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

 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（由申请人提供）：蛋白酶是开发强大的治疗药物的丰富来源，可以灭活引起疾病的蛋白质，而大多数药物都是结合剂并作用于细菌外毒素。在化学计量比下，蛋白酶能够催化周转。治疗性蛋白酶目前用于治疗血栓形成、脓毒症和凝血、神经肌肉。值得注意的是，所有这些应用都需要鉴定自然界中具有所需活性的蛋白酶，不幸的是，大多数潜在的治疗靶点目前无法确定，因为尚未鉴定出具有合适特异性的蛋白酶。两种独立且协同的方法将有助于定制治疗性蛋白酶的工程。我们的第一个目标是开发一种用于测定蛋白酶特异性的高通量方法，该技术对于扫描天然蛋白酶的活性非常有价值。那可以 此外，目前的方法无法可靠地预测人类蛋白质组中的脱靶切割，但是，下一步，我们独特的方法将通过结合我们的 mRNA 显示技术在单个实验中筛选所有可能的八肽底物。来自潜在数百万个裂解序列的数据将被编译成高分辨率特异性图谱，该图谱将有助于准确预测和随后消除蛋白酶。在我们的第二个目标中，我们将通过应用我们开发的酶定向进化的 mRNA 展示技术，从超过 1012 个独特的蛋白质突变体库中鉴定出新型外毒素裂解蛋白酶。同时对多个底物残基进行蛋白水解特异性工程，我们将能够改进针对任何潜在蛋白质靶点的活性，同时最大限度地减少脱靶活性。我们的总体目标是建立分析和创建新的蛋白酶特异性的方法，以中和化脓性链球菌超抗原外毒素 SpeA，该毒素与链球菌中毒性休克综合征、坏死性筋膜炎、川崎病等疾病有关。开辟了通向新型蛋白酶疗法的道路，该疗法具有广泛的应用，从中和传染病中的各种细菌外毒素到治疗多种疾病涉及异常蛋白质的其他疾病。