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（通过应用证明）：蛋白酶是可以使疾病的疗法的丰富来源，这些疗法可以使疾病 - 可苏依蛋白te蛋白创造出在Infectuary中的therfertagin不幸的是，所有的应用都需要确定所需的活动。 Custim Therapeutites的工程。有效地对人类蛋白质组进行治疗从电气化的数百万裂解序列将汇编为分辨率特异性图。超过1012个蛋白质，将mRNA y技术应用于我们开发的酶的定向酶，将对多个底物残留物进行工程OU方法的第一个应用，我们将设计为中和化脓性链球菌超毒素Exotoxin EA，这与链球菌有毒综合征，坏死性筋膜炎，川崎样疾病和创建更多的疾病有关。新型的蛋白酶特异性，这可能为新的蛋白酶开辟道路，蛋白酶疗法具有广泛的应用，从中和广泛的细菌外毒素在可感染性疾病中，以便针对其他疾病的其他疾病涉及异常蛋白质。