High-throughput screening of one-bead-one-compound libraries: identification of cyclic peptidyl inhibitors against calcineurin/NFAT interaction.
High-throughput screening of one-bead-one-compound libraries: identification of cyclic peptidyl inhibitors against calcineurin/NFAT interaction.
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DOI：
10.1021/co200101w
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发表时间：
2011-09-12
影响因子：
--
通讯作者：
Pei, Dehua
中科院分区：
文献类型：
--
作者：
Liu, Tao;Qian, Ziqing;Xiao, Qing;Pei, Dehua
关键词：
单珠单化合物（OBOC）文库为药物发现和生物医学研究提供了强大的工具。然而，筛选大量珠粒/化合物（>1百万）并根据其效力对初始命中物（其共价连接到固体支持物）进行排序仍然面临重大的技术挑战。在这项工作中，我们整合了我们自己和其他实验室的一些最新技术进展，以开发一种快速筛选大型OBOC文库的通用方法。该方法已被应用于合成和筛选环肽文库，其特征在于：（1）空间分离的珠，其表面层含有环肽，其内部含有线性编码肽;（2）文库的快速珠上筛选（> 100万）通过多阶段程序（磁珠分选、酶联测定和基于荧光的筛选）;（3）从单个阳性珠粒选择性释放环肽，用于液相测定它们的结合亲和力;（4）通过部分Edman降解/质谱（PED/MS）进行命中鉴定。针对蛋白磷酸酶钙调磷酸酶（Cn）筛选文库，鉴定出一系列与活化T细胞核因子（NFAT）的底物对接位点结合的环肽，KD值约为1 μM。这些化合物的亲和力和特异性的进一步改进可能导致一类新的免疫抑制剂，其选择性更高，因此毒性比环孢素A和FK 506低。
One-bead-one-compound (OBOC) libraries provide a powerful tool for drug discovery as well as biomedical research. However, screening a large number of beads/compounds (>1 million) and rank ordering the initial hits (which are covalently attached to a solid support) according to their potencies still post significant technical challenges. In this work, we have integrated some of the latest technical advances from our own as well as other laboratories to develop a general methodology for rapidly screening large OBOC libraries. The methodology has been applied to synthesize and screen a cyclic peptide library that features: (1) spatially segregated beads containing cyclic peptides on the surface layer and linear encoding peptides in their interior; (2) rapid on-bead screening of the library (>1 million) by a multi-stage procedure (magnetic bead sorting, enzyme-linked assay, and fluorescence based screening); (3) selective release of cyclic peptides from single positive beads for solution-phase determination of their binding affinities; and (4) hit identification by partial Edman degradation/mass spectrometry (PED/MS). Screening of the library against protein phosphatase calcineurin (Cn) identified a series of cyclic peptides that bind to the substrate-docking site for nuclear factor of activated T cells (NFAT) with KD values of ~1 μM. Further improvement of the affinity and specificity of these compounds may lead to a new class of immunosuppressive agents that are more selective and therefore less toxic than cyclosporine A and FK506.
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影响因子：
64.5
作者：
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通讯作者：
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影响因子：
--
作者：
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通讯作者：
Pei, Dehua