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

摘要 在基因组医学时代，我们能够准确地识别一系列 人类的病理，包括癌症。然而，癌症转录因子的许多真正驱动因素， 肿瘤抑制因子和染色质重构体(如p53、myc和SWI/SNF)不容易被 传统的小分子活性部位抑制剂，因为它们的功能受蛋白质相互作用的调节。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%，然而多肽抑制剂- 这些药物有效地针对这些相互作用-仅占FDA批准的药物的2%。多肽疗法 面临着重大挑战，包括昂贵的合成，体内因蛋白酶降解而不稳定，以及 生物利用度。为了解决这些问题，人们提出了“装订多肽”来提高这两种药物的效力 以及此类疗法的药代动力学。不幸的是，这些装订的多肽-含有非天然的 氨基酸以共价方式维持螺旋结构--不能进行基因组编码，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产品的能力 仿生肽疗法和工具。因此，迭代设计、基因编码和 在体内可靠地合成一类稳定的这些分子将为各种不同的 癌症中的蛋白质-蛋白质相互作用。 这项提议寻求对治疗相关的、细胞可渗透的订书钉的生产进行基因组编码 细菌有机体中的多肽。这将允许产生可筛选的多肽库，大大地 降低合成成本，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)， 将开发一种体内机制来产生细胞透过性的生物模拟肽。首先，一种新颖的硫代- 使用一种新的非正则氨基酸[目标1]，将在体外表征乙醚的装订机制。 其次，这种氨基酸在体内有效地结合到蛋白质中，将在高通量的情况下进化 机器人空间[目标2]。最后，一种混杂的细菌合成酶将被进化为有效地催化 用于基因编码已装订多肽产品的装订机制[目标3]。总而言之，这 提案将扩展NCAA设计和合并的广度和吞吐量，并最终开发一个 用于治疗和表征目前“不能用药的”治疗靶点的活体多肽--订书机 在癌症中。

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.