Directed Evolution of Selective Protease Inhibitors with an Expanded Genetic Code

具有扩展遗传密码的选择性蛋白酶抑制剂的定向进化

• 批准号: 8325229
• 负责人: Jennifer L. Furman
• 金额: $ 4.92万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8325229
• 关键词: Acids; Active Sites; Affinity; Amber; Amino Acids; Amino Acyl-tRNA Synthetases; Antibodies; Architecture; Bacillus amyloliquefaciens ribonuclease; Bacteriophages; Binding; Boronic Acids; Breast Cancer Cell; Cancer cell line; Capsid Proteins; Cardiovascular Diseases; Cell surface; Chloramphenicol; Consensus; Cyclic Peptides; DNA Sequence; Development; Diagnosis; Disulfides; Enzyme-Linked Immunosorbent Assay; Esters; Evolution; Extracellular Domain; Generations; Genetic Code; Genetic Engineering; Goals; Human; Label; Libraries; Ligase; Malignant Neoplasms; Mass Spectrum Analysis; Methanococcus; Methods; Mutate; Mutation; Neoplasm Metastasis; Organism; Peptide Hydrolases; Peptide Library; Peptide Synthesis; Peptides; Phage Display; Pharmacologic Substance; Phase; Physiological Processes; Population; Post-Translational Protein Processing; Protease Inhibitor; Proteins; Reagent; Recombinants; Scaffolding Protein; Screening procedure; Serine; Serine Protease; Serine Proteinase Inhibitors; Site; Solid; Specificity; System; Techniques; Technology; Terminator Codon; Testing; Therapeutic Intervention; Time; Tissues; Transfer RNA; Translating; Trypsin; Tyrosine-Specific tRNA; base; cofactor; combinatorial; directed evolution; fitness; gel electrophoresis; inhibitor/antagonist; matriptase; member; mutant; novel; novel diagnostics; novel therapeutics; overexpression; pyrrolysine

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.

描述（由申请人提供）：该提案详细介绍了针对不断发展的基于蛋白质的抑制剂的一般噬菌体显示策略的发展，该抑制剂包含不自然的氨基酸“弹头”，用于选择性地靶向已知在癌症中过表达的细胞表面蛋白酶。所有已知生物都编码相同的20个氨基酸。但是，考虑到内源性蛋白质具有改变功能的众多辅助因子和翻译后修饰，扩展的遗传密码可能会通过具有新功能或增强适应性的蛋白质产生的蛋白质来提供进化的优势。最近已经证明，经过修改的TRNA和氨基酰基-TRNA合成酶对能够将非天然氨基酸掺入大型抗体库中，以进行功能选择。此处描述了一种初始的噬菌体显示系统，用于将非天然氨基酸掺入细胞表面蛋白酶MT-SP1（Matriptase）的环状肽抑制剂中。具体目的如下：1）遗传编码用于靶向MT-SP1活性位点的非自然氨基酸弹头，以及2）使用扩展的遗传代码的循环肽的噬菌体显示，用于定向进化。最终，该策略对于在肽或较大的蛋白质支架的背景下的非自然氨基酸的定向演变应该是一般的，以靶向任何相关的生物分子。