Multivalent protein-DNA nanostructures as synthetic blocking antibodies

多价蛋白质-DNA 纳米结构作为合成阻断抗体

• 批准号: 10587455
• 负责人: Nicholas Stephanopoulos
• 金额: $ 29.6万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587455
• 关键词: 3-Dimensional; ACE2; Affinity; Algorithms; Antibodies; Basic Science; Beds; Binding; Binding Proteins; Binding Sites; Biological; Biology; Blocking Antibodies; Coagulation Process; Computer Simulation; Cyclic Peptides; DNA; DNA Structure; Disease; Docking; Evolution; Feedback; Fibrin; Fibrinogen; Geometry; Grain; Health; Image; Immobilization; Individual; Libraries; Ligand Binding; Ligands; Light; Literature; Location; Measures; Membrane Proteins; Methods; Molecular; Multiprotein Complexes; Mutate; Mutation; Nanostructures; Peptides; Phage Display; Polymers; Positioning Attribute; Process; Protein Engineering; Proteins; Reporting; SARS-CoV-2 inhibitor; SARS-CoV-2 spike protein; Series; Shapes; Site; Specificity; Structure; Surface; System; Testing; Translations; Variant; Work; design; flexibility; hybrid protein; imaging agent; in silico; monomer; mutant; nano; nanobodies; nanoscale; neutralizing antibody; next generation; novel; novel therapeutics; polymerization; polypeptide; process optimization; protein complex; protein protein interaction; receptor; scaffold; simulation; small molecule; synthetic antibodies; targeted agent

Project Summary Protein-protein interactions (PPIs) drive countless processes in biology. The ability to block these interactions with high specificity is crucial for probing the basic science of these processes, as well as for developing imaging agents or novel therapeutics. However, most traditional molecules for blocking protein-protein interactions—like small molecules, peptides, or antibodies—rely on the precise targeting of the crucial interface or binding pocket, which can be difficult for some targets. Furthermore, none of these approaches can be easily tuned to match the valency or size of the target, and binding to patches on the protein not directly involved in PPIs can fail to block activity. Here, we propose to develop a nanoscale synthetic antibody (“nano-synbody”) consisting of a tunable DNA nanostructure bearing 2-3 peptide/protein ligands that can bind to distinct surfaces of a target protein and block its association with its partner through the steric bulk of the DNA structure. The individual peptide/protein binding agents will be derived from either known molecules, or found independently through methods like phage display. Critically, our method merges computational simulation—and in silico “evolution”—of these hybrid protein-DNA nano-synbodies, creating a library of structures and probing their association with the target. We aim to create a feedback loop, whereby the computational simulations yield candidate nano-synbodies that can be experimentally tested, further informing the next round of simulations. We will first apply this pipeline to a homo-trimeric nano-synbody against the SARS-CoV-2 spike protein trimer (Aim 1). This test bed will allow us to optimize the process and find a high-affinity blocking structure. Then, we will apply our method to nano-synbodies for blocking the assembly of fibrinogen into fibrin clots (Aim 2). The second Aim will involve phage display against fibrin to find novel binding agents, and thereby convert them into high-affinity hetero-trivalent structures. In both Aims, we will demonstrate the advantage of nano-structuring ligand presentation over simple oligomerization with flexible linkers. Taken together, our work will generate a new computational-experimental paradigm for the design of tunable, user-defined nanostructures that can present three or more peptides/proteins for binding to any protein, and blocking its association with its target. Crucially, our approach does not require binding directly to the interface, which should enable it to target a much larger range of proteins that may not be amenable to traditional approaches, large protein complexes, or mutants/variants of the targets that might escape single binding agents.

项目摘要 蛋白质 - 蛋白质相互作用（PPI）驱动生物学中的无数过程。阻止这些互动的能力 具有高特异性对于探测这些过程的基础科学以及开发成像至关重要 代理或新疗法。但是，大多数用于阻断蛋白质蛋白质相互作用的传统分子 - 小分子，胡椒粉或抗体 - 很大程度上是基于关键界面或结合口袋的精确靶向 对于某些目标来说可能很困难。此外，这些方法都无法轻松调整以匹配 目标的价值或大小，并与不直接参与PPI的蛋白质上的斑块结合可能无法阻止 活动。在这里，我们建议开发由可调的纳米级合成抗体（“纳米同胞”） DNA纳米结构轴承2-3个肽/蛋白质配体，可以与靶蛋白的不同表面结合和 通过DNA结构的空间大块阻止其与伴侣的关联。单个肽/蛋白质 结合剂将源自已知的分子，或通过噬菌体等方法独立发现 展示。至关重要的是，我们的方法合并了这些混合动力的计算模拟以及硅的“进化” 蛋白质-DNA纳米合成体，创建一个结构库并探测其与靶标的关联。我们 旨在创建一个反馈循环，计算模拟产生候选纳米合成体 经过实验测试，进一步告知下一轮仿真。我们将首先将此管道应用于 对SARS-COV-2尖峰蛋白蛋白质剂的均匀纳米同步体（AIM 1）。这个测试床将使我们能够 优化过程并找到高亲和力的阻塞结构。然后，我们将我们的方法应用于纳米合成体 将纤维蛋白原的组装阻塞到纤维夹中（AIM 2）。第二个目标将涉及噬菌体显示 纤维蛋白以找到新型的结合剂，从而将其转换为高亲和力的异差数结构。在这两个中 目的，我们将证明纳米结构配体呈现优于简单寡聚的优势 具有灵活的链接器。综上所述，我们的工作将为 设计可调的，用户定义的纳米结构，可以呈现三种或更多的胡椒粉/蛋白 任何蛋白质，并阻止其与目标的关联。至关重要的是，我们的方法不需要直接绑定 到界面，这应该使其能够靶向更大范围的蛋白质范围 传统方法，大蛋白质复合物或可能逃脱单个目标的靶标的突变体/变体 结合剂。