Activity-based selections and amplification of non-natural protease substrates

描述（由申请人提供）：蛋白酶催化肽和蛋白质中酰胺键的水解，在大多数生物学过程中发挥重要作用，并且是许多疾病（包括癌症）的非常重要的治疗和诊断靶标。在这个建议中，提出了有效的和系统的组合工具的发展，以确定在前列腺癌中涉及的蛋白酶的底物特异性配置文件。这三种蛋白酶的高度选择性和特异性底物的产生将有助于设计更具选择性的诊断工具，并有可能有助于设计选择性抑制剂。 K99阶段：该提案的博士后阶段（具体目标I和II）将在加州大学伯克利分校Jonathan Ellman教授的指导下进行。K99阶段的目标是开发一种产生完全特异性蛋白酶底物的方法。底物将是肽，并且将对DNA结合的文库进行使用硫酯易断裂键的基于活性的选择。DNA展示技术将用于文库的编码、路由和扩增。Jon Ellman是组合化学及其在蛋白酶底物应用领域的领先专家。这一时期将提供组合化学和蛋白酶化学领域的基本培训。所获得的知识将证明是非常宝贵的完成具体目标III和IV和过渡到一个成功的职业生涯作为一个独立的科学家。 独立的R00阶段：具体目标III和IV将侧重于具体目标I和II中开发的技术的应用。靶向参与前列腺癌的蛋白酶的肽底物将作为前列腺癌的前药疗法进行评估（具体目标III）。从这些实验中提供的底物也将提供用于前列腺肿瘤的体内成像的蛋白酶激活的近红外探针的产生的基础。 相关性：这一建议有可能大大改善公众健康，通过推进的合成和评估（使用基于活动的选择）的组合库。拟议的研究开发的方法，将提供完全选择性的蛋白酶底物。所获得的基质将用作药物递送剂和选择性成像剂的基础。

DESCRIPTION (provided by applicant): Proteases, which catalyze the hydrolysis of amide bonds in peptides and proteins, play essential roles in most biological processes and are very important therapeutic and diagnostic targets for a multitude of diseases, including cancer. In this proposal, the development of efficient and systematic combinatorial tools is proposed to determine the substrate specificity profiles of proteases involved in prostate cancer. Generation of highly selective and specific substrates for these three proteases will aid in designing more selective diagnostic tools as well as have the potential to aid in designing selective inhibitors. Mentored K99 Phase: The postdoctoral phase (specific aims I and II) of the proposal will be carried out under the mentorship of Professor Jonathan Ellman at the University of California-Berkeley. The K99 phase goal is to develop a method for the generation of completely specific protease substrates. The substrates will be peptidic and activity-based selections using a thioester scissile bond will be performed on the DNA-bound library. DNA display technology will be utilized for encoding, routing, and amplification of the library. Jon Ellman is a leading expert in the field of combinatorial chemistry and its application to protease substrates. This period will provide essential training in the fields of combinatorial chemistry and protease chemistry. The acquired knowledge will prove invaluable for completion of specific aims III and IV and transition into a successful career as an independent scientist. Independent R00 Phase: Specific aims III and IV will focus on the application of the technology developed in specific aims I and II. Peptidic substrates targeting proteases involved in prostate cancer will be evaluated as pro-drug therapeutics for prostate cancer (specific aim III). The substrates afforded from these experiments will also provide the basis for the generation of protease-activated near IR probes for the in vivo imaging of prostate tumors. Relevance: This proposal has the potential to greatly improve public health through advancement of the synthesis and evaluation (using activity-based selections) of combinatorial libraries. The proposed research develops methodology that will provide completely selective protease substrates. The substrates obtained will be used as the basis for drug delivery agents and selective imaging agents.