Elucidation of the Contribution of Active Site and Exosite Interactions to Affinity and Specificity of Peptidylic Serine Protease Inhibitors Using Non-Natural Arginine Analogs
Elucidation of the Contribution of Active Site and Exosite Interactions to Affinity and Specificity of Peptidylic Serine Protease Inhibitors Using Non-Natural Arginine Analogs
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DOI：
10.1124/mol.111.072280
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发表时间：
2011-10-01
影响因子：
3.6
通讯作者：
Jensen, Knud J.
中科院分区：
文献类型：
--
作者：
Hosseini, Masood;Jiang, Longguang;Jensen, Knud J.
有越来越多的兴趣开发肽与丝氨酸蛋白酶的病理生理功能的药理学干预。从噬菌体展示的肽库中，我们先前分离出尿激酶型纤溶酶原激活物的肽抑制剂，这是干预癌症侵袭的潜在靶点。这两种肽，upain-1 （CSWRGLENHRMC）和mupain-1 (CPAYSRYLDC)，分别是人类和小鼠尿激酶型纤溶酶原激活剂的竞争性抑制剂。两者都有一个Arg作为P1残基，插入酶活性位点的S1口袋中，但它们的特异性在很大程度上取决于酶活性位点外的相互作用，即所谓的外源相互作用。在这里，我们描述了upain-2 （CSWRGLENHAAC）和一些upain-2和mupain-1变体的合成，其中P1精氨酸被新的非天然精氨酸类似物取代，并显著提高了肽对靶点的亲和力。利用人类和小鼠尿激酶型纤溶酶原激活物的嵌合体以及x射线晶体学，我们描绘了P1残基和外源相互作用对抑制剂对其靶酶的亲和力和特异性的相对贡献。将特定的非天然氨基酸插入P1位置的效果是由以下事实决定的：S1口袋中P1残基相互作用的改变导致外源相互作用的改变，反之亦然。当考虑到用于药理干预的丝氨酸蛋白酶抑制剂的亲和力和特异性时，这些发现具有普遍的意义，并可能为治疗癌症的潜在候选药物铺平道路。
There is increasing interest in developing peptides for pharmacological intervention with pathophysiological functions of serine proteases. From phage-displayed peptide libraries, we previously isolated peptidylic inhibitors of urokinase-type plasminogen activator, a potential target for intervention with cancer invasion. The two peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), are competitive inhibitors of human and murine urokinase-type plasminogen activator, respectively. Both have an Arg as the P1 residue, inserting into the S1 pocket in the active site of the enzymes, but their specificity depends to a large extent on interactions outside the enzymes' active sites, so-called exosite interactions. Here we describe upain-2 (CSWRGLENHAAC) and the synthesis of a number of upain-2 and mupain-1 variants in which the P1 Arg was substituted with novel non-natural Arg analogs and achieved considerable improvement in the affinity of the peptides to their targets. Using chimeras of human and murine urokinase-type plasminogen activator as well as X-ray crystallography, we delineated the relative contribution of the P1 residue and exosite interactions to the affinity and specificity of the inhibitors for their target enzyme. The effect of inserting a particular non-natural amino acid into the P1 position is determined by the fact that changes in interactions of the P1 residue in the S1 pocket lead to changed exosite interactions and vice versa. These findings are of general interest when the affinities and specificities of serine protease inhibitors to be used for pharmacological intervention are considered and could pave the way for potential drug candidates for the treatment of cancer.