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: 10097168
• 负责人: Farren J. Isaacs
• 金额: $ 58.8万
• 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/10097168
• 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

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.

项目概要 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，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) 决定其结构、功能并赋予其特异性 细胞信号传导。目前阐明 PTM 介导的信号传导和功能的进展是 由于研究细胞中瞬时 PTM 的挑战和生产蛋白质的方法有限而受到阻碍 含有修饰氨基酸的特定组合。合成生物学和化学生物学的最新进展。 已成功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，尤其是基因组开发的最新进展。 重新编码生物（GRO）——具有开放编码通道的重新编码大肠杆菌菌株——以及工程化翻译 编码 PTM（例如磷酸丝氨酸）的系统已允许激活人类磷蛋白。 能力精确定义了活性蛋白质状态，绘制底物网络图，并暗示了新功能 然而，出现了两个重要的挑战，阻碍了全面的研究。 对这些蛋白质网络的理解并限制了将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 其次，在分子水平上靶向 PTM 的小分子的开发。 精确调节蛋白质活性是新药开发的一个决定性挑战。 在本提案中，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记录生物体的初步数据 (GRO) 与大肠杆菌中的三个开放密码子（目标 1），设计重新分配意义和终止的翻译机器 用于编码翻译后氨基酸的多个非标准氨基酸的位点特异性掺入的密码子 修饰蛋白质（目标 2），并利用这些技术开发合成生物学平台 综合激活疾病相关蛋白质网络，用于分离新候选药物（目标 3）。 意义：这项工作意义重大，因为它将实现生理学的合成激活 GRO 中分子水平的相关蛋白质网络可以阐明复杂的蛋白质激活系统。 疾病背后的生物分子相互作用和难以概括的人类蛋白质网络 在其天然环境中研究和操作这些激活的蛋白质网络以小分子。 文库建立了一种快速、简便的新方法来探测分子特异性的生物标志物，并设定了 基于新合成生物学的药物发现平台的阶段。