DRUG DISCOVERY BY DIRECTED EVOLUTION IN MAMMALIAN CELLS

项目摘要 定向进化将自然进化的原理引入实验室，其独特之处在于它对 分子工程学。作为一个例子，它负责产生大多数已批准的 治疗性抗体和那些仍在临床开发中的抗体。尽管取得了进展，但一个有价值的治疗利基 仍然不在它的范围内：哺乳动物细胞。而在体外系统中，噬菌体、细菌和酵母 在实验室的操纵下，哺乳动物细胞被证明不那么容易驯服。因此，权力 寻求调节哺乳动物细胞的药物开发管道仍然无法接触到进化的过程 发信号。此外，许多定向进化运动导致生物分子在以下情况下以关键方式失效 移植到人类细胞上。为了解决药物发现中的这些限制和先进方法，我将 探索和挖掘病毒多样性，以创建一种新的哺乳动物细胞分子进化系统。下一首, 专注于5-HT2A型5-羟色胺受体，我将创建细胞外纳米小体来作为药物发现的模板 通过结构确定和电子对接开展运动。通过这些研究获得的见解将是 应用于针对暗GPCR的特定国家纳米实体的定向进化。这项工作将导致 哺乳动物细胞定向进化的一般方法、针对HTR2A的化学物质和新途径 推动实现GPCRs的去孤立化。

Project Summary Directed evolution, which adopts principles of natural evolution to the laboratory, is singular in its impact on molecular engineering. As one example, it is responsible for the generation of the majority of approved therapeutic antibodies and those still in clinical development. Despite its progress, a valuable therapeutic niche remains outside its scope: the mammalian cell. While in vitro systems, phage, bacteria, and yeast have lent themselves to laboratory manipulation, mammalian cells have proven less tractable. Consequently, the power of evolution remains inaccessible to drug development pipelines that seek to modulate mammalian cell signaling. Further, many directed evolution campaigns result in biomolecules that fail in critical ways when transplanted to human cells. To address these limitations and advance methods in drug discovery, I will explore and mine viral diversity to create a novel system for molecular evolution in mammalian cells. Next, focusing on the 5-HT2A serotonin receptor, I will create extracellular nanobodies to template a drug discovery campaign via structural determination and in silico docking. The insights gained by these studies will be applied toward the directed evolution of state-specific nanobodies against dark GPCRs. This work will result in a general method for directed evolution in mammalian cells, chemical matter against HTR2A, and new paths forward for the deorphanization of GPCRs.