An Intracellular Helix-constrained Peptide Library Screening Platform to Derive Functional Transcription Factor Antagonists

60%的多蛋白复合物具有螺旋界面，20%的多蛋白复合物参与基因调控。因此，螺旋相互作用抑制剂具有成为一类有用的转录调节剂的巨大潜力。小分子通常不能消除这些相互作用，因为它们扩展到相互作用热点之外的能力有限。相比之下，基于肽的抑制剂可以阻断更大的相互作用区域，使其成为蛋白质-蛋白质相互作用（PPI）抑制的首选方法。然而，与蛋白质结合位点相对应的肽序列在分离时可能会失去其结构。对于螺旋肽，这可以通过使用“螺旋诱导约束”来补偿，这可以被认为是将肽固定在一起以保持螺旋结构的安全别针。然而，约束的有益效果很难预测，这会导致大量的时间和费用。我们将通过筛选细胞内的整个肽库来解决这个具有挑战性的问题，其中每个成员都被锁定为螺旋构象。通过在文库筛选过程中限制所有肽，我们将确定那些约束提供改进的靶标亲和力，对蛋白酶的抗性，以及潜在的跨生物膜的潜力。我们将通过使用转录阻断存活（TBS）试验筛选活细胞内的螺旋约束文库，确保拮抗剂在阻断转录因子- dna结合方面真正发挥作用。我们将充分表征肽，以确定他们如何实现其有利的性质，使用一系列生化分析。最后，利用获得的知识，我们将创建广泛使用的isPCA/isCAN肽库筛选软件的更新版本，该软件搜索大量肽，以识别最有可能以高亲和力和选择性结合到给定目标的肽（例如Chen等人，Nature 2019， Aupic等人，Nat comm 2021， Daudey等人，Chem Sci 2021）。在此基础上，我们将进一步确定可以引入“模块”的序列，这些“模块”既可以容忍约束，更重要的是可以促进螺旋度、结合、生物稳定性和潜在的膜通透性。该软件预计将被广泛应用于社区，以促进选择性抑制广泛的疾病相关的PPIs，其中螺旋界面存在。这些计算工具将免费提供给科学界，并可在线访问。我们设想它们将被蛋白质生化学家、细胞生物学家和合成生物学家广泛采用。

>60% of all multi-protein complexes feature helical interfaces, with >20% participating in gene regulation. Helical interaction inhibitors therefore have enormous potential to become a useful class of transcriptional modulator. Small molecules typically fail to abrogate these interactions owing to their limited ability to extend beyond interaction hotspots. In contrast, peptide-based inhibitors can block much larger interaction areas, making them a preferred approach for protein-protein interaction (PPI) inhibition.However, peptide sequences corresponding to binding sites within a protein can lose their structure when created in isolation. For helical peptides, this can be compensated for with the use of 'helix-inducing constraints' which can be thought of as safety pins that hold the peptide together to retain helical structure. However, the beneficial effect of a constraint can be very hard to predict, leading to considerable time and expense. We will tackle this challenging problem by screening entire libraries of peptides inside cells where each members has been locked into a helical conformation. By constraining all peptides during the library screen we will identify those in which the constraint provides improved target affinity, resistance to proteases, and potentially the potential to cross biological membranes. We will ensure that antagonists are truly functional in blocking transcription factor-DNA binding by screening the helix-constrained libraries within living cells using our Transcription Block Survival (TBS) Assay. We will fully characterise peptides to establish how they acheive their favourble properties using a range of biochemical assays. Finally, using knowledge gained we will create updated versions of our widely used isPCA/isCAN peptide library screening software that searches vast numbers of peptides to identify those most likely to bind with high affinity and selectivity to a given target (e.g. Chen et al, Nature 2019, Aupic et al, Nat Commun 2021, Daudey et al, Chem Sci 2021). Building on these we will further identify sequences within which 'modules' can be introduced that both tolerate constraints but that more importantly promote increased helicity, binding, biostability, and potentially membrane permeability. The software is anticipated to be widely used by the community to facilitate selective inhibiton of a wide range of disease relevant PPIs in which helical interfaces present. The computational tools will be made freely available and accessible online to the scientific community. We envisage that they will be widely adopted by protein biochemists, cell biologists and synthetic biologists.