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，我们能够准确地确定一系列的分子敏感性人类病理，包括癌症。但是，许多真正的癌症驱动因素 - 转录因素，肿瘤补充剂和染色质改造（例如p53，MYC和SWI/SNF）不能轻易地针对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几乎占所有感兴趣的医学靶标的90％，但肽抑制剂 - 有效地针对这些相互作用 - 仅占FDA批准的药物的2％。肽疗法面临主要挑战，包括昂贵的合成，蛋白酶降解的体内不稳定性和差生物利用度。要记住这些问题，已经提出了“钉肽”来提高效力和这种疗法的药代动力学。不幸的是，这些滞留的肽 - 包含非天然的肽氨基酸以共价维持螺旋结构 - 不能在基因组上编码生产需要其他化学步骤，这大大限制了发现和合成新的能力仿生肽疗法和工具。因此，迭代设计的能力，基因编码和可靠地在体内合成稳定的这些分子类别将产生新的化学问题癌症中的蛋白质蛋白质相互作用。该提案旨在基因组化治疗相关的，可渗透的钉子细菌生物中的肽。这将允许生成可筛选的肽纤维，急剧降低合成成本，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，机器人噬菌体辅助连续的定向进化（Robopace），将开发出一种生产可渗透生物模拟胡椒体的体内机制。首先，一个新颖的硫将使用一种新型的非典型氨基酸（AIM 1]，将在体外表征醚固定机制。其次，在该氨基酸中掺入蛋白质中的体内有效的体内将在高通量中进化 Robopace [AIM 2]。最后，将进化为有效催化的混杂细菌合成酶固定机制是为了基于基因模型上分阶段的肽产生[AIM 3]。总的来说，这提案将扩大NCAA设计和公司的广度和吞吐量，并最终发展为了治疗和特征当前“不良”的治疗靶标的体内胡椒盖机制在癌症中。