Screening and selection of specific protein-protein antagonists using ultrastable microprotein scaffolds

 描述（由申请人提供）：基于蛋白质的治疗方法的成功正在彻底改变药物开发。与小分子药物不同，基于肽和蛋白质的治疗剂可以以高选择性和特异性靶向与人类疾病有关的蛋白质-蛋白质相互作用缺陷。然而，尽管它们取得了成功，但仍然存在许多与它们作为治疗剂的用途相关的稳定性和递送问题。例如，单克隆抗体（一种最成功的基于蛋白质的治疗方法，市场上有几种重磅炸弹药物，临床开发中还有更多）由于无法穿过生物膜，只能靶向细胞外分子靶标。它们的生产成本也极其昂贵，并且由于对蛋白水解降解敏感而不具有生物利用度。这些问题导致了对替代蛋白质支架的探索，作为新型基于蛋白质的治疗剂的来源。为了应对这一重要挑战，我们提出使用遗传和化学编码的cyclotides库来选择能够调节蛋白质-蛋白质相互作用的特定cyclotide序列。环肽是一个新出现的大家族的植物源性主链环化多肽（约30个氨基酸长），其具有3个二硫键稳定的核心，其特征在于不寻常的打结结构。它们具有使其成为理想的药物开发工具的几个特性。为了实现这一目标，我们建议使用Camarero实验室开发的蛋白质剪接技术在活细菌细胞内产生大型的遗传编码的cyclotide文库。这些基于细胞的文库将使用不同的细胞内报告基因进行筛选，以鉴定编码活性环肽序列的细菌。此外，我们还将开发新的策略，用于生成和快速筛选针对膜相关细胞外受体的化学合成环肽。将在哺乳动物细胞中表征和测定选定的环肽，以测试它们拮抗选定的细胞内或细胞外蛋白质靶标的能力。我们还将探索这些有趣的微蛋白的细胞穿透特性以及改善它的方法。最后，我们还想研究它们的药代动力学（PK）特性，并探索不同的方法来提高它们的生物利用度。

 DESCRIPTION (provided by applicant): The success of protein-based therapeutics is revolutionizing drug development. Unlike small molecule drugs, peptide and protein-based therapeutics can target with high selectivity and specificity defective protein-protein interaction involved in human disease. Despite their success, however, there are still numerous stability and delivery issues associated with their use as therapeutic agents. For example, monoclonal antibodies (one the most successful protein-based therapeutics with several blockbuster drugs on the market and many more in clinical development) can only target extracellular molecular targets due to their inability to cross biological membranes. They are also extremely expensive to produce and are not bioavailable due to their susceptibility to proteolytic degradation. These issues have led to the exploration of alternative protein scaffolds as a source for novel types of protein-based therapeutics. In response to this important challenge, we propose the use of genetically- and chemically-encoded libraries of cyclotides for selecting specific cyclotide sequences able to modulate protein-protein interactions. Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides (˜30 amino acids long) that share a 3 disulfide-stabilized core characterized by an unusual knotted structure. They have several characteristics that make them ideal drug development tools. To achieve this objective we propose to use protein-splicing technology developed in the Camarero lab to generate large, genetically encoded cyclotide libraries inside live bacterial cells. These cell-based libraries wil be screened using different in-cell reporters to identify bacteria encoding active cyclotide sequences. In addition, we will also develop novel strategies for the generation and rapid screening of chemically synthesized cyclotides targeted against membrane associated extracellular receptors. Selected cyclotides will be characterized and assayed in mammalian cells to test their ability to antagonize the selected intracellular or extracellular protein targes. We will also explore the cell penetrating properties of these interesting microproteins as well as ways to improve it. Finally, we also want to study their pharmacokinetic (PK) properties and explore different approaches to improve their bioavailability.