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

 描述(由申请人提供)：基于蛋白质的疗法的成功正在给药物开发带来革命性的变化。与小分子药物不同，基于多肽和蛋白质的治疗药物可以高选择性和特异性地靶向参与人类疾病的蛋白质-蛋白质相互作用缺陷。然而，尽管它们取得了成功，但它们作为治疗剂的使用仍然存在许多稳定性和递送问题。例如，单抗(最成功的基于蛋白质的治疗药物之一，市场上有几种重磅炸弹药物，还有更多正在临床开发中的药物)只能针对细胞外分子靶点，因为它们不能穿过生物膜。它们的生产成本也非常高，而且由于其对蛋白质降解的敏感性，无法在生物上使用。这些问题导致了对替代蛋白质支架的探索，作为基于蛋白质的新型疗法的来源。为了应对这一重要挑战，我们建议使用遗传和化学编码的环肽文库来选择能够调节蛋白质-蛋白质相互作用的特定环肽序列。环肽是一类新出现的由植物衍生的大骨架环化多肽(˜，全长30个氨基酸)，具有3个二硫键稳定的核心，具有不寻常的打结结构。它们有几个特点，使它们成为理想的药物开发工具。为了实现这一目标，我们建议使用卡马雷罗实验室开发的蛋白质剪接技术在活细菌细胞内生成大型的、遗传编码的环肽文库。这些基于细胞的文库将使用不同的细胞内报告程序进行筛选，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来产生和快速筛选以膜相关细胞外受体为靶点的化学合成环肽。选定的环肽将在哺乳动物细胞中进行表征和测试，以测试它们对抗选定的细胞内或细胞外蛋白靶标的能力。我们还将探索这些有趣的微蛋白的细胞穿透特性以及改进它的方法。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.