Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays

A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor (GPCR) activation and internalization was applied to the human β2AR. This technology avoids microscopy and antibody-based detection methods. A major goal for the project was to identify G-protein independent/β2AR ligands or β2AR biased ligands that induce β2AR internalization. Analysis of the most potent hits in the primary project revealed that they interfered with fluorogen activation by the FAP rather than interacting with the receptor itself. These molecules were pursued further because they had the potential to enable improved assay protocols to monitor receptor trafficking and receptor location in real time. A highly potent compound (ML342, CID 2953239) was declared as a Molecular Libraries Probe Center Network (MLPCN) probe molecule.