Resetca Diana, Haftchenary Sina, Gunning Patrick T, Wilson Derek J
From the Center for Research in Mass Spectrometry, Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada and.
Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada.
J Biol Chem. 2014 Nov 21;289(47):32538-47. doi: 10.1074/jbc.M114.595454. Epub 2014 Oct 6.
The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization.
转录因子信号转导子与转录激活子3（STAT3）的活性在多种血液系统恶性肿瘤和实体瘤中失调。开发药理学上的STAT3 Src同源2（SH2）结构域相互作用抑制剂在癌症治疗方面具有巨大潜力，并且最近已经开发出一类新型的基于水杨酸的STAT3二聚化抑制剂，其中包括口服生物利用度良好的候选药物。化合物SF-1-066和BP-1-102预计会与STAT3 SH2结构域结合。然而，鉴于SH2结构域高度无序和动态的性质，对这一预测进行实验证实颇具难度。我们通过时间分辨电喷雾电离氢-氘交换质谱法研究了STAT3与这些小分子抑制剂之间的蛋白质-配体相互作用。分析在生理条件下STAT3与SF-1-066或BP-1-102络合诱导的氘摄取的位点特异性演变，能够将计算机模拟预测的抑制剂结合位点定位到STAT3 SH2结构域。两种抑制剂与SH2结构域的结合均导致动力学显著局部降低，这与抑制剂结合位点的溶剂排斥以及抑制剂复合的SH2结构域刚性增加一致。有趣的是，抑制剂结合在SH2结构域之外诱导了变构扰动热点，主要表现为在对DNA结合和核定位重要的STAT3区域中氘摄取增加。
