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: 9333090
• 负责人: Nicholas Alan Sawyer
• 金额: $ 5.67万
• 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/9333090
• 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; Impairment; In Vitro; Laboratories; Luciferases; Malignant Neoplasms; Measurement; Measures; Mediating; Molecular Conformation; Mus; NMR Spectroscopy; Peptides; Play; Protein Conformation; Protein Family; Proteins; Quantitative Reverse Transcriptase PCR; Research; Role; STAT2 gene; Side; Signal Transduction; Specificity; Structure; TP53 gene; Therapeutic; Therapeutic Intervention; Transcription Coactivator; Transcriptional Activation; Transcriptional Activation Domain; Transcriptional Regulation; Tumor Volume; Up-Regulation; Xenograft procedure; alpha helix; angiogenesis; base; biological systems; covalent bond; design; experimental study; flexibility; functional group; genome-wide; hypoxia inducible factor 1; improved; in vitro activity; in vivo; interest; mimetics; mimicry; novel; peptide structure; peptidomimetics; protein E; protein aminoacid sequence; protein folding; protein function; protein protein interaction; protein structure; 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.

项目总结/摘要 内含子无序蛋白（IDP）是一类普遍存在的蛋白质，其结构 可塑性使它们在蛋白质-蛋白质相互作用（PPI）和信号传导中起枢纽作用 网络.因此，IDP PPI受到严格调控，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， 它的天然结合伴侣，诱导天然样IDP构象的小分子， 这是研究IDP功能的理想方法。然而，复杂的IDP PPI接口是难以实现的目标。 传统的小分子相反，使用稳定的α-螺旋的非传统方法 肽，例如在Arora中开发的氢键替代物（HBS）方法， 实验室，有希望创造小分子，核天然样IDP折叠与 既有高亲和力又有高特异性。因此，我假设HBS肽可以模拟关键， IDP结合配偶体的部分在体外使IDP折叠成核并竞争性抑制IDP PPI 和体内。总体目标是创建HBS衍生的人工文件夹，诱导IDP 偏袒一方 经典的IDP PPI实例是通用转录共激活因子p300与 缺氧诱导因子C端转录激活结构域 1 α（HIF 1 α C3）。这种相互作用对于低氧诱导的转录是至关重要的，从而导致 许多参与血管生成、侵袭和转移的癌症相关基因的上调， 增殖阿罗拉实验室已经证明，模拟HIF 1 α C3螺旋片段 在小鼠中下调缺氧诱导的转录并减小异种移植肿瘤的大小。 然而，靶向p300可能会损害其与众多结合伴侣相互作用的能力。 在目前的提案中，我建议开发一种互补策略，使用HBS肽， 诱导HIF 1 α CX 3折叠并竞争性抑制p300-HIF 1 α CX 3相互作用。到 为了实现这一目标，我将首先设计和合成模拟p300螺旋的HBS肽， 与HIF 1 α C3相互作用的片段。我将评估HBS肽结合亲和力， 体外对HIF 1 α C3的特异性。然后，我将表征HIF 1 α CTAD-HBS的结构 复合物，以评估HBS肽诱导的HIF 1 α Cf 3折叠。我也会评估哈佛商学院 肽调节HIF 1 α介导的转录的能力。HBS肽 以高亲和性和特异性诱导天然样HIF 1 α Cf 3折叠可作为有用的 研究HIF 1 α功能的工具和HIF 1 α抑制的潜在癌症治疗线索。这 诱导IDP折叠策略也可以提供一般方法的具体应用， 使用结构化肽调节IDP结构和活性。