权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

用于衍生功能性转录因子拮抗剂的细胞内螺旋限制肽库筛选平台

基本信息

批准号：
BB/X001849/1
负责人：
Jody Mason
金额：
$ 65.9万
依托单位：
University of Bath
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2023
资助国家：
英国
起止时间：
2023 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FX001849%2F1
关键词：
Intracellular Helix constrained Peptide Library

项目摘要

>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.

&gt；60%的多蛋白复合体具有螺旋界面，其中20%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。小分子通常无法消除这些相互作用，因为它们的能力有限，无法延伸到相互作用热点之外。相反，基于多肽的抑制剂可以阻断更大的相互作用区域，使其成为抑制蛋白质-蛋白质相互作用(PPI)的首选方法。然而，与蛋白质内结合部位相对应的多肽序列在分离创建时可能会失去其结构。对于螺旋多肽，这可以通过使用“螺旋诱导约束”来补偿，这可以被认为是将多肽固定在一起以保持螺旋结构的安全别针。然而，约束的有益效果可能很难预测，这会导致相当多的时间和费用。我们将通过筛选细胞内每个成员被锁定为螺旋构象的整个多肽文库来解决这一具有挑战性的问题。通过在文库筛选期间限制所有多肽，我们将识别其中限制提供了更好的靶向亲和力、对蛋白水解酶的抵抗力以及潜在地跨越生物膜的那些。我们将通过使用我们的转录阻断生存(TBS)分析来筛选活细胞中的螺旋受限文库，以确保拮抗剂在阻断转录因子-DNA结合方面具有真正的功能。我们将用一系列的生化分析来确定多肽的特性，以确定它们是如何获得它们最喜欢的特性的。最后，利用所获得的知识，我们将创建我们广泛使用的ISPCA/Iscan多肽文库筛选软件的更新版，该软件搜索大量多肽，以确定那些最可能与给定目标具有高亲和力和选择性结合的多肽(例如，Chen等人，自然，2019，Aupic等人，NAT Commun 2021，Daudey等人，化学科学2021)。在此基础上，我们将进一步确定可以在其中引入‘模块’的序列，这些‘模块’既能容忍限制，更重要的是促进螺旋性、结合、生物稳定性和潜在的膜通透性的增加。该软件预计将被社区广泛使用，以促进对存在螺旋界面的广泛疾病相关PPI的选择性抑制。这些计算工具将向科学界免费提供和在线获取。我们预计它们将被蛋白质生物化学家、细胞生物学家和合成生物学家广泛采用。