Genomic incorporation of stapled peptides for cost effective discovery and synthesis of novel therapeutics

钉合肽的基因组整合，以经济有效的方式发现和合成新疗法

基本信息

批准号：
10360415
负责人：
Emma J Chory
金额：
$ 6.98万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10360415
关键词：
Active Sites Amino Acids Amino Acyl-tRNA Synthetases Bacteria Bacteriophages Biological Assay Biological Availability Biomimetics Capsid Proteins Cells Characteristics Chemicals Codon Nucleotides Dehydration Directed Molecular Evolution Drug Kinetics Enzymes Evolution FDA approved Face Generations Genome Genomics Goals Human Pathology Hydro-Lyases In Vitro Libraries Ligase Malignant Neoplasms Medicine Methods Minor Molecular Organism Peptide Hydrolases Peptide Library Peptide Synthesis Peptides Permeability Pharmaceutical Preparations Phase Predisposition Prochlorococcus Production Property Proteins RNA, Transfer, Amino Acid-Specific Reaction Resistance Robotics Scheme Serine Site Solid Specificity Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Structure Sulfhydryl Compounds TP53 gene Therapeutic Threonine Transfer RNA Tumor Suppressor Proteins Validation base chromatin remodeling cost cost effective design improved in vivo inhibitor interest iterative design mutant novel novel therapeutics peptide A peptide drug protein protein interaction small molecule small molecule inhibitor stapled peptide therapeutic target thioether tool transcription factor unnatural amino acids

项目摘要

ABSTRACT In the era of genome medicine, we are able to precisely identify the molecular susceptibilities of a range of human pathologies, including cancer. However, many of the bona fide drivers of cancer—transcription factors, tumor suppressors, and chromatin remodelers (such as p53, myc, and SWI/SNF) cannot be readily targeted by traditional small-molecule active-site inhibitors, as their functions are modulated by protein interactions. Indeed, protein-protein interactions constitute nearly 90% of all medicinal targets of interest, yet peptides inhibitors – which effectively target these interactions – account for only 2% of FDA-approved drugs. Peptide therapies face major challenges including costly synthesis, in vivo instability from protease degradation, and poor bioavailability. To remedy these issues, “stapled-peptides” have been proposed to improve both the potency and pharmacokinetics of such therapies. Unfortunately, these stapled peptides— which contain non-natural amino acids to covalently maintain a helical structure— cannot be genomically encoded because their production requires additional chemical steps, which drastically limits the ability to discover and synthesize new biomimetic peptide therapies and tools. Therefore, the ability to iteratively design, genomically encode, and reliably synthesize a stable class of these molecules in vivo would yield novel chemical probes for a variety of protein-protein interactions in cancer. This proposal seeks to genomically-encode the production of therapeutically relevant, cell-permeable stapled peptides in a bacterial organism. This would allow for the generation of screenable peptide-libraries, drastically reduce the cost of synthesis, and ultimately provide a discovery platform for an entirely new class of protein- protein inhibitors. Utilizing high-throughput, robotic phage-assisted continuous directed evolution (roboPACE), an in vivo mechanism to produce cell-permeable bio-mimetic peptides will be developed. First, a novel thio- ether stapling mechanism will be characterized in vitro utilizing a novel non-canonical amino acid [Aim 1]. Second, efficient in vivo incorporation of this amino acid into proteins will be evolved in high-throughput with roboPACE [Aim 2]. Finally, a promiscuous bacterial synthetase enzyme, will be evolved to efficiently catalyze the stapling mechanism in order to genomically-encode stapled-peptide production [Aim 3]. Collectively, this proposal will extend the breadth and throughput of ncAA design and incorporation, and ultimately develop an in vivo peptide-stapling mechanism in order to treat and characterize presently “undruggable” therapeutic targets in cancer.

抽象的在基因组医学时代，我们能够精确识别一系列疾病的分子敏感性然而，许多癌症的真正驱动因素——转录因子，肿瘤抑制因子和染色质重塑因子（例如 p53、myc 和 SWI/SNF）不能轻易被靶向传统的小分子活性位点抑制剂，因为它们的功能是通过蛋白质相互作用调节的。蛋白质-蛋白质相互作用构成了所有感兴趣的医学靶标的近 90%，但肽抑制剂 – 有效针对这些相互作用的药物——仅占 FDA 批准的肽疗法药物的 2%。面临重大挑战，包括合成成本高、蛋白酶降解造成的体内不稳定以及质量差为了解决这些问题，人们提出了“钉合肽”来提高这两种功效。不幸的是，这些钉合肽含有非天然物质。共价维持螺旋结构的氨基酸——不能被基因组编码，因为它们生产需要额外的化学步骤，这极大地限制了发现和合成新材料的能力因此，迭代设计、基因组编码和开发的能力。在体内可靠地合成一类稳定的分子将产生针对多种分子的新型化学探针癌症中的蛋白质-蛋白质相互作用。该提案旨在对治疗相关的、细胞渗透性缝合的生产进行基因组编码这将允许产生可筛选的肽库，这是戏剧性的。降低合成成本，并最终为全新类别的蛋白质提供发现平台利用高通量、机器人噬菌体辅助连续定向进化（roboPACE），首先，将开发一种生产可渗透细胞的仿生肽的体内机制。将利用一种新型非规范氨基酸在体外表征醚装订机制[目标 1]。其次，这种氨基酸在体内有效掺入蛋白质的方法将以高通量的方式发展 roboPACE [目标 2] 最终，将进化出一种混杂的细菌合成酶，以有效催化。装订机制，以便对装订肽生产进行基因组编码[目标 3]。该提案将扩大 NCAA 设计和整合的广度和吞吐量，并最终开发出一个体内肽装订机制，以治疗和表征目前“不可成药”的治疗靶点在癌症中。