Developing methods to engineer therapeutic proteases

开发治疗性蛋白酶的工程方法

• 批准号: 8890930
• 负责人: Burckhard Seelig
• 金额: $ 21.94万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-05 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8890930
• 关键词: Bacteria; Bacterial exotoxin; Biological Assay; Blood Circulation; Caspase; Cleaved cell; Coagulation Process; Communicable Diseases; Complex; Computer Analysis; Custom; Data; Databases; Development; Digestion; Disease; Engineering; Enzymes; Evolution; Exotoxins; Future; Generations; Goals; Human; Human Genome; Human body; Immune System Diseases; In Vitro; Infection; Inflammatory; Libraries; Link; Maps; Messenger RNA; Methods; Mining; Mutation; Nature; Necrotizing fasciitis; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Procedures; Proteins; Proteome; Research; Resolution; Scanning; Scheme; Sepsis; Source; Specificity; Staphylococcus aureus; Streptococcus pyogenes; Structure; Superantigens; Techniques; Technology; Therapeutic; Thrombosis; Toxic Shock Syndrome; Toxin; Variant; blood filtration; cross reactivity; design; directed evolution; mutant; neuromuscular; next generation sequencing; novel; novel therapeutics; prototype; public health relevance; research study; small molecule; streptopain; therapeutic target; tool; Bacteria; Bacterial exotoxin; Biological Assay; Blood Circulation; Caspase; Cleaved cell; Coagulation Process; Communicable Diseases; Complex; Computer Analysis; Custom; Data; Databases; Development; Digestion; Disease; Engineering; Enzymes; Evolution; Exotoxins; Future; Generations; Goals; Human; Human Genome; Human body; Immune System Diseases; In Vitro; Infection; Inflammatory; Libraries; Link; Maps; Messenger RNA; Methods; Mining; Mutation; Nature; Necrotizing fasciitis; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Procedures; Proteins; Proteome; Research; Resolution; Scanning; Scheme; Sepsis; Source; Specificity; Staphylococcus aureus; Streptococcus pyogenes; Structure; Superantigens; Techniques; Technology; Therapeutic; Thrombosis; Toxic Shock Syndrome; Toxin; Variant; blood filtration; cross reactivity; design; directed evolution; mutant; neuromuscular; next generation sequencing; novel; novel therapeutics; prototype; public health relevance; research study; small molecule; streptopain; therapeutic target; tool

 DESCRIPTION (provided by applicant): Proteases are a rich source for the development of powerful therapeutics that can inactivate disease-causing proteins. We will create the first therapeutic protease targeted at infectious disease by neutralizing bacterial exotoxins. While most drugs are binders and act at a stoichiometric ratio, proteases are capable of catalytic turnover. Therapeutic proteases are currently used to treat thrombosis, sepsis, and coagulation, neuromuscular and digestion disorders. Significantly, all of these applications required identification of an existing protease in nature with the desired activity. Unfortunately, most potential therapeutic targets are not currently addressable because proteases with suitable specificities have not been identified. We will overcome this limitation by establishing two independent and synergistic methods that will enable the engineering of custom therapeutic proteases. In our first aim, we will develop a high-throughput method for assaying protease specificity. This technique will be invaluable for scanning natural proteases for activity that can be efficiently repurposed for therapeutic applications. Additionally, current methods are unable to reliably predict off-target cleavage within the human proteome. However, our unique approach will screen all possible octa-peptide substrates in a single experiment by combining our mRNA display technology, next-generation sequencing, LC-MS/MS, and computational analysis. The data from potentially millions of cleavage sequences will be compiled into a high-resolution specificity map. This map will facilitate the accurate prediction and subsequent elimination of protease cross-reactivity with human proteins. In our second aim, we will identify novel exotoxin-cleaving proteases from a library of over 1012 unique protein mutants by applying the mRNA display technology for the directed evolution of enzymes which we developed. This approach will enable the engineering of proteolytic specificity for multiple substrate residues simultaneously. As a result, we will be able to refine activity against potentially any protein target while minimizing off-target activity. In the first application of ou approach, we will design a protease to neutralize the Streptococcus pyogenes superantigen exotoxin SpeA, which has been linked to streptococcal toxic shock syndrome, necrotizing fasciitis, Kawasaki-like diseases, and many more diseases. Our overall goal is to establish methods to analyze and create novel protease specificities, which may open the path to a new class of protease therapeutics with broad applications from neutralizing the wide array of bacterial exotoxins in infectious disease to treating a variety of additional disorders that involv aberrant proteins.

 描述（由适用提供）：蛋白酶是开发强大疗法的丰富来源，可以使引起疾病的蛋白质失活。我们将通过中和细菌外毒素来创建第一个针对传染病的治疗蛋白。虽然大多数药物是粘合剂，并且以化学计量比作用，但蛋白酶的催化转换能力。目前，治疗性蛋白酶用于治疗血栓形成，败血症和凝结，神经肌肉和消化疾病。值得注意的是，所有这些应用都需要在本质上鉴定现有的蛋白酶，并具有所需的活动。不幸的是，当前大多数潜在的治疗靶标都无法解决，因为尚未确定具有合适规格的蛋白酶。我们将通过建立两种独立和协同的方法来克服这一限制，这些方法将使自定义治疗蛋白酶的工程化。在我们的第一个目标中，我们将开发一种用于分析蛋白酶特异性的高通量方法。该技术对于扫描自然蛋白酶的活动将是无价的 为治疗应用重新利用了效率。此外，当前的方法无法可靠地预测人蛋白质组内的脱靶裂解。但是，我们独特的方法将通过组合我们的mRNA显示技术，下一代测序，LC-MS/MS和计算分析来筛选所有可能的八肽底物。来自潜在的数百万裂解序列的数据将编译为高分辨率特异性图。该地图将促进与人蛋白质蛋白质交叉反应的准确性预测和随后消除。在我们的第二个目标中，我们将通过将mRNA显示技术应用于我们开发的酶的定向演化中，从而从1012多个独特蛋白突变体的库中鉴定出新型的外毒素裂解的蛋白质。这种方法将使多个底物的蛋白水解特异性能够简单地保留。结果，我们将能够在最小化靶向靶向活性的同时，优化潜在的任何蛋白质靶标的活性。在OU方法的首次应用中，我们将设计一种蛋白质，以中和化脓性链球菌超抗毒素Exotoxin SPEA，该蛋白质已与链球菌毒性休克综合征，坏死性筋膜炎，川崎样疾病以及更多疾病有关。我们的总体目标是建立分析和创建新型蛋白质规范的方法，这可能为新的蛋白质治疗提供了一类新的蛋白质治疗方法，并从中和传染病中的广泛细菌外毒素到治疗涉及异常蛋白质的多种其他疾病。