Thiosemicarbazones Functioning as Zinc Metallochaperones to Reactivate Mutant p53

Abstract

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.

Multifunctional Compounds for Activation of the p53‐Y220C Mutant in Cancer

“…Mutations to p53C can disrupt protein stability and/or cause loss of Zn . Consequently, the discovery of small molecule Zn chaperones aimed to restore wild‐type function in mutant p53 has generated much attention . For example, the thiosemicarbazone ligand ZMC1 (Scheme b) induces conformational “wild‐type‐like” change in the common Zn‐binding p53 mutation (R175 H), and restores p53 transactivation function .…”

“…A series of ligands ( L1 ‐ L5 ) that are designed to restore wild‐type function in p53‐Y220C were synthesized and characterized (Scheme ). The ligand series was designed with two motifs in mind: (1) a p53‐Y220C binding diiodophenol core, and (2) Zn‐binding groups to promote metallochaperone activity . Metal‐binding groups were installed at the 2‐position, following rationales for related p53‐binding molecules .…”

“…This value derives from the low intracellular levels of free zinc, and the fact that under physiological conditions, WTp53 is predominantly in the holo (zinc‐bound) form . Metallochaperones designed to rescue zinc‐binding in p53 mutants should therefore have Zn 2+ affinities that are less than that of the native site ( K d1 ), yet higher than that of non‐native sites ( K d2 ), estimated to be about 10 −6 m for WTp53 . The p53‐Y220C mutant is prone to the loss of Zn 2+ due to local unfolding and increased aggregation, so the exact value of K d1 is unknown.…”

“…Given the high level of in vitro cytotoxicity of L5 on both p53‐Y220C and WTp53 cell lines, it likely that the activity of L5 is due to both p53‐dependent and p53‐independent pathways. Indeed, recent studies on p53 activating scaffolds show that increased ROS generation is an important component of the observed cytotoxicity, in addition to p53 activation . In the case of metal‐binding agents, ROS‐associated toxicity could be due to the in situ formation of redox‐active Cu complexes .…”

“…Further studies indicate that subtle tuning of the Zn‐binding affinity of the metallochaperones is critical for p53 reactivation, as it functions by repopulating Zn‐deficient p53C with Zn 2+ . More broadly, targeted metal ion chelation and redistribution has been shown as a promising anti‐cancer strategy, and a number of recent studies have highlighted both the novelty and complexity of this approach …”