Aggarwal Saurabh, Harden James L, Denmeade Samuel R
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University Whiting School of Engineering, Baltimore, Maryland 21231, USA.
Bioconjug Chem. 2006 Mar-Apr;17(2):335-40. doi: 10.1021/bc0502659.
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.
随机肽的大型组合文库已被用于多种应用，包括蛋白质-蛋白质相互作用分析、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中丢失特异性但低亲和力的结合肽。这些特异性结合剂的丢失常常导致分离出与筛选过程中使用的成分非特异性结合的肽。此前已证明，由于亲和力效应，使肽二聚化或多聚化可显著提高其结合动力学10至1000倍。为利用这一发现，我们通过改进“一珠一化合物”方法，在聚乙二醇丙烯酰胺（PEGA）珠上构建了一个12氨基酸二聚体肽的随机文库。由于空间位阻和聚集效应以及存在许多难以合成的肽序列，化学合成100,000个二聚体肽可能存在问题。我们设计了一种方法（在此详细描述），通过改进现有的“分割与混合”合成方法，将二聚体文库合成中固有的问题降至最低。使用这种方法，二聚体文库被用于分离一系列选择性结合上皮癌细胞的肽。一种氨基酸序列为QMARIPKRLARH的肽以二聚体形式结合到掺入血液中的前列腺癌细胞，但不结合循环造血细胞。然而，该肽的单体形式与同一LNCaP细胞系结合不佳。这些数据表明，从这种“一珠一二聚体”文库中获得的“命中”肽可直接用于最终应用，或用作构建第二代文库的先导。
