Astex Pharmaceuticals , 436 Cambridge Science Park , Cambridge CB4 0QA , United Kingdom.
Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School , Newcastle University , Framlington Place , Newcastle-upon-Tyne NE2 4HH , United Kingdom.
Bioconjug Chem. 2018 Jun 20;29(6):2100-2106. doi: 10.1021/acs.bioconjchem.8b00315. Epub 2018 Jun 12.
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.
MDM2 是 p53 肿瘤抑制因子的关键负调控因子。MDM2 与 p53 的直接结合抑制了该蛋白的转录活性，并诱导其多泛素化，使其成为蛋白酶体降解的目标。因此，拮抗 MDM2-p53 结合的小分子抑制剂，如 RG7388，已进入临床开发阶段，旨在恢复 TP53 野生型肿瘤中 p53 的功能。在这里，我们描述了 RG7388 的反式环辛烯标记衍生物 RG7388-TCO 的设计、合成和生物学评价，该化合物对 MDM2 具有高细胞效力和特异性。RG7388-TCO 与四嗪标记的 BODIPY 染料的细胞内反应使我们能够对 SJSA-1 和 T778 肿瘤细胞中的内源性 MDM2 进行荧光成像。RG7388-TCO 还可通过与 SJSA-1 裂解物中的四嗪标记琼脂糖珠反应来下拉 MDM2。所呈现的数据表明，RG733-TCO 能够精确地对细胞中的 MDM2 进行成像，并可用于体外评估靶标占有率和 MDM2-p53 拮抗作用。
