Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94158, United States.
J Am Chem Soc. 2012 Dec 19;134(50):20214-7. doi: 10.1021/ja310659j. Epub 2012 Dec 4.
Src-family tyrosine kinases play pivotal roles in human physiology and disease, and several drugs that target members of this family are in clinical use. None of these drugs appear to discriminate among closely related kinases. However, assessing their selectivity toward endogenous kinases in living cells remains a significant challenge. Here, we report the design of two Src-directed chemical probes, each consisting of a nucleoside scaffold with a 5'-electrophile. A 5'-fluorosulfonylbenzoate (1) reacts with the conserved catalytic lysine (Lys295) and shows little discrimination among related kinases. By contrast, a 5'-vinylsulfonate (2) reacts with a poorly conserved, proximal cysteine (Cys277) found in three Src-family and six unrelated kinases. Both 1 and 2 bear an alkyne tag and efficiently label their respective endogenous kinase targets in intact cells. Using 1 as a competitive probe, we determined the extent to which ponatinib, a clinical Bcr-Abl inhibitor, targets Src-family kinases. Remarkably, while ponatinib had little effect on endogenous Fyn or Src, it potently blocked the critical T-cell kinase, Lck. Probes 1 and 2 thus enable competitive profiling versus distinct kinase subsets in living cells.
Src 家族酪氨酸激酶在人类生理和疾病中发挥着关键作用,几种针对该家族成员的药物已在临床应用。这些药物似乎都不能区分密切相关的激酶。然而,评估它们在活细胞中对内源性激酶的选择性仍然是一个重大挑战。在这里,我们报告了两种 Src 定向化学探针的设计,每个探针都由一个带有 5'-亲电体的核苷支架组成。5'-氟磺酰苯酯 (1) 与保守的催化赖氨酸 (Lys295) 反应,对相关激酶的选择性很小。相比之下,5'-乙烯砜 (2) 与三个 Src 家族和六个不相关激酶中发现的一个保守性差的近位半胱氨酸 (Cys277) 反应。1 和 2 都带有炔基标记,并在完整细胞中有效地标记其各自的内源性激酶靶标。我们使用 1 作为竞争性探针,确定了临床 Bcr-Abl 抑制剂 ponatinib 靶向 Src 家族激酶的程度。值得注意的是,虽然 ponatinib 对内源性 Fyn 或 Src 几乎没有影响,但它强烈抑制了关键的 T 细胞激酶 Lck。探针 1 和 2 因此能够在活细胞中针对不同的激酶亚群进行竞争性分析。