Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach
Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach
复制标题
DOI：
10.1021/bc0502659
复制
发表时间：
2006-03-01
影响因子：
4.7
通讯作者：
Denmeade, SR
中科院分区：
文献类型：
--
作者：
Aggarwal, S;Harden, JL;Denmeade, SR
随机肽的大型组合文库已用于多种应用，包括蛋白质-蛋白质相互作用的分析、表位作图和药物靶向。筛选这些文库的主要障碍是在洗涤步骤期间特异性但低亲和力结合肽的损失。这些特异性结合剂的丧失通常导致与选择过程中使用的组分非特异性结合的肽的分离。先前，已经证明，由于亲合力效应，二聚化或多聚化肽可以显著地将其结合动力学提高10至1000倍。为了利用这一观察结果，我们通过修改“一珠一化合物”的方法在聚乙二醇丙烯酰胺（PEGA）珠上构建了12个氨基酸二聚体肽的随机文库。由于空间和聚集效应以及存在许多难以合成的肽序列，100 000个肽作为二聚体的化学合成可能是有问题的。我们已经设计了一种方法，在这里详细描述，通过修改现有的“分裂和池”合成方法，以最大限度地减少合成二聚体库中固有的问题。使用这种方法，二聚体文库用于分离一系列选择性结合上皮癌细胞的肽。一种具有氨基酸序列QMARIPKRLARH的肽作为二聚体结合到掺入血液中的前列腺癌细胞，但不结合到循环造血细胞。然而，该肽的单体形式不能很好地结合到相同的LNCaP细胞系。这些数据表明，从这种“一珠一二聚体”文库获得的“命中”可直接用于最终应用或用作构建第二代文库的先导。
Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100 000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.