Phage Display with Two Genetically Incorporated Noncanonical Amino Acids

描述(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，噬菌体展示文库的结构多样性有限，通过组合化学产生的小分子通常不能大量汇集在一起进行有效筛选。我们的长期目标是结合噬菌体展示的快速筛选功能和合成化学的多样性生成能力，组装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、成像和治疗。这一特殊应用的主要目的是开发构建具有扩大化学多样性的噬菌体展示文库的方法，并筛选这些文库以确定血管内皮生长因子(VEGF)的特异性配体，血管内皮生长因子是抗血管生成药物的关键靶点。我们的中心假设是，通过将两种不同的非规范氨基酸(NAA)基因整合到噬菌体展示文库中，改变NAA的身份，和/或对所结合的NAA进行化学修饰，可以显著扩大噬菌体展示文库的化学多样性。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，就可以快速筛选出具有不同化学结构的大量非天然多肽，以对抗许多癌症治疗靶点，从而识别用于癌症治疗和预防的新疗法。在强大的初步数据指导下，本应用的目标将通过追求三个具体目标来实现：1)优化M13KE噬菌体，以无偏展示20个天然氨基酸和2个NAA；2)构建与2个NAA不同组合并入的非自然噬菌体展示文库，并筛选这些文库以识别血管内皮生长因子特异性配体；以及3)通过扩大基因编码的NAA库并选择性地对所纳入的NAA进行修饰，扩大非自然噬菌体展示文库的化学多样性。本申请中提出的研究具有创新性，因为它将极大地扩展噬菌体展示技术，通过修饰展示的多肽具有多样化的结构部分，从而有利于药物发现。这项拟议的研究具有重要意义，因为它将加快目前的药物发现进展，并有助于完成NIH在促进健康和抗击疾病方面的使命。

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.