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用于鉴定癌症靶向治疗剂的两种高通量方法。然而，噬菌体展示文库具有有限的结构多样性，并且通过组合化学产生的小分子通常不能大量汇集用于有效筛选。我们的长期目标是将噬菌体展示的快速筛选特征和合成化学的多样性产生能力相结合，以组装用于高通量鉴定小分子抗血管生成剂的方法，并将这些分子应用于癌症诊断、分析、成像和治疗。该特定应用的主要目的是开发用于构建具有扩增的化学二聚体的噬菌体展示文库并筛选这些文库以鉴定对血管内皮生长因子（VEGF）特异性的配体（VEGF是抗血管生成药物的关键靶标）的方法。我们的中心假设是，噬菌体展示库的化学多样性可以显着扩大通过遗传将两种不同的非典型氨基酸（NAAs）到库中，不同的身份的NAAs，和/或化学修饰的注册NAAs。这项研究的基本原理是，一旦构建了这些非天然噬菌体展示文库，就可以针对许多癌症治疗靶点快速筛选具有多样化化学结构的各种非天然肽，从而鉴定出用于癌症治疗和预防的新疗法。在强有力的初步数据的指导下，本申请的目的将通过追求三个具体目标来实现：1）优化M13 KE噬菌体以无偏展示20种天然氨基酸和2种NAA; 2）构建掺入2种NAA的不同组合的非天然噬菌体展示文库并筛选这些文库以鉴定VEGF特异性配体;和3）通过扩大遗传编码的NAA库和选择性修饰掺入的NAA来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药物发现所需的多样化结构部分的展示肽来显著扩展噬菌体展示技术。这项研究具有重要意义，因为它将加速当前药物发现的进展，并有助于实现NIH促进健康和对抗疾病的使命。

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.