A G-protein-coupled receptor-based sensor design platform for high throughput drug screening

建议书摘要 G蛋白偶联受体是最大的跨膜受体家族，由800多个成员组成。 人体内的受体类型。GPCRs在多种生理过程中发挥着重要作用。因此， 在FDA批准的所有药品中，有34%是GPCRs的目标。尽管有这么多人，但超过 一半的GPCR仍然没有被FDA批准的药物靶向。这在一定程度上是由于缺乏具有成本效益的 用于监测GPCR后即时信号事件的高通量药物筛选平台 激活。为了进一步探索FDA药物靶向和非靶向GPCRs的治疗潜力，它 有必要开发一种新型的高通量高信噪比的GPCR药物筛选平台。 基于gpcr的荧光传感器对于高通量药物筛选是有利的，因为它们不 需要昂贵的试剂或底物，能够以低成本筛选数百种化合物。我瞄准了 设计基于gpcr的荧光传感器，可用于为Gαi/o-Couted和GαS筛选药物- 成对的GPCR。为了设计和优化这个平台，我将为kappa阿片受体Mu设计传感器。 阿片受体、β-2-肾上腺素能受体和多巴胺D1受体。我将使用有理的组合 蛋白质设计和定向进化来优化这些传感器。然后，我将进行原则性药物的证明 与加州大学化学基因组学中心合作，用这些传感器进行筛查 密歇根州。我希望这个平台比目前的GPCR配体药物筛选成本更低，更容易使用 站台。这项提议的完成将允许为高电压设计一系列广泛的传感器 吞吐量GPCR药物筛选。这一新的荧光传感器设计平台将使新的 可用于治疗目的的GPCRs药物。

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.