A Lexicon of Stapled Peptide Helices Engineered to Capture the Protein Interactom

描述（由申请人提供）：无论是短暂的还是稳定的，正常的还是异常的，蛋白质相互作用及其接触部位都是发现生物学途径、疾病机制和治疗干预机会的基础。该提案的目标是将化学、生物学和医学相结合，创造一种变革性的高通量技术，精确识别蛋白质靶标及其明确的相互作用位点。就像一把钥匙的牙齿完全适合锁一样，互补蛋白质的形状对生物相互作用的执行至关重要。蛋白质的分子握手依赖于其离散的亚结构，并且这些接触点通常嵌入在复杂蛋白质内，该复杂蛋白质提供维持基本生物活性折叠的基础结构。理想情况下，这些进化磨练的亚结构可以用于捕获并从而对其蛋白质靶点进行分类;然而，脱离整个蛋白质的背景，生物活性亚结构域通常会展开，导致生物形状，效力和特异性的丧失。为了回收结构化肽选择性结合和捕获其蛋白质靶标的巨大能力，我们将首先恢复其生物活性形状，然后将其化学衍生化以进行非共价和共价捕获。在本提案中，我们重点关注肽1-螺旋，可以说是细胞利用的最普遍和最通用的生物形状。我们将应用我们强大的“碳氢化合物钉合”技术来合成多种生物活性的1-螺旋，然后通过化学方法安装新的固定和嵌入功能，通过捕获全方位的稳定到瞬时蛋白质相互作用物来扩大我们对相互作用物组的掌握。我们的共价捕获化学和蛋白质组学分析提供了一个二合一的优势：识别蛋白质靶点及其相互作用位点。由于蛋白质相互作用位点是药物设计的地形模板，我们相信，我们的方法的结合位点识别功能将提供一个关键的相互作用组发现和临床翻译之间的联系。为了实现我们的目标，我们将采取逐步的方法：（1）结构稳定，（2）定向亲和捕获，（3）共价捕获，和（4）结合位点鉴定。每一步都将适用于高通量，并使用概念验证生物实验进行验证。一旦确定和编目，蛋白质相互作用必须进行生物学验证。我们的方法的一个开创性的特点是，我们用来捕获蛋白质相互作用组的1-螺旋可以用来验证和药物在细胞和体内研究中的相互作用。因此，我们相信，用于蛋白质捕获的工程化钉合肽将创造一种强大而通用的方法来阐明相互作用组，并大规模扩大发现新相互作用的潜力以及它们如何影响健康和疾病。 公共卫生相关性：蛋白质相互作用介导健康和疾病中无数的细胞活动;我们的目标是创建一种变革性的高通量技术，快速准确地识别蛋白质靶标及其明确的相互作用位点。我们多学科方法的新奇始于介导蛋白质相互作用的蛋白质亚结构的化学重建，将自然界的进化磨练结合基序转化为发现工具箱;接下来，我们在这些生物活性结构中化学植入分子功能，用于固定和不可逆的蛋白质嵌入。通过在化学，生物学和医学的界面上操作，我们的目标是开发和部署一种技术，克服识别，区分和药物化广泛的人类蛋白质靶点的艰巨挑战。

DESCRIPTION (provided by applicant): Whether fleeting or stable, normal or aberrant, protein interactions and their sites of contact form the basis for discovery of biological pathways, disease mechanisms, and opportunities for therapeutic intervention. The goal of this proposal is to intertwine chemistry, biology, and medicine to create a transformative high- throughput technology that precisely identifies protein targets and their explicit sites of interaction. Like the teeth of a key that perfectly fit into a lock, complementary protein shape is critical to the execution of biological interactions. The molecular handshakes of proteins rely on their discrete substructures and these contact points are typically embedded within a complex protein that provides the infrastructure to maintain the essential bioactive fold. Ideally, these evolutionarily honed substructures could be used to capture and thereby catalogue their protein targets; however, out of context from the whole protein, bioactive subdomains often unfold, resulting in loss of biological shape, potency, and specificity. To reclaim the enormous capacity of structured peptides to selectively bind and capture their protein targets, we will first restore their bioactive shape and then chemically derivatize them for both non-covalent and covalent capture. In this proposal, we focus on the peptide 1-helix, arguably the most ubiquitous and versatile biological shape harnessed by the cell. We will apply our robust "hydrocarbon stapling" technology to synthesize a diversity of bioactive 1-helices and then chemically install new immobilization and intercalating functionalities to expand our grasp of the interactome by trapping the full-range of stable to transient protein interactors. Our covalent capture chemistry and proteomic analyses afford a two-for-one advantage: identification of protein targets and their sites of interaction. Since protein interaction sites are the topographic templates for drug design, we believe that the binding site identification feature of our approach will provide a critical link between interactome discovery and clinical translation. To accomplish our goals, we will take a step-wise approach: (1) structural stabilization, (2) directional affinity capture, (3) covalent capture, and (4) binding site identification. Each step will be adapted for high-throughput and validated using proof-of-concept biological experiments. Once identified and catalogued, protein interactions must be validated biologically. A seminal feature of our approach is that the very 1-helices we use to capture the protein interactome can be used to validate and drug the interactions in cellular and in vivo studies. Thus, we believe that engineering stapled peptides for protein capture will create a powerful and versatile approach to elucidating the interactome, and massively expand the potential for discovery of novel interactions and how they impact health and disease. PUBLIC HEALTH RELEVANCE: Protein interactions mediate innumerable cellular activities in health and disease; our goal is to create a transformative high-throughput technology that rapidly and precisely identifies protein targets and their explicit sites of interaction. The novelty of our multidisciplinary approach begins with the chemical recreation of protein substructures that mediate protein interaction, transforming Nature's evolutionarily-honed binding motifs into a discovery toolbox; next, we chemically implant in these bioactive structures molecular functionalities for immobilization and irreversible protein intercalation. By operating at the interface of chemistry, biology, and medicine, we aim to develop and deploy a technology that surmounts the formidable challenge of identifying, distinguishing, and drugging the broad array of human protein targets.