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

项目摘要 健康和患病的生理状态由相互作用的蛋白质的复杂网络控制， 在细胞中观察到的集体行为。翻译后蛋白质的精确定位和化学组成 跨蛋白质修饰的修饰（PTM）决定了它们的结构，功能，并赋予特异性， 细胞信号目前对PTM介导的信号传导和功能的阐明进展是 受到研究细胞中瞬时PTM的挑战和有限的生产蛋白质的方法的阻碍 含有修饰的氨基酸的特定组合。合成与化学生物学的最新进展 已经成功地证明了编码多种非标准氨基酸（nsAA）的能力，包括 生理相关的PTM转化为蛋白质。特别是，基因组学发展的最新进展 再编码菌（GRO）-再编码的E.开放编码通道的大肠杆菌-和工程翻译 编码PTM的系统（例如，磷酸丝氨酸）已经允许人磷蛋白的活化。这些 这些能力精确地定义了活性蛋白质状态，绘制了底物网络，并暗示了蛋白质的新功能。 疾病相关突变。然而，出现了两个重要挑战， 这些蛋白质网络的理解和限制这些见解转化为有针对性的临床解决方案。 首先，导致活性蛋白质状态的不同PTM的精确排列和贡献通常是 未知且难以破译。第二，在分子水平上靶向PTM的小分子的开发， 调节蛋白质活性的精确性是新药开发的决定性挑战。具体目标： 在这项提案中，我们寻求利用基因组，生物分子和蛋白质组学技术的强大基础， 系统和合成生物学方面的专业知识，以及构建基因组编码生物体的初步数据 (GRO)在E.大肠杆菌（Aim 1），工程翻译机器，重新分配正义和停止 用于位点特异性掺入多个非标准氨基酸的密码子 修饰成蛋白质（目标2），并利用这些技术开发合成生物学平台， 合成激活靶向分离新药候选物的疾病相关蛋白质网络（目的3）。 意义：这项工作将是有意义的，因为它将使合成激活生理 GRO中分子水平的相关蛋白质网络。这些活化的蛋白质系统可以阐明复杂的 生物分子相互作用是疾病的基础，并重现了人类蛋白质网络， 在他们的母语环境中学习和操作。将这些活化的蛋白质网络组装成小分子 库建立了一种快速简便的新方法来探测分子特异性的生物标志物，并将 一个新的基于合成生物学的药物发现平台。

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.