Directed Evolution of Selective Protease Inhibitors with an Expanded Genetic Code

描述（由申请人提供）：该提案详细介绍了一种通用噬菌体展示选择策略的发展，该策略用于进化的基于蛋白质的抑制剂，该抑制剂含有非天然氨基酸“弹头”，用于选择性靶向已知在癌症中过表达的细胞表面蛋白酶。所有已知的生物体都编码相同的20种氨基酸。然而，考虑到大量的辅助因子和翻译后修饰使内源性蛋白质具有改变的功能，扩展的遗传密码可能通过产生具有新功能或增强适应性的蛋白质而提供进化优势。最近有研究表明，经过修饰的trna和氨基酰基- trna合成酶对能够将非天然氨基酸整合到大规模抗体文库中，以进行功能选择。本文描述了一种初始噬菌体展示系统，用于将非天然氨基酸掺入细胞表面蛋白酶MT-SP1（基质酶）的环肽抑制剂中。具体目的如下：1)基因编码一个针对MT-SP1活性位点的非天然氨基酸战斗部；2)利用扩展的遗传密码展示环状肽的噬菌体，用于定向进化。最终，这种策略应该适用于针对任何相关生物分子的肽或更大的蛋白质支架的非天然氨基酸的定向进化。公共卫生相关性：蛋白酶参与许多不同的生理过程。因此，蛋白酶功能的失调可能与从心血管疾病到癌症的各种病理状况有关。因此，蛋白酶是治疗干预或诊断的极好靶点，目前占药物靶点的5-10%。MT-SP1最初是从乳腺癌细胞中分离出来的，并与转移相关的组织重塑有关。选择性环肽亲和试剂应该为评估该蛋白酶的过表达提供有价值的方法，并可能最终为发现新的诊断和治疗方法提供基础。

DESCRIPTION (provided by applicant): This proposal details the development of a general phage display selection strategy for evolving protein-based inhibitors that contain an unnatural amino acid "warhead" for selectively targeting cell surface proteases known to be overexpressed in cancer. All known organisms encode the same 20 amino acids. However, considering the vast array of cofactors and posttranslational modifications that endow endogenous proteins with altered functionalities, it is possible that an expanded genetic code may provide an evolutionary advantage through the generation of proteins with novel functions or enhanced fitness. It has recently been demonstrated that modified tRNAs and aminoacyl-tRNA synthetase pairs are capable of incorporating unnatural amino acids into large scale antibody libraries for the purpose of carrying out functional selections. Herein is described an initial phage display system for the incorporation of unnatural amino acids into cyclic peptide inhibitors of a cell surface protease, MT-SP1 (matriptase). The specific aims are as follows: 1) Genetically encoding an unnatural amino acid warhead for targeting the MT-SP1 active site and 2) Phage display of cyclic peptides for directed evolution using an expanded genetic code. Ultimately this strategy should be general for the directed evolution of unnatural amino acids in the context of peptides or larger protein scaffolds for targeting any relevant biomolecule. PUBLIC HEALTH RELEVANCE: Proteases are involved in many diverse physiological processes. As a consequence, misregulation of protease function may correlate with a variety of pathological conditions ranging from cardiovascular disorders to cancer. Therefore, proteases are excellent targets for therapeutic intervention or diagnosis, and currently represent 5-10% of pharmaceutical targets. MT-SP1 was initially isolated from breast cancer cells and has been implicated in tissue remodeling associated with metastasis. Selective cyclic peptide affinity reagents should provide a valuable method for evaluating the overexpression of this protease, and may ultimately provide a basis for the discovery of novel diagnostics and therapeutics. PUBLIC HEALTH RELEVANCE: Proteases, such as MT-SP1, are involved in many diverse physiological processes. As a consequence, misregulation of function may correlate with a variety of pathological conditions ranging from cardiovascular disorders to cancer. Selective cyclic peptide affinity reagents targeted against MT-SP1 should provide a valuable method for evaluating the overexpression of this protease, and may ultimately provide a basis for the discovery of novel diagnostics and therapeutics.