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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.

摘要在基因组医学的时代，我们能够精确地识别一系列的分子亲和性。人类病理学，包括癌症。然而，许多真正的癌症转录因子驱动因子，肿瘤抑制因子和染色质重塑因子（如p53、myc和SWI/SNF）不能被传统的小分子活性位点抑制剂，因为它们的功能是由蛋白质相互作用调节的。的确，蛋白质-蛋白质相互作用构成了所有感兴趣的药物靶点的近90%，然而肽类抑制剂- 有效靶向这些相互作用的药物-仅占FDA批准药物的2%。多肽疗法面临的主要挑战包括昂贵的合成，来自蛋白酶降解的体内不稳定性，以及差的生物利用度为了解决这些问题，已经提出了“钉合肽”来改善免疫调节剂的效力和免疫调节剂的效力。和药代动力学。不幸的是，这些含有非天然多肽的钉合肽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，这大大限制了发现和合成新的仿生肽疗法和工具。因此，迭代设计、基因组编码和在体内可靠地合成一类稳定的这些分子将产生用于多种生物学和分子生物学的新型化学探针。癌症中的蛋白质相互作用。该提议寻求对治疗相关的、细胞可渗透的钉合蛋白的产生进行基因组编码。细菌有机体中的肽。这将允许产生可筛选的肽库，大大地降低合成成本，并最终为一类全新的蛋白质提供发现平台- 蛋白质抑制剂利用高通量、机器人噬菌体辅助的连续定向进化（roboPACE），将开发一种产生细胞可渗透的生物模拟肽的体内机制。首先，一种新颖的硫- 醚钉合机制将利用新型非规范氨基酸在体外表征[目的1]。第二，将该氨基酸有效地体内掺入蛋白质中将以高通量进行， roboPACE [Aim 2]。最后，一种混杂的细菌合成酶将被进化成有效地催化钉合机制，以便基因组编码钉合肽生产[目的3]。总的来说，这该提案将扩大ncAA设计和合并的广度和吞吐量，并最终开发一种新的为了治疗和表征目前“不可用药的”治疗靶点，在癌症中。