Tor Vergata University of Rome, 00133 Rome, Italy.
University of Padova, 35131 Padova, Italy.
J Phys Chem Lett. 2024 Oct 10;15(40):10252-10257. doi: 10.1021/acs.jpclett.4c01767. Epub 2024 Oct 3.
Peptide-based drugs are powerful inhibitors of therapeutically relevant protein-protein interactions. Their affinity and selectivity for target proteins are commonly assessed using fluorescence-based assays such as anisotropy/polarization or quantitative microarrays. This study reveals that labeling can perturb peptide/protein binding by more than 1 order of magnitude. We have recently developed inhibitors targeted to the N-terminal Src homology 2 (SH2) domain of oncogenic phosphatase SHP2. Despite their high activity and selectivity, these molecules demonstrated an undesired interaction with the SH2 domain of another protein, known as APS, in a fluorescence microarray assay. Fluorescence anisotropy measurement in solution showed that the dissociation constant was significantly influenced by labeling (∼10 times), and the effect depended on the specific fluorophore and SH2 domain. Notably, displacement assays performed with unlabeled peptides were successfully used to eliminate these artifacts, demonstrating that the inhibitors' affinity for their target is over 1,000 times higher than for APS.
基于肽的药物是治疗相关蛋白-蛋白相互作用的有效抑制剂。它们对靶蛋白的亲和力和选择性通常使用基于荧光的测定法（如各向异性/偏振或定量微阵列）进行评估。本研究表明，标记可以使肽/蛋白质的结合受到超过 1 个数量级的干扰。我们最近开发了针对致癌磷酸酶 SHP2 的 N 端Src 同源 2（SH2）结构域的抑制剂。尽管这些分子具有高活性和选择性，但在荧光微阵列测定中，它们与另一种称为 APS 的蛋白质的 SH2 结构域表现出不理想的相互作用。在溶液中的荧光各向异性测量表明，标记显著影响了解离常数（约 10 倍），并且这种影响取决于特定的荧光团和 SH2 结构域。值得注意的是，使用未标记的肽进行的置换测定成功地消除了这些假象，表明抑制剂对其靶标的亲和力比 APS 高 1000 多倍。
