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.

Chemotherapy and radiation are more effective in wild-type (WT) p53 tumors due to p53 activation. This is one rationale for developing drugs that reactivate mutant p53 to synergize with chemotherapy and radiation. Zinc metallochaperones (ZMC) are a new class of mutant p53 reactivators that restore WT structure and function to zinc-deficient p53 mutants. We hypothesized that the thiosemicarbazone, ZMC1, would synergize with chemotherapy and radiation. Surprisingly, this was not found. We explored the mechanism of this and found the reactive oxygen species (ROS) activity of ZMC1 negates the signal on p53 that is generated with chemotherapy and radiation. We hypothesized that a zinc scaffold generating less ROS would synergize with chemotherapy and radiation. The ROS effect of ZMC1 is generated by its chelation of redox active copper. ZMC1 copper binding (K Cu) studies reveal its affinity for copper is approximately 10 8 greater than Zn 2þ. We identified an alternative zinc scaffold (nitrilotriacetic acid) and synthesized derivatives to improve cell permeability. These compounds bind zinc in the same range as ZMC1 but bound copper much less avidly (10 6-to 10 7-fold lower) and induced less ROS. These compounds were synergistic with chemotherapy and radiation by inducing p53 signaling events on mutant p53. We explored other combinations with ZMC1 based on its mechanism of action and demonstrate that ZMC1 is synergistic with MDM2 antagonists, BCL2 antagonists, and molecules that deplete cellular reducing agents. We have identified an optimal Cu 2þ :Zn 2þ binding ratio to facilitate development of ZMCs as chemotherapy and radiation sensitizers. Although ZMC1 is not synergistic with chemotherapy and radiation, it is synergistic with a number of other targeted agents.