MDM2 is a key negative regulator of the p53 tumor suppressor. Direct binding of MDM2 to p53 represses the protein’s transcriptional activity and induces its polyubiquitination, targeting it for degradation by the proteasome. Consequently, small molecule inhibitors that antagonize MDM2-p53 binding, such as RG7388, have progressed into clinical development aiming to reactivate p53 function in TP53 wild-type tumors. Here, we describe the design, synthesis, and biological evaluation of a trans-cyclooctene tagged derivative of RG7388, RG7388-TCO, which showed high cellular potency and specificity for MDM2. The in-cell reaction of RG7388-TCO with a tetrazine-tagged BODIPY dye enabled fluorescence imaging of endogenous MDM2 in SJSA-1 and T778 tumor cells. RG7388-TCO was also used to pull down MDM2 by reaction with tetrazine-tagged agarose beads in SJSA-1 lysates. The data presented show that RG733-TCO enables precise imaging of MDM2 in cells and can permit a relative assessment of target engagement and MDM2-p53 antagonism in vitro.

中文翻译：

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用于MDM2细胞成像和评估对MDM2-p53抑制的动态响应的高效可点击探针

MDM2是p53肿瘤抑制因子的关键负调控因子。MDM2与p53的直接结合会抑制蛋白质的转录活性，并诱导其多聚泛素化，将其靶向以被蛋白酶体降解。因此，对抗MDM2-p53结合的小分子抑制剂，例如RG7388，已进入临床研究，旨在重新激活TP53中的p53功能。野生型肿瘤。在这里，我们描述了RG7388，RG7388-TCO的反式环辛烯标记衍生物的设计，合成和生物学评估，该衍生物对MDM2具有很高的细胞效力和特异性。RG7388-TCO与四嗪标记的BODIPY染料的细胞内反应使SJSA-1和T778肿瘤细胞中内源性MDM2的荧光成像成为可能。RG7388-TCO还用于通过与SJSA-1裂解物中的四嗪标记的琼脂糖珠反应来拉低MDM2。所提供的数据表明，RG733-TCO能够对细胞中的MDM2进行精确成像，并可以在体外相对评估靶标参与度和MDM2-p53拮抗作用。