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: 8528629
• 负责人: Jennifer L. Furman
• 金额: $ 3.35万
• 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 2014-03-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8528629
• 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; 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; screening

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）：该提案详细描述了用于进化基于蛋白质的抑制剂的一般噬菌体展示选择策略的开发，所述抑制剂含有用于选择性靶向已知在癌症中过表达的细胞表面蛋白酶的非天然氨基酸“弹头”。所有已知的生物体都编码相同的20种氨基酸。然而，考虑到大量的辅因子和翻译后修饰赋予内源性蛋白质改变的功能，这是可能的，扩展的遗传密码可能会提供一个进化优势，通过产生新的功能或增强健身的蛋白质。最近已经证明，修饰的tRNA和氨酰-tRNA合成酶对能够将非天然氨基酸掺入大规模抗体文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（间质蛋白酶）的环肽抑制剂中的初始噬菌体展示系统。具体目标如下：1）遗传编码用于靶向MT-SP1活性位点的非天然氨基酸弹头和2）使用扩展的遗传密码进行定向进化的环肽的噬菌体展示。最终，这种策略应该是通用的非天然氨基酸的肽或更大的蛋白质支架靶向任何相关的生物分子的背景下的定向进化。

项目成果

A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli.

• DOI: 10.1021/bi4000244
• 发表时间: 2013-03-12
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Chatterjee, Abhishek;Sun, Sophie B.;Furman, Jennifer L.;Xiao, Han;Schultz, Peter G.
• 通讯作者: Schultz, Peter G.