! Project Summary The objective of this SBIR Phase II project is to optimize and validate a prototype assay for ligand bias to be used in screening drugs that target seven transmembrane G- protein coupled receptors (GPCRs). The assay is based on genetically-encoded fluorescent biosensors that report signaling with a change in fluorescence intensity. Aim 1 of this project involves engineering and testing to increase the change in fluorescence intensity of biosensors for β arrestin1 and β arrestin2. This involves using an established workflow and screening process to iteratively improve linkers in between a very bright green fluorescent protein and β-arrestin. This proprietary process consists of randomly mutagenizing key amino acids to create low diversity libraries of candidate biosensors that are screened for improved fluorescence properties. Aim 2 involves optimizing assay parameters and validating the multiplex assay comprised of the optimized green fluorescent biosensor for β arrestin, co-expressed with existing red fluorescent biosensors for diacylglycerol (DAG) or cyclic AMP. The red sensor indicates G-protein mediated activity while the green sensor indicates arrestin activity. The assay will be validated with a select panel of GPCRs and small molecule and peptide agonists. GPCRs, are one of the largest family of drug targets. Their activity is mediated by both G proteins and β-arrestins which activate a network of distinct signaling pathways. Depending upon the GPCR and cell type, biased agonism can be therapeutically beneficial or produce unwanted or harmful side effects (Violin and Lefkowitz, 2007). Quantifying bias in GPCR signaling could help to identify new therapeutic drugs that avoid adverse effects. Detecting agonist bias early in the screening process, in cell types that are relevant to disease, has the potential to reduce the risk and cost of drug discovery.