Phage Display with Two Genetically Incorporated Noncanonical Amino Acids

描述（由申请人提供）：噬菌体展示和组合化学是鉴定癌症靶向治疗的两种高通量方法。然而，噬菌体展示文库具有有限的结构多样性，组合化学产生的小分子通常不能大量汇集以进行有效筛选。我们的长期目标是结合噬菌体展示的快速筛选特性和合成化学的多样性产生能力，组装高通量鉴定小分子抗血管生成药物的方法，并将这些分子应用于癌症诊断，分析，成像和治疗。这项特殊应用的主要目的是开发构建具有扩展化学多样性的噬菌体展示文库的方法，并筛选这些文库以识别血管内皮生长因子（VEGF）特异性配体，VEGF是抗血管生成药物的关键靶点。我们的中心假设是，噬菌体展示文库的化学多样性可以通过基因整合两种不同的非规范氨基酸（NAAs）进入文库，改变NAAs的身份，和/或化学修饰整合的NAAs来显着扩展。本研究的基本原理是，一旦构建这些非天然噬菌体展示文库，就可以快速筛选出具有多种化学结构的大量非天然肽，以对抗许多癌症治疗靶点，从而鉴定出治疗和预防癌症的新疗法。在强有力的初步数据的指导下，本项目将实现三个具体目标：1)优化M13KE噬菌体，使其能够公正地展示20种天然氨基酸和2种NAAs；2)构建包含2种NAAs不同组合的非自然噬菌体展示文库，并对文库进行筛选，鉴定vegf特异性配体；3)通过扩大基因编码的NAAs库和选择性修饰纳入的NAAs，扩大非天然噬菌体展示文库的化学多样性。本申请中提出的研究是创新的，因为它将通过修改具有多种结构片段的噬菌体展示肽来显著扩展噬菌体展示技术，以用于药物发现。拟议的研究具有重要意义，因为它将加速当前药物发现的进程，并有助于实现NIH在促进健康和对抗疾病方面的使命。

DESCRIPTION (provided by applicant): Phage display and combinatorial chemistry are two high throughput approaches for identification of targeted therapeutics of cancer. However, a phage display library has a limited structural diversity and small molecules generated by combinatorial chemistry generally cannot be pooled in large numbers for efficient screening. Our long term goal is to combine the rapid-screening feature of phage display and the diversity- generating power of synthetic chemistry to assemble approaches for high throughput identification of small- molecule antiangiogenic agents and apply these molecules to cancer diagnosis, profiling, imaging, and therapy. The primary objective of this particular application is to develop methods for constructing phage display libraries with expanded chemical diversities and screening these libraries to identify ligands specific for vascular endothelial growth factor (VEGF), a key target of antiangiogenic drugs. Our central hypothesis is that the chemical diversity of a phage display library can be significantly expanded by genetically incorporating two different noncanonical amino acids (NAAs) into the library, varying the identities of NAAs, and/or chemically modifying the incorporated NAAs. The rationale for the proposed research is that, once these unnatural phage display libraries are constructed, a large variety of unnatural peptides with diversified chemical structures can be rapidly screened against many cancer therapeutic targets, leading to identification of new therapeutics for cancer treatment and prevention. Guided by strong preliminary data, the objective of this application will be attained by pursuing three specific aims: 1) Optimize M13KE phage for the unbiased display of 20 natural amino acids and 2 NAAs; 2) Construct unnatural phage display libraries incorporated with different combinations of 2 NAAs and screen these libraries to identify VEGF-specific ligands; and 3) Expand the chemical diversity of unnatural phage display libraries by expanding the pool of genetically encoded NAAs and selectively modifying the incorporated NAAs. The research proposed in this application is innovative, because it will expand the phage display technique significantly by amending the displayed peptides with diversified structure moieties desirable for drug discovery. The proposed research is significant because it will accelerate the current drug discovery progresses and contribute to fulfilling the NIH mission in promoting health and combating diseases.