Developing next-generation genomically recoded organisms to synthetically activate biomarkers for drug discovery

项目总结 健康和患病的生理状态由相互作用的蛋白质组成的复杂网络控制，这些蛋白质赋予 在细胞中观察到的集体行为。翻译后的精确位置和化学成分 跨蛋白质修饰的修饰(PTM)决定了它们的结构、功能和赋予蛋白质的特异性 细胞信号。目前在阐明PTM介导的信号和功能方面的进展是 受制于在细胞中研究瞬时PTMS的挑战和有限的蛋白质生产方法 含有特定的修饰氨基酸组合。合成与化学生物学的最新进展 已经成功地展示了编码各种非标准氨基酸(NsAA)的能力，包括 生理上相关的PTM，转化为蛋白质。特别是，基因组学发展的最新进展 编码有机体(GROS)-具有开放编码通道的编码大肠杆菌菌株-和工程翻译 编码PTM(例如，磷酸丝氨酸)的系统已经允许激活人的磷酸蛋白。这些 功能精确定义了活性蛋白质状态，绘制了底物网络图，并暗示了新的功能 与疾病相关的突变。然而，已经出现了两个重要的挑战，这排除了全面的 了解这些蛋白质网络，并限制将这些见解转化为有针对性的临床解决方案。 首先，导致蛋白质活性状态的不同PTM的精确排列和贡献通常是 未知且难以破译。第二，以PTM为靶点的小分子的发展 精确地调节蛋白质活性是新药开发的决定性挑战。具体目标： 在这项提议中，我们寻求利用基因组、生物分子和蛋白质组技术的坚实基础， 系统和合成生物学方面的专业知识，以及构建基因组编码有机体的初步数据 (GRO)在大肠杆菌中有三个开放密码子(目标1)，设计重新分配感觉和停止的翻译机械 编码翻译后的多种非标准氨基酸的位点特异性结合密码子 修饰成蛋白质(目标2)，并利用这些技术开发一个合成生物学平台， 人工激活与疾病相关的蛋白质网络，目标是分离新的候选药物(目标3)。 意义：这项工作将具有重大意义，因为它将使生理上的合成激活 GROS在分子水平上的相关蛋白质网络。这些激活的蛋白质系统可以阐明复合体 疾病背后的生物分子相互作用并概括了人类蛋白质网络，这些网络很难 在他们的母语环境中学习和操控。向小分子挑战这些激活的蛋白质网络 文库建立了一种快速而简便的新方法来探测分子特异性的生物标记物，并设置了 为一个基于合成生物学的新药物发现平台做准备。

PROJECT SUMMARY Healthy and diseased physiological states are governed by a complex web of interacting proteins that confer the collective behavior observed in cells. The precise placement and chemical composition of post-translational modifications (PTMs) decorated across proteins determines their structure, function, and impart specificity for cellular signaling. Current progress toward the elucidation of PTM-mediated signaling and function is hampered by the challenge of studying transient PTMs in cells and limited methods to produce proteins containing specific combinations of modified amino acids. Recent advances in synthetic and chemical biology have successfully demonstrated the ability to encode diverse nonstandard amino acids (nsAAs), including physiologically relevant PTMs, into proteins. In particular, recent advances in the development of genomically recoded organism (GROs) – recoded strains of E. coli with open coding channels – and engineered translation systems that encode PTMs (e.g., phosphoserine) have allowed activation of human phosphoproteins. These capabilities have precisely defined active protein states, map substrate networks, and implicate new function for disease-relevant mutations. However, two important challenges have emerged that preclude a comprehensive understanding of these protein networks and limit the translation of such insights into targeted clinical solutions. First, the precise arrangement and contributions of distinct PTMs that lead to active protein states is often unknown and hard to decipher. Second, the development of small molecules that target PTMs at molecular precision to modulate protein activity is a defining challenge for the development of new drugs. Specific Aims: In this proposal, we seek to leverage a strong foundation of genomic, biomolecular and proteomic technologies, expertise in systems and synthetic biology, and preliminary data to construct a genomically recoded organism (GRO) with three open codons in E. coli (Aim 1), engineer translational machinery that reassigns sense and stop codons for site-specific incorporation of multiple nonstandard amino acids that encode post-translational modifications into proteins (Aim 2), and utilize these technologies to develop a synthetic biology platform that synthetically activates disease-relevant protein networks targeted for isolation of new drug candidates (Aim 3). Significance: This work will be significant because it will enable the synthetic activation of physiologically relevant protein networks at the molecular level in GROs. These activated protein systems can elucidate complex biomolecular interactions that underlie disease and recapitulate human protein networks that are difficult to study and manipulate in their native contexts. Challenging these activated protein networks to small molecule libraries establishes a rapid and facile new approach to probe biomarkers at molecular specificity and sets the stage for a new synthetic-biology based drug discovery platform.