Li Chan, Reddy Tummala R K, Fischer Peter M, Dekker Lodewijk V
School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, United Kingdom.
Assay Drug Dev Technol. 2010 Feb;8(1):85-95. doi: 10.1089/adt.2009.0218.
Protein-protein interactions are increasingly of interest as targets in small-molecule drug discovery. The interaction between the Ca2+- and phospholipid-binding protein Annexin A2 and its binding partner S100A10 has been implicated in angiogenesis and cancer metastasis. Here, we present a methodology to screen for inhibitors of this protein interaction. We developed a Cy5-labeled S100A10 tracer and showed by circular dichroism spectroscopy that the secondary structure is indistinguishable from that of non-labeled S100A10. This tracer was used to develop a binding assay based upon fluorescence resonance energy transfer to a Cy3-labeled Annexin A2 peptide ligand. The binding parameters matched those for unlabeled components as observed by equilibrium dialysis, which we determined separately, as well as those determined by isothermal titration calorimetry. Binding of labeled and unlabeled peptide was specific and mutually competitive. We used this assay for screening a small compound library derived by computational interrogation of the S100A10-binding pocket. Hits were obtained with IC(50) values in range of the IC(50) of the cognate Annexin A2 peptide ligand. Hits were subjected to an exact parallel assay measuring an unrelated protein-protein interaction (antigen-antibody). In this way, we identified genuine hits that inhibited the interaction between S100A10 and Annexin A2 but do not affect the fluorescence readout. These compounds are potentially of interest as candidates for further analysis and medical chemistry exploration. The simple assay format described here can be employed in early-stage exploration of other protein-protein interaction targets.
蛋白质-蛋白质相互作用作为小分子药物发现的靶点越来越受到关注。钙离子和磷脂结合蛋白膜联蛋白A2与其结合伴侣S100A10之间的相互作用与血管生成和癌症转移有关。在此，我们提出一种筛选该蛋白质相互作用抑制剂的方法。我们开发了一种Cy5标记的S100A10示踪剂，并通过圆二色光谱表明其二级结构与未标记的S100A10无法区分。该示踪剂用于开发一种基于荧光共振能量转移至Cy3标记的膜联蛋白A2肽配体的结合测定法。结合参数与通过平衡透析单独测定的未标记组分的参数相匹配，也与通过等温滴定量热法测定的参数相匹配。标记和未标记肽的结合具有特异性且相互竞争。我们使用该测定法筛选通过对S100A10结合口袋进行计算查询而衍生的小化合物库。获得的命中化合物的IC(50)值在同源膜联蛋白A2肽配体的IC(50)范围内。对命中化合物进行精确的平行测定，测量一种不相关的蛋白质-蛋白质相互作用（抗原-抗体）。通过这种方式，我们鉴定出真正抑制S100A10与膜联蛋白A2之间相互作用但不影响荧光读数的命中化合物。这些化合物作为进一步分析和药物化学探索的候选物可能具有研究价值。本文所述的简单测定形式可用于其他蛋白质-蛋白质相互作用靶点的早期探索。
