Inducing Native-like Folding of Intrinsically Disordered Proteins using Stabilized a-Helical Peptides

使用稳定的α-螺旋肽诱导本质无序蛋白质的类似天然折叠

• 批准号: 9190844
• 负责人: Nicholas Alan Sawyer
• 金额: $ 5.25万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9190844
• 关键词: Affinity; Binding; Binding Proteins; Biological Assay; C-terminal; Cells; Circular Dichroism; Complement; Complex; Computer Simulation; Disease; EP300 gene; Evaluation; Fluorescence; Fluorescence Polarization; Genes; Genetic Transcription; Goals; HIF1A gene; Hydrogen Bonding; Hypoxia; In Vitro; Laboratories; Luciferases; Malignant Neoplasms; Measurement; Measures; Mediating; Molecular Conformation; Mus; NMR Spectroscopy; Peptides; Play; Protein Conformation; Protein Family; Proteins; Research; Role; STAT2 gene; Side; Signal Transduction; Specificity; Structure; TP53 gene; Therapeutic; Therapeutic Intervention; Transcription Coactivator; Transcriptional Activation; Transcriptional Regulation; Tumor Volume; Up-Regulation; Xenograft procedure; alpha helix; angiogenesis; base; biological systems; covalent bond; design; flexibility; functional group; genome-wide; hypoxia inducible factor 1; improved; in vitro activity; in vivo; interest; mimetics; mimicry; novel; peptide structure; peptidomimetics; protein aminoacid sequence; protein folding; protein function; protein protein interaction; protein structure; research study; small molecule; tool; tumor microenvironment; tumor xenograft

PROJECT SUMMARY/ABSTRACT Intrinsically disordered proteins (IDPs) are a ubiquitous class of proteins whose structural plasticity allows them to function as hubs in protein-protein interaction (PPI) and signaling networks. IDP PPIs are therefore tightly regulated, and aberrant IDP PPIs are associated with many disease states. As IDP folding and activity are dependent on the conformations induced by its native binding partners, small molecules that induce native-like IDP conformations are highly desirable for studying IDP function. However, complex IDP PPI interfaces are difficult targets for traditional small molecules. Instead, non-traditional approaches using stabilized α-helical peptides, such as the hydrogen bond surrogate (HBS) approach developed in the Arora laboratory, are promising for creating small molecules that nucleate native-like IDP folding with both high affinity and high specificity. Thus, I hypothesize that HBS peptides can mimic key portions of IDP binding partners to nucleate IDP folding and competitively inhibit IDP PPIs in vitro and in vivo. The overall goal is to create HBS-derived artificial folders that induce IDPs to favor one fold over others. A classic IDP PPI example is the interaction of the general transcriptional coactivator p300 with the intrinsically disordered C-terminal transcription-activation domain of hypoxia-inducible factor 1 α (HIF1α CTAD). This interaction is critical for hypoxia-inducible transcription, leading to the upregulation of many cancer-associated genes involved in angiogenesis, invasion, and proliferation. The Arora lab has shown that mimicry of a HIF1α CTAD helical fragment downregulates hypoxia-inducible transcription and decreases xenograft tumor size in mice. However, targeting p300 may impair its ability to interact with its numerous binding partners. In the current proposal, I propose to develop a complementary strategy to use HBS peptides to induce HIF1α CTAD folding and competitively inhibit the p300-HIF1α CTAD interaction. To achieve this goal, I will first design and synthesize HBS peptides that mimic a p300 helical fragment that interacts with HIF1α CTAD. I will evaluate HBS peptide binding affinity and specificity for HIF1α CTAD in vitro. I will then characterize the structures of HIF1α CTAD-HBS complexes to evaluate HBS peptide-induced folding of HIF1α CTAD. I will also evaluate HBS peptides’ ability to modulate HIF1α-mediated transcription using cell-based assays. HBS peptides that induce native-like HIF1α CTAD folding with high affinity and specificity may serve as useful tools for studying HIF1α function and potential cancer therapeutic leads for HIF1α inhibition. This strategy for inducing IDP folding may also provide a specific application of a general approach to modulate IDP structures and activities using structured peptides.

项目概要/摘要 本质无序蛋白 (IDP) 是一类普遍存在的蛋白质，其结构 可塑性使它们能够充当蛋白质-蛋白质相互作用 (PPI) 和信号传导的枢纽 网络。因此，IDP PPI 受到严格监管，异常的 IDP PPI 与 许多疾病状态。由于 IDP 折叠和活性依赖于诱导的构象 它的天然结合伙伴，诱导类似天然 IDP 构象的小分子是高度 适合研究 IDP 功能。然而，复杂的 IDP PPI 接口很难成为目标 传统的小分子。相反，使用稳定 α 螺旋的非传统方法 肽，例如 Arora 开发的氢键替代 (HBS) 方法 实验室有望创造出类似天然 IDP 折叠的小分子 兼具高亲和力和高特异性。因此，我假设 HBS 肽可以模仿关键的 IDP 结合伴侣的部分使 IDP 折叠成核并在体外竞争性抑制 IDP PPI 和体内。总体目标是创建源自哈佛商学院的人工文件夹，诱导国内流离失所者 比其他人更喜欢其中的一倍。 一个经典的 IDP PPI 例子是通用转录辅激活因子 p300 与 缺氧诱导因子本质上无序的C端转录激活结构域 1 α (HIF1α CTAD)。这种相互作用对于缺氧诱导转录至关重要，从而导致 许多癌症相关基因的上调，涉及血管生成、侵袭和 增殖。 Arora 实验室已证明 HIF1α CTAD 螺旋片段的拟态 下调缺氧诱导的转录并减小小鼠异种移植肿瘤的大小。 然而，针对 p300 可能会削弱其与其众多结合伙伴相互作用的能力。 在当前的提案中，我建议开发一种补充策略，使用 HBS 肽来 诱导 HIF1α CTAD 折叠并竞争性抑制 p300-HIF1α CTAD 相互作用。到 为了实现这个目标，我将首先设计并合成模仿p300螺旋的HBS肽 与 HIF1α CTAD 相互作用的片段。我将评估 HBS 肽结合亲和力并 HIF1α CTAD 体外特异性。然后我将描述 HIF1α CTAD-HBS 的结构 复合物来评估 HBS 肽诱导的 HIF1α CTAD 折叠。我也会评价HBS 肽使用基于细胞的检测来调节 HIF1α 介导的转录的能力。 HBS肽 诱导类似天然 HIF1α CTAD 折叠的高亲和力和特异性可能有用 研究 HIF1α 功能和抑制 HIF1α 的潜在癌症治疗线索的工具。这 诱导 IDP 折叠的策略也可能提供通用方法的具体应用 使用结构化肽调节 IDP 结构和活性。