Yu Xin, Blanden Adam, Tsang Ashley T, Zaman Saif, Liu Yue, Gilleran John, Bencivenga Anthony F, Kimball S David, Loh Stewart N, Carpizo Darren R
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.).
Rutgers Cancer Institute of New Jersey (X.Y., A.T.T., S.Z., Y.L., D.R.C.), Department of Surgery, Rutgers Robert Wood Johnson Medical School (X.Y., A.T.T, Y.L., D.R.C.), Rutgers Translational Sciences, Department of Chemistry and Chemical Biology (S.D.K.), Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy (J.G., A.F.B., S.D.K.), Rutgers University, New Brunswick, New Jersey; Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York (A.B., S.N.L.); and Mount Sinai St. Luke's Roosevelt General Surgery Residency Program, New York, New York (A.T.T.)
Mol Pharmacol. 2017 Jun;91(6):567-575. doi: 10.1124/mol.116.107409. Epub 2017 Mar 20.
Small-molecule restoration of wild-type structure and function to mutant p53 (so-called mutant reactivation) is a highly sought-after goal in cancer drug development. We previously discovered that small-molecule zinc chelators called (ZMCs) reactivate mutant p53 by restoring zinc binding to zinc-deficient p53 mutants. The lead compound identified from the NCI-60 human tumor cell lines screen, NSC319726 (ZMC1), belongs to the thiosemicarbazone (TSC) class of metal ion chelators that bind iron, copper, magnesium, zinc, and other transition metals. Here, we have investigated the other TSCs, NSC319725 and NSC328784, identified in the same screen, as well as the more well studied TSC, 3-AP (Triapine), to determine whether they function as ZMCs. We measured the zinc zinc ionophore activity, ability to restore zinc to purified p53 DNA binding domain (DBD), and ability to restore site-specific DNA binding to purified R175H-DBD in vitro. We tested all four TSCs in a number of cell-based assays to examine mutant p53 reactivation and the generation of reactive oxygen species (ROS). We found that NSC319725 and NSC328784 behave similarly to ZMC1 in both biophysical and cell-based assays and are heretofore named ZMC2 (NSC319725) and ZMC3 (NSC328784). 3-AP generates a ROS signal similar to ZMC1-3, but it fails to function as a ZMC both in vitro and in cells and ultimately does not reactivate p53. These findings indicate that not all TSCs function as ZMCs, and much of their activity can be predicted by their affinity for zinc.
在癌症药物研发中，将野生型结构和功能恢复至突变型p53（即所谓的突变体重激活）是一个备受追捧的目标。我们之前发现，一类名为锌螯合剂（ZMCs）的小分子通过恢复锌与锌缺乏型p53突变体的结合来重新激活突变型p53。从NCI - 60人类肿瘤细胞系筛选中鉴定出的先导化合物NSC319726（ZMC1），属于硫代氨基脲（TSC）类金属离子螯合剂，可结合铁、铜、镁、锌及其他过渡金属。在此，我们研究了在同一筛选中鉴定出的其他TSC，NSC319725和NSC328784，以及研究更为深入的TSC，3 - AP（曲阿替班），以确定它们是否具有ZMCs的功能。我们测量了锌离子载体活性、将锌恢复至纯化的p53 DNA结合结构域（DBD）的能力，以及在体外恢复对纯化的R175H - DBD的位点特异性DNA结合的能力。我们在一系列基于细胞的实验中测试了所有这四种TSC，以检测突变型p53的重新激活以及活性氧（ROS）的产生。我们发现，在生物物理和基于细胞的实验中，NSC319725和NSC328784的行为与ZMC1相似，因此在此将它们命名为ZMC2（NSC319725）和ZMC3（NSC328784）。3 - AP产生的ROS信号与ZMC1 - 3相似，但它在体外和细胞中均不能发挥ZMC的功能，最终也无法重新激活p53。这些发现表明，并非所有的TSC都具有ZMC的功能，并且它们的许多活性可以通过其对锌的亲和力来预测。
