PROPOSAL SUMMARY G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors, comprising over 800 receptor types in the human body. GPCRs play crucial roles in various physiological processes. Consequently, GPCRs are the target of 34% of all FDA approved pharmaceuticals. Despite this significant number, more than half of GPCRs remain untargeted by FDA approved drugs. This is partially due to the lack of a cost-effective, high-throughput drug screening platform for monitoring the immediate signaling events following GPCR activation. To further explore the therapeutic potential of both the FDA drug-targeted and untargeted GPCRs, it is necessary to develop a new high-throughput GPCR drug screening platform with a high signal-to-noise. GPCR-based fluorescent sensors are advantageous for high-throughput drug screening because they do not require expensive reagents or substrates, enabling the screening of hundreds of compounds at low costs. I aim to engineer GPCR-based fluorescent sensors that can be used to screen drugs for both Gαi/o-coupled and Gαs- coupled GPCRs. To design and optimize this platform, I will engineer sensors for the kappa opioid receptor, mu opioid receptor, beta-2-adrenergic receptor, and the dopamine D1 receptor. I will use a combination of rational protein design and directed evolution to optimize these sensors. Then, I will perform a proof of principle drug screening with these sensors in collaboration with the Center of Chemical Genomics at the University of Michigan. I expect this platform to cost less and be easier to use than the current GPCR ligand drug screening platforms. The completion of this proposal will allow the engineering of a wide array of sensors for high throughput GPCR drug screening. This new fluorescent sensor design platform will enable the screening of novel drugs for GPCRs that can be used for therapeutic purposes.