Effective Disruption of Phosphoprotein−Protein Surface Interaction Using Zn(II) Dipicolylamine-Based Artificial Receptors via Two-Point Interaction

Protein phosphorylation is ubiquitously involved in living cells, and it is one of the key events controlling protein−protein surface interactions, which are essential in signal transduction cascades. We now report that the small molecular receptors bearing binuclear Zn(II)-Dpa can strongly bind to a bis-phosphorylated peptide in a cross-linking manner under neutral aqueous conditions when the distance between the two Zn(II) centers can appropriately fit in that of the two phosphate groups of the phosphorylated peptide. The binding property was quantitatively determined by ITC (isothermal titration calorimetry), induced CD (circular dichroism), and NMR. On the basis of these findings, we demonstrated that these types of small molecules were able to effectively disrupt the phosphoprotein−protein interaction in a phosphorylated CTD peptide and the Pin1 WW domain, a phosphoprotein binding domain, at a micromolar level. The strategy based on a small molecular disruptor that directly interacts with phosphoprotein is unique and should be promising in developing a designer inhibitor for phosphoprotein−protein interaction.蛋白质磷酸化（Protein phosphorylation）广泛存在于活细胞中，是调控蛋白质-蛋白质表面相互作用的关键事件之一，而此类相互作用是信号转导级联反应（signal transduction cascades）不可或缺的组成部分。本研究报道，携带双核Zn(II)-二吡啶甲基胺（binuclear Zn(II)-Dpa）的小分子受体，在中性水溶液环境中，当两个锌(II)中心的间距与磷酸化肽段的两个磷酸基团间距适配时，能够以交联模式强效结合双磷酸化肽段。该结合特性通过等温滴定量热法（ITC，isothermal titration calorimetry）、诱导圆二色谱（CD，circular dichroism）以及核磁共振波谱（NMR）完成了定量表征。基于上述研究结果，本研究证实此类小分子可在微摩尔浓度级别有效阻断磷酸化CTD肽段与磷酸化蛋白质结合结构域Pin1 WW结构域之间的磷酸化蛋白质-蛋白质相互作用。这种以直接靶向磷酸化蛋白质的小分子干扰剂为核心的策略独具特色，有望为开发针对磷酸化蛋白质-蛋白质相互作用的定制化抑制剂提供可行前景。