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: 8198219
• 负责人: Jennifer L. Furman
• 金额: $ 4.63万
• 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/8198219
• 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. 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.

描述（由申请人提供）：该提案详细介绍了用于进化基于蛋白质的抑制剂的通用噬菌体展示选择策略的开发，该抑制剂含有非天然氨基酸“弹头”，用于选择性地靶向已知在癌症中过度表达的细胞表面蛋白酶。所有已知的生物都编码相同的 20 种氨基酸。然而，考虑到赋予内源性蛋白质改变功能的大量辅因子和翻译后修饰，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来提供进化优势。最近已经证明，修饰的 tRNA 和氨酰基-tRNA 合成酶对能够将非天然氨基酸掺入大规模抗体库中，以进行功能选择。本文描述了用于将非天然氨基酸掺入细胞表面蛋白酶MT-SP1（matriptase）的环肽抑制剂中的初始噬菌体展示系统。具体目标如下：1）基因编码非天然氨基酸弹头，用于靶向MT-SP1活性位点；2）噬菌体展示环肽，使用扩展的遗传密码进行定向进化。最终，这种策略应该适用于在肽或更大的蛋白质支架的背景下针对任何相关生物分子的非天然氨基酸的定向进化。公众健康相关性：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等多种病理状况有关。因此，蛋白酶是治疗干预或诊断的极好靶标，目前占药物靶标的 5-10%。 MT-SP1 最初是从乳腺癌细胞中分离出来的，并且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这种蛋白酶的过度表达提供有价值的方法，并可能最终为新的诊断和治疗方法的发现提供基础。 公共卫生相关性：MT-SP1 等蛋白酶参与许多不同的生理过程。因此，功能失调可能与从心血管疾病到癌症等多种病理状况相关。针对 MT-SP1 的选择性环肽亲和试剂应该为评估这种蛋白酶的过度表达提供有价值的方法，并可能最终为新的诊断和治疗方法的发现提供基础。