Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan.
PLoS One. 2013 Aug 16;8(8):e71646. doi: 10.1371/journal.pone.0071646. eCollection 2013.
Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.
一些信号转导和转录激活因子（STAT）家族成员在各种人类肿瘤中持续激活。STAT 的活性取决于其Src 同源 2（SH2）结构域介导的与含有磷酸化酪氨酸的序列结合。因此，拮抗这种结合是抑制 STAT 激活的一种可行方法。我们基于放大发光接近均相分析（Alpha）技术开发了一种用于 STAT3 和 STAT5b-SH2 结合的新型多重分析。AlphaLISA 和 AlphaScreen 珠被组合在单个孔测定中，这使得能够同时监测 STAT3 和 STAT5b-SH2 与磷酸化酪氨酸肽的结合。生物素标记的重组人 STAT 蛋白作为 N-和 C-末端缺失突变体获得。优化了生物素化肽的 DIG 标记的间隔长度、反应时间和氯化钠浓度，以建立 STAT3 和 STAT5b-SH2 结合的 Z'值大于 0.6 的 HTS 系统。我们使用这种多重分析方法对化学文库进行了 HTS 筛选，并鉴定了命中化合物。一种 2-氯-1,4-萘二酮衍生物，化合物 1，优先在体外抑制 STAT3-SH2 结合，并且 HeLa 细胞中 STAT3 的核易位。使用多重分析进行的初步结构活性关系（SAR）研究表明，3-取代基对 STAT3 和 STAT5b 抑制的活性和选择性都有影响。因此，这种多重分析不仅可用于寻找潜在的先导化合物，还可用于获得开发新的 STAT3/STAT5b 抑制剂的 SAR 数据。
