Department of Chemistry, National University of Singapore , 3 Science Drive 3, Singapore 117543.
J Am Chem Soc. 2014 Jul 16;136(28):9990-8. doi: 10.1021/ja502780z. Epub 2014 Jul 3.
Target identification of bioactive compounds within the native cellular environment is important in biomedical research and drug discovery, but it has traditionally been carried out in vitro. Information about how such molecules interact with their endogenous targets (on and off) is currently highly limited. An ideal strategy would be one that recapitulates protein-small molecule interactions in situ (e.g., in living cells) and at the same time enables enrichment of these complexes for subsequent proteome-wide target identification. Similarly, small molecule-based imaging approaches are becoming increasingly available for in situ monitoring of a variety of proteins including enzymes. Chemical proteomic strategies for simultaneous bioimaging and target identification of noncovalent bioactive compounds in live mammalian cells, however, are currently not available. This is due to a lack of photoaffinity labels that are minimally modified from their parental compounds, yet chemically tractable using copper-free bioorthogonal chemistry. We have herein developed novel minimalist linkers containing both an alkyl diazirine and a cyclopropene. We have shown chemical probes (e.g., BD-2) made from such linkers could be used for simultaneous in situ imaging and covalent labeling of endogenous BRD-4 (an important epigenetic protein) via a rapid, copper-free, tetrazine-cyclopropene ligation reaction (k2 > 5 M(-1) s(-1)). The key features of our cyclopropenes, with their unique C-1 linkage to BRD-4-targeting moiety, are their tunable reactivity and solubility, relative stability, and synthetic accessibility. BD-2, which is a linker-modified analogue of (+)-JQ1 (a recently discovered nanomolar protein-protein-interaction inhibitor of BRD-4), was subsequently used in a cell-based proteome profiling experiment for large-scale identification of potential off-targets of (+)-JQ1. Several newly identified targets were subsequently confirmed by preliminary validation experiments.
在生物医学研究和药物发现中，鉴定天然细胞环境中生物活性化合物的靶标非常重要，但传统上是在体外进行的。目前，关于这些分子如何与内源性靶标（开/关）相互作用的信息非常有限。理想的策略应该是一种能够在原位（例如，在活细胞中）重现蛋白质-小分子相互作用的策略，同时能够富集这些复合物，以便随后进行蛋白质组范围内的靶标鉴定。同样，基于小分子的成像方法越来越多地用于原位监测各种蛋白质，包括酶。然而，目前还没有用于同时在活哺乳动物细胞中对非共价生物活性化合物进行生物成像和靶标鉴定的化学蛋白质组学策略。这是由于缺乏经过最小修饰的光亲和标签，使其与其母体化合物保持最小差异，但仍可使用无铜生物正交化学进行化学处理。我们在此开发了含有烷基重氮和环丙烯的新型最小化接头。我们已经表明，由这种接头制成的化学探针（例如 BD-2）可用于通过快速、无铜、四嗪-环丙烯连接反应（k2 > 5 M(-1) s(-1)）对内源性 BRD-4（一种重要的表观遗传蛋白）进行原位成像和共价标记。我们的环丙烯的关键特点是其独特的 C-1 与 BRD-4 靶向部分的连接，具有可调反应性和溶解性、相对稳定性和合成可及性。BD-2 是（+）-JQ1（一种最近发现的 BRD-4 纳米摩尔蛋白-蛋白相互作用抑制剂）的接头修饰类似物，随后用于基于细胞的蛋白质组谱分析实验，以大规模鉴定（+）-JQ1 的潜在脱靶。随后通过初步验证实验证实了几个新鉴定的靶标。
