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Developing methods to engineer therapeutic proteases

开发治疗性蛋白酶的工程方法

基本信息

批准号：
8991707
负责人：
Burckhard Seelig
金额：
$ 18.13万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-05 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8991707
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 Directed Molecular Evolution Disease Engineering Enzymes Evolution Exotoxins Future Generations Goals Health 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 mutant neuromuscular next generation sequencing novel novel therapeutics prototype research study small molecule streptopain therapeutic target tool

项目摘要

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）：蛋白酶是开发能够抑制致病蛋白的强效治疗剂的丰富来源。我们将通过中和细菌外毒素来创造第一种针对传染病的治疗性蛋白酶。虽然大多数药物是结合剂并以化学计量比起作用，但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、神经肌肉和消化障碍。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现有蛋白酶。不幸的是，大多数潜在的治疗靶点目前还无法确定，因为尚未鉴定出具有合适特异性的蛋白酶。我们将通过建立两种独立和协同的方法来克服这种限制，这将使定制治疗性蛋白酶的工程化成为可能。在我们的第一个目标，我们将开发一个高通量的方法来测定蛋白酶的特异性。这项技术对于扫描天然蛋白酶的活性将是非常宝贵的，可以有效地重新用于治疗应用。此外，目前的方法不能可靠地预测人类蛋白质组内的脱靶切割。然而，我们独特的方法将通过结合我们的mRNA展示技术，下一代测序，LC-MS/MS和计算分析，在一个实验中筛选所有可能的八肽底物。来自潜在的数百万个切割序列的数据将被编译成高分辨率的特异性图谱。该图谱将有助于准确预测和随后消除蛋白酶与人类蛋白质的交叉反应性。在我们的第二个目标，我们将确定新的外毒素裂解蛋白酶从超过1012个独特的蛋白质突变体库，通过应用mRNA展示技术，我们开发的酶的定向进化。这种方法将能够同时工程化多个底物残基的蛋白水解特异性。因此，我们将能够改进针对潜在任何蛋白质靶标的活性，同时最大限度地减少脱靶活性。在ou方法的第一个应用中，我们将设计一种蛋白酶来中和化脓性链球菌超抗原外毒素SpeA，该外毒素与链球菌中毒性休克综合征、坏死性筋膜炎、川崎病样疾病和许多其他疾病有关。我们的总体目标是建立分析和创造新的蛋白酶特异性的方法，这可能会开辟一条新的蛋白酶治疗方法的道路，从中和感染性疾病中广泛的细菌外毒素到治疗各种其他涉及异常蛋白质的疾病。