Teruya Kanae, Rankin Gregory M, Chrysanthopoulos Panagiotis K, Tonissen Kathryn F, Poulsen Sally-Ann
Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111, Australia.
School of Natural Sciences, Griffith University, Nathan, Queensland, 4111, Australia.
Chembiochem. 2017 Apr 18;18(8):739-754. doi: 10.1002/cbic.201600598. Epub 2017 Mar 22.
Chemical probes are small-molecule reagents used by researchers for labelling and detection of biomolecules. We present the design, synthesis, and characterisation of a panel of 11 structurally diverse photoaffinity labelling (PAL) probes as research tools for labelling the model enzyme carbonic anhydrase (CA) in challenging environments, including in protein mixtures and cell lysates. We targeted the ubiquitous CA II as well as the two cancer-associated CAs (CA IX and CA XII) that are of high priority as potential biomarkers of aggressive and/or multidrug-resistant cancer. We utilise an atypical biophysical approach, native state mass spectrometry, to monitor the initial protein-probe binding and subsequent UV crosslinking efficiency of the protein:probe complex. This mass spectrometry methodology represents a new approach for chemical probe optimisation and development that might have broader applications to chemical probe characterisation beyond this study. This also represents one of the first studies, to the best of our knowledge, in which a comprehensive set of PAL probes has been used to establish the relationship between probe structure, noncovalent protein-probe binding, and covalent protein-probe crosslinking efficiency. Our results demonstrate the benefits of a comprehensive analysis of chemical probe structure-activity relationships to support the development of optimum chemical probes.
化学探针是研究人员用于标记和检测生物分子的小分子试剂。我们展示了一组11种结构多样的光亲和标记（PAL）探针的设计、合成和表征，这些探针作为研究工具，用于在具有挑战性的环境中标记模型酶碳酸酐酶（CA），包括在蛋白质混合物和细胞裂解物中。我们的目标是普遍存在的CA II以及两种与癌症相关的CA（CA IX和CA XII），它们作为侵袭性和/或多药耐药性癌症潜在生物标志物具有高度优先性。我们采用一种非典型的生物物理方法——天然状态质谱法，来监测蛋白质 - 探针复合物的初始蛋白质 - 探针结合以及随后的紫外线交联效率。这种质谱方法代表了一种化学探针优化和开发的新方法，可能在本研究之外对化学探针表征有更广泛的应用。据我们所知，这也是首批研究之一，其中使用了一套全面的PAL探针来建立探针结构、非共价蛋白质 - 探针结合以及共价蛋白质 - 探针交联效率之间的关系。我们的结果证明了对化学探针构效关系进行全面分析以支持最佳化学探针开发的益处。
