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

开发下一代基因组重新编码的生物体以合成激活药物发现的生物标志物

• 批准号: 10263259
• 负责人: Farren J. Isaacs
• 金额: $ 58.34万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10263259
• 关键词: Acetylation; Address; Amino Acids; Arginine; Behavior; Binding; Biochemical; Biological Markers; Biology; Cells; Chemicals; Clinical; Code; Codon Nucleotides; Complex; DNA biosynthesis; Data; Development; Disease; Disease Progression; Disease model; Drug Targeting; Engineering; Enzymes; Escherichia coli; Essential Genes; Foundations; Genetic Code; Genome; Genome engineering; Genomics; Human; Internet; Knowledge; Lead; Length; Maps; Mediating; Metabolic; Methods; Modality; Molecular; Mutation; Organism; Organism Strains; Pathway interactions; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphotransferases; Physiological; Positioning Attribute; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Recombinants; Regulation; Research; Role; Sense Codon; Series; Signal Transduction; Signaling Protein; Site; Specificity; Structure; System; Systems Biology; Technology; Terminator Codon; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transfer RNA; Translations; Validation; Work; base; drug candidate; drug development; drug discovery; human disease; in vivo; insight; interest; new technology; new therapeutic target; next generation; novel strategies; novel therapeutics; programs; pyrrolysine; release factor; scaffold; small molecule; small molecule libraries; synthetic biology; therapeutic protein; tool; Acetylation; Address; Amino Acids; Arginine; Behavior; Binding; Biochemical; Biological Markers; Biology; Cells; Chemicals; Clinical; Code; Codon Nucleotides; Complex; DNA biosynthesis; Data; Development; Disease; Disease Progression; Disease model; Drug Targeting; Engineering; Enzymes; Escherichia coli; Essential Genes; Foundations; Genetic Code; Genome; Genome engineering; Genomics; Human; Internet; Knowledge; Lead; Length; Maps; Mediating; Metabolic; Methods; Modality; Molecular; Mutation; Organism; Organism Strains; Pathway interactions; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphotransferases; Physiological; Positioning Attribute; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Recombinants; Regulation; Research; Role; Sense Codon; Series; Signal Transduction; Signaling Protein; Site; Specificity; Structure; System; Systems Biology; Technology; Terminator Codon; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transfer RNA; Translations; Validation; Work; base; drug candidate; drug development; drug discovery; human disease; in vivo; insight; interest; new technology; new therapeutic target; next generation; novel strategies; novel therapeutics; programs; pyrrolysine; release factor; scaffold; small molecule; small molecule libraries; synthetic biology; therapeutic protein; tool

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.

项目摘要 健康且解散的身体状态受到复杂的相互作用蛋白质的控制 在细胞中观察到的集体行为。翻译后的精确放置和化学组成 跨蛋白质装饰的修改（PTM）决定了它们的结构，功能和赋予特异性 细胞信号传导。阐明PTM介导的信号传导和功能的当前进展是 受细胞中瞬时PTM的挑战和产生蛋白质的有限方法的挑战阻碍 包含修饰氨基酸的特定组合。合成和化学生物学的最新进展 已经成功证明了编码非标准氨基酸（NSAA）的能力，包括 生理上相关的PTMS，分为蛋白质。特别是，基因发展的最新进展 重新编码的有机体（GRO） - 带有开放编码通道的大肠杆菌的重新编码菌株和工程翻译 编码PTM的系统（例如，磷酸碱）允许激活人磷蛋白。这些 功能具有精确定义的活性蛋白质状态，地图基板网络，并为 与疾病有关的突变。但是，出现了两个重要的挑战，这些挑战排除了全面的挑战 了解这些蛋白质网络，并限制将这种见解转化为有针对性的临床解决方案。 首先，导致活性蛋白质态的不同PTM的精确排列和贡献通常是 未知且难以破译。其次，靶向PTMS分子的小分子的发展 调节蛋白质活性的精度是新药开发的决定性挑战。具体目的： 在此提案中，我们试图利用基因组，生物分子和蛋白质组学技术的强大基础， 在系统和合成生物学方面的专业知识，以及构建基因组生物的初步数据 （GRO）带有大肠杆菌中的三个开放密码子（AIM 1），工程师翻译机械重新分配并停止 多种非标准氨基酸的特定地点保险的密码子编码翻译后的密码子 修改蛋白质（AIM 2），并利用这些技术开发一个合成生物学平台，该平台 合成激活与疾病相关的蛋白质网络，该蛋白网络针对隔离新药候选物（AIM 3）。 意义：这项工作将很重要，因为它将实现物理的合成激活 GROS分子水平的相关蛋白网络。这些激活的蛋白质系统可以阐明复合物 生物分子相互作用是基于疾病和概括的人类蛋白网络的基础 在其本地环境中进行研究和操纵。将这些激活的蛋白质网络挑战到小分子 图书馆建立了一种快速而便捷的新方法，以分子特异性探测生物标志物，并将其设置为 新的基于合成生物的药物发现平台的阶段。