Computational de novo design of a disulfide-rich miniprotein synthetic library and its application to engineer binders to neutralizing epitopes on Clostridium difficile toxins TcdA and TcdB

富含二硫键的微型蛋白合成文库的计算从头设计及其在工程粘合剂中的应用，以中和艰难梭菌毒素 TcdA 和 TcdB 上的表位

• 批准号: 10318198
• 负责人: Christopher David Bahl
• 金额: $ 20.53万
• 依托单位: INSTITUTE FOR PROTEIN INNOVATION, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-10 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318198
• 关键词: Affinity; Algorithm Design; Algorithms; American; Amino Acid Sequence; Amino Acids; Antibiotics; Antibodies; Area; Bacteria; Bacterial Toxins; Binding; Cell surface; Cessation of life; Chronic; Clostridium difficile; Collection; Defensins; Dehydration; Development; Diagnostic; Disulfides; Engineering; Enterotoxins; Epitopes; Equipment; Exhibits; Exotoxins; FDA approved; Fluorescence-Activated Cell Sorting; Gene Library; Genes; Gram-Positive Bacteria; Graph; Healthcare; Hydrophobicity; Infection; Insecta; Intestinal Mucosa; Intestines; Intravenous Immunoglobulins; Kidney Failure; Laboratories; Libraries; Mediating; Membrane Proteins; Monitor; Mutation; Oligonucleotides; Oral; Oral Administration; Pathogenesis; Pathogenicity; Peptides; Process; Proliferating; Protein Engineering; Protein Family; Proteins; Randomized; Reagent; Reproduction spores; Saccharomyces cerevisiae; Scaffolding Protein; Sorting - Cell Movement; Source; Specific qualifier value; Structure; Surface; Targeted Toxins; Techniques; Technology; Testing; Therapeutic; Time; Toxic Megacolon; Toxin; Treatment Efficacy; United States; Venoms; Work; Yeasts; design; effective therapy; enteric infection; frontier; healthcare-associated infections; high reward; high risk; improved; innovation; member; model design; neutralizing antibody; new technology; next generation sequencing; novel; novel therapeutics; protein folding; protein protein interaction; protein structure; rational design; scaffold; screening; small molecule; synthetic construct; therapeutic candidate; therapeutic development; tool; vector

Project Summary Disulfide-rich miniproteins are a powerful yet underutilized protein family for reagent, diagnostic and therapeutic applications. They are hyperstable like small molecules, yet are large enough to bind specifically to protein targets with high affinity and thus can be readily used to inhibit protein-protein interactions. Disulfide-rich miniproteins occur naturally, but the natural molecules are challenging to engineer. There is a pressing need for new technologies which enable disulfide-rich miniproteins to be engineered to bind to arbitrary protein targets, as is routine for other protein scaffolds like antibodies. Here, we propose a computational de novo design strategy using Rosetta to build a synthetic miniprotein library that will be generally useful for screening protein affinity reagents. The typical approach to creating protein libraries is to generate a large amount of random sequence diversity in a localized area of a single protein structure. Most of these sequences will be unstable or unable to bind any target (e.g. too polar or too nonpolar). Rather than use random sequences, we propose to explicitly design each member of a synthetic disulfide-rich miniprotein library. Our design library will contain 106 different miniproteins that display the widest possible variety of binding surfaces. The genes encoding this library will be synthesized using oligo pools. To perform computational de novo design at this scale, we extended the SEWING algorithm in Rosetta to generate hundreds of thousands of unique miniprotein structures and sequences. We developed novel filters to assess design model quality and to quantify and compare protein structures and surfaces. As a test case of this approach, we will screen our disulfide-rich miniprotein library via yeast display for binders to Clostridium difficile enterotoxins TcdA and TcdB. C. difficile is the leading cause of healthcare-related infections in the USA, and these two proteins mediate its pathogenicity. At present, the only available treatments for C. difficile enteric infection that target these toxin proteins are antibodies, which must be injected and have poor efficacy. A hyperstable miniprotein binder to the same neutralizing epitope could be administered orally. Therefore, this work presents a new frontier in de novo miniprotein engineering and library design. It will also result in a source of novel, therapeutically interesting molecules for the treatment of C. difficile infection.

项目摘要 富含二硫键的微蛋白是一种功能强大但充分利用的蛋白质家族，用于试剂，诊断和治疗 申请。它们像小分子一样令人震撼，但足够大，可以专门结合蛋白质 具有高亲和力的靶标可以很容易地用于抑制蛋白质蛋白质相互作用。二硫键丰富 微动蛋白自然发生，但天然分子对工程师来说具有挑战性。迫切需要 使富含二硫键的微蛋白能够设计成与任意蛋白质靶标结合的新技术， 与其他蛋白质支架（如抗体）一样。在这里，我们提出了一个从头设计 使用Rosetta构建合成小巧蛋白库的策略通常可用于筛选蛋白质 亲和力试剂。 创建蛋白质库的典型方法是在中产生大量的随机序列多样性 单个蛋白质结构的局部区域。这些序列中的大多数将不稳定或无法绑定任何 目标（例如，太极或太极极了）。而不是使用随机序列，我们建议明确设计 富含合成二硫键的小型蛋白质库的每个成员。我们的设计库将包含106个不同的 微动蛋白显示出最宽的结合表面。编码此库的基因将是 使用Oligo池合成。为了按照此规模执行从头设计，我们扩展了 在罗塞塔（Rosetta 序列。我们开发了新颖的过滤器来评估设计模型质量并量化和比较蛋白质 结构和表面。 作为这种方法的测试案例，我们将通过Binders的酵母显示器筛选富含二硫键的微蛋白库 艰难梭菌肠毒素TCDA和TCDB。艰难梭菌是医疗保健相关的主要原因 美国的感染，这两种蛋白质介导了其致病性。目前，唯一可用的 靶向这些毒素蛋白的艰难梭菌肠道感染的治疗是抗体，必须注射 并且功效不佳。催眠的微蛋白粘合剂到相同的中和表位的粘合剂可能是 口服。因此，这项工作为从头微蛋白工程和图书馆提供了新的边界 设计。这也将导致一种新颖的治疗性有趣分子来治疗C。 艰难的感染。