY1R Specific Agonists to Treat Alcohol and Drug Addiction

National Center For Advancing Translational Sciences

Investigators

Abstract

To date, the team have tested two different assays that measure intracellular cAMP level after NPY stimulation in 1536-well plate format using HEK293 cells stably expressing NPY1R. In the Homogeneous Time-Resolved Fluorescence (HTRF) cAMP assay, NPY activates the NPYR1 on the cell membrane that couples to the Gq/Gi G-protein to suppress the forskolin stimulated increase in cAMP. In the ACTone assay, the cells contain a Cyclic Nucleotide-Gated (CNG) channel (a reporter), which is activated by elevated intracellular cAMP, resulting in cell membrane depolarization that is detected by a fluorescent membrane potential dye. For each of the two assays, the team optimized several conditions, including cell density as well as concentrations of NPY1 peptide, forskolin (increases cAMP), and IBMX (prevents degradation of cAMP in response to forskolin stimulation). The team recently completed a drug repurposing screen of 2,800 compounds using the HTRF cAMP assay with four compound concentrations. Twenty-two (22) NPYR1 activator hits were identified from this screen and will be confirmed to eliminate potential false positive compounds.