Heinis Christian, Rutherford Trevor, Freund Stephan, Winter Greg
Laboratory of Molecular Biology, Medical Research Council, Cambridge, UK.
Nat Chem Biol. 2009 Jul;5(7):502-7. doi: 10.1038/nchembio.184.
Here we describe a phage strategy for the selection of ligands based on bicyclic or linear peptides attached covalently to an organic core. We designed peptide repertoires with three reactive cysteine residues, each spaced apart by several random amino acid residues, and we fused the repertoires to the phage gene-3-protein. Conjugation with tris-(bromomethyl)benzene via the reactive cysteines generated repertoires of peptide conjugates with two peptide loops anchored to a mesitylene core. Iterative affinity selections yielded several enzyme inhibitors; after further mutagenesis and selection, we were able to chemically synthesize a lead inhibitor (PK15; Ki =1.5 nM) specific to human plasma kallikrein that efficiently interrupted the intrinsic coagulation pathway in human plasma tested ex vivo. This approach offers a powerful means of generating and selecting bicyclic macrocycles (or if cleaved, linear derivatives thereof) as ligands poised at the interface of small-molecule drugs and biologics.
在此，我们描述了一种基于共价连接到有机核心的双环或线性肽来选择配体的噬菌体策略。我们设计了具有三个反应性半胱氨酸残基的肽库，每个残基之间间隔几个随机氨基酸残基，并将这些肽库与噬菌体基因3蛋白融合。通过反应性半胱氨酸与三（溴甲基）苯共轭，生成了肽共轭物库，其中两个肽环锚定在均三甲苯核心上。通过迭代亲和选择得到了几种酶抑制剂；经过进一步的诱变和选择，我们能够化学合成一种对人血浆激肽释放酶具有特异性的先导抑制剂（PK15；Ki = 1.5 nM），该抑制剂在体外测试中能有效中断人血浆中的内源性凝血途径。这种方法为生成和选择双环大环化合物（或如果被切割，其线性衍生物）作为处于小分子药物和生物制品界面的配体提供了一种强大的手段。
