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个功能强大但未充分利用的蛋白质家族，用于试剂，诊断和治疗 应用.它们像小分子一样超稳定，但又大到足以特异性地与蛋白质结合。 这些靶点具有高亲和力，因此可以容易地用于抑制蛋白质-蛋白质相互作用。富二硫化物 微蛋白是天然存在的，但天然分子的工程设计具有挑战性。迫切需要 新技术使富含二硫键的微蛋白能够被工程化以结合任意蛋白质靶， 这对于其他蛋白质支架如抗体是常规的。在这里，我们提出了一个计算从头设计 使用Rosetta构建合成微蛋白文库策略，该文库通常可用于筛选蛋白质 亲和试剂 创建蛋白质文库的典型方法是在蛋白质文库中产生大量随机序列多样性。 单一蛋白质结构的局部区域。这些序列中的大多数将是不稳定的或不能结合任何 目标（例如，极性太强或非极性太强）。而不是使用随机序列，我们建议显式设计 每一个都是合成的富含二硫化物的微蛋白文库的成员。我们的设计库将包含106个不同的 显示最广泛的结合表面的小蛋白。编码这个文库的基因将 使用寡聚物池合成。为了在这种规模下进行计算从头设计，我们扩展了 Rosetta中的SEWING算法生成数十万个独特的微型蛋白质结构， 序列的我们开发了新的过滤器来评估设计模型的质量，并量化和比较蛋白质 结构和表面。 作为这种方法的一个测试案例，我们将通过酵母展示筛选我们的富含二硫化物的微蛋白库， 艰难梭菌肠毒素TcdA和TcdB。C.艰难梭菌是导致医疗相关疾病的主要原因 感染在美国，这两种蛋白质介导其致病性。目前，唯一可用 C.针对这些毒素蛋白质的艰难梭菌肠道感染是抗体，必须注射 且功效差。同一中和表位的超稳定微蛋白结合剂可以是 口服给药因此，本工作为微蛋白质工程和文库的研究开辟了新的领域 设计这也将导致一个新的来源，治疗有趣的分子治疗C。 艰难感染