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构成了发现生物途径、疾病机制和治疗干预机会的基础。这项提议的目标是将化学、生物学和医学相互交织在一起，创造一种变革性的高通量技术，准确地识别蛋白质靶标及其明确的相互作用部位。就像钥匙的齿可以完美地插入锁一样，互补蛋白质的形状对于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，这些接触点通常嵌入复杂的蛋白质中，为维持必要的生物活性折叠提供基础设施。理想情况下，这些经过进化磨练的亚结构可以用来捕获它们的蛋白质靶标，从而对其进行分类；然而，在脱离整个蛋白质的背景下，生物活性亚域通常会展开，导致生物形状、效力和特异性的丧失。为了恢复结构多肽选择性结合和捕获蛋白质靶标的巨大能力，我们将首先恢复它们的生物活性形状，然后对它们进行化学衍生化，以获得非共价和共价捕获。在这项提议中，我们将重点放在多肽1-螺旋上，这可能是细胞利用的最普遍和最多功能的生物形状。我们将应用我们强大的“碳氢化合物装订”技术来合成各种生物活性1-螺旋，然后通过化学安装新的固定化和插层功能，通过捕获全方位的稳定到瞬时蛋白质相互作用来扩大我们对相互作用组的掌握。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：识别蛋白质靶标及其相互作用位置。由于蛋白质相互作用位点是药物设计的地形模板，我们相信我们方法的结合位点识别特征将在相互作用组发现和临床翻译之间提供关键的联系。为了实现我们的目标，我们将采取一种循序渐进的方法：(1)结构稳定，(2)定向亲和力捕获，(3)共价捕获，(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学实验进行验证。一旦确定并编目，蛋白质的相互作用必须经过生物学验证。我们方法的一个开创性特征是，我们用来捕获蛋白质相互作用组的1-螺旋可以用来验证细胞和体内研究中的相互作用并对其进行药物治疗。因此，我们相信，设计用于蛋白质捕获的装订多肽将创造一种强大而通用的方法来阐明相互作用组，并极大地扩大发现新的相互作用及其如何影响健康和疾病的潜力。 与公共健康相关：蛋白质相互作用调节了健康和疾病中无数的细胞活动；我们的目标是创造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部位。我们多学科方法的新颖性始于调节蛋白质相互作用的蛋白质亚结构的化学重建，将大自然进化磨练的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用于固定和不可逆蛋白质嵌入的分子功能。通过在化学、生物和医学的界面上进行操作，我们的目标是开发和部署一种技术，以克服识别、区分和下药广泛的人类蛋白质靶标的艰巨挑战。

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.