DRUG DISCOVERY BY DIRECTED EVOLUTION IN MAMMALIAN CELLS

项目摘要 定向进化将自然进化的原理应用于实验室， 分子工程学作为一个例子，它负责产生大多数批准的 治疗性抗体和仍在临床开发中的抗体。尽管取得了进展，一个有价值的治疗利基 仍然在其研究范围之外：哺乳动物细胞。虽然在体外系统中，噬菌体，细菌和酵母已经提供了 虽然哺乳动物细胞本身已被实验室操作，但已证明它们不太容易处理。因此，功率 对于寻求调节哺乳动物细胞的药物开发管道来说， 信号此外，许多定向进化运动导致生物分子在关键方面失败， 移植到人体细胞。为了解决这些局限性和药物发现的先进方法，我将 探索和挖掘病毒的多样性，为哺乳动物细胞的分子进化创造一个新的系统。接下来， 专注于5-HT2A血清素受体，我将创建细胞外纳米抗体，以模板药物发现 通过结构测定和计算机对接进行的活动。通过这些研究获得的见解将是 应用于针对暗GPCR的状态特异性纳米抗体的定向进化。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，以及新的途径 为GPCR的去病毒化而努力。

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.