Developing methods to engineer therapeutic proteases

 描述(申请人提供)：蛋白水解酶是开发强大的治疗方法的丰富来源，可以灭活致病蛋白。我们将通过中和细菌外毒素来创造第一个针对传染病的治疗性蛋白酶。虽然大多数药物是粘合剂，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、败血症、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现有的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还不能寻址，因为具有适当特异性的蛋白酶还没有被确定。我们将通过建立两种独立和协同的方法来克服这一限制，这两种方法将使定制治疗性蛋白酶的工程成为可能。在我们的第一个目标中，我们将开发一种高通量的检测蛋白酶特异性的方法。这项技术对于扫描天然蛋白酶的活性将是无价的， 被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非靶标切割。然而，我们独特的方法将结合我们的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。来自可能数以百万计的切割序列的数据将被汇编成高分辨率的特异图。这一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。在我们的第二个目标中，我们将通过应用我们开发的酶的定向进化的mRNA展示技术，从超过1012个独特的蛋白质突变体库中鉴定新的外毒素裂解酶。这种方法将能够同时对多个底物残基进行蛋白质降解专一性工程。因此，我们将能够针对潜在的任何蛋白质靶标精炼活性，同时将靶外活性降至最低。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，该毒素已与链球菌中毒性休克综合征、坏死性筋膜炎、川崎样疾病等许多疾病有关。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，这可能开辟一类新的蛋白酶疗法的道路，具有广泛的应用，从中和传染病中广泛的细菌外毒素到治疗涉及异常蛋白质的各种额外疾病。

 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.