Zinc Metallochaperones as Mutant p53 Reactivators: A New Paradigm in Cancer Therapeutics

Abstract

Restoration of wild-type structure and function to mutant p53 with a small molecule (hereafter referred to as "reactivating" mutant p53) is one of the holy grails in cancer therapeutics. The majority of TP53 mutations are missense which generate a defective protein that is targetable. We are currently developing a new class of mutant p53 reactivators called zinc metallochaperones (ZMCs) and, here, we review our current understanding of them. The p53 protein requires the binding of a single zinc ion, coordinated by four amino acids in the DNA binding domain, for proper structure and function. Loss of the wild-type structure by impairing zinc binding is a common mechanism of inactivating p53. ZMCs reactivate mutant p53 using a novel two-part mechanism that involves restoring the wild-type structure by reestablishing zinc binding and activating p53 through post-translational modifications induced by cellular reactive oxygen species (ROS). The former causes a wild-type conformation change, the later induces a p53-mediated apoptotic program to kill the cancer cell. ZMCs are small molecule metal ion chelators that bind zinc and other divalent metal ions strong enough to remove zinc from serum albumin, but weak enough to donate it to mutant p53. Recently we have extended our understanding of the mechanism of ZMCs to the role of cells' response to this zinc surge. We found that cellular zinc homeostatic mechanisms, which normally function to maintain free intracellular zinc levels in the picomolar range, are induced by ZMCs. By normalizing zinc levels, they function as an OFF switch to ZMCs because zinc levels are no longer sufficiently high to maintain a wild-type structure. This on/off switch leads to a transient nature to the mechanism of ZMCs in which mutant p53 activity comes on in a few hours and then is turned off. This finding has important implications for the translation of ZMCs to the clinic because it indicates that ZMC concentrations need not be maintained at high levels for their activity. Indeed, we found that short exposures (as little as 15 min) were adequate to observe the mutant p53 reactivating activity. This switch mechanism imparts an advantage over other targeted therapeutics in that efficacy can be accomplished with minimal exposure which minimizes toxicity and maximizes the therapeutic window. This on/off switch mechanism is unique in targeted cancer therapeutics and will impact the design of human clinical trials.

Keywords: mutant p53; on/off switch mechanism; pancreatic cancer; zinc homeostasis; zinc metallochaperones.

Figure 1

Model of the ZMC1 dual mechanism. (a) The p53^R175H is misfolded owing to impaired zinc binding (apo form) and, as a result, no site-specific DNA binding occurs (and no transcription of p53 targets). ZMC1 functions as a zinc metallochaperone (ZMC) by providing a source of zinc to facilitate refolding of the p53^R175H to a WT-like structure (holo form). (b) ZMC1 boosts reactive oxygen species (ROS) levels, which activates a stress response (i.e., ATM) that transactivates the p53^R175H through amino-terminal phosphorylation (P) and acetylation (A) events. (c) The newly conformed and transactivated p53^R175H can now bind DNA in a site-specific manner, causing transcription of apoptotic effectors that leads to tumor cell death. K_d1 = K_d of the native binding site, K_d2 = K_d of the non-native binding site.

Figure 2

The ZMC Switch mechanism. (A) At physiologic zinc levels (10⁻⁹ M), p53^R175H is in its Apo (zinc free) state because the mutation weakens its binding at the native ligation site (p53^R175H Zn K_d2 × 10⁻⁶). This causes the protein to misfold and lose its wild-type transcriptional function. (B) Upon treatment with a ZMC, intracellular zinc levels increase approximately 1000 fold (1.5 × 10⁻⁵ M), and this allows zinc to bind in the p53^R175H native ligation site and the protein adopts a wild-type conformation (on switch). (C) Once the protein undergoes a wild-type conformation change, the p53^R175H exhibits wild-type transcriptional activity and an apoptotic mechanism is induced. (D) Within hours the cell responds to this zinc surge by activating zinc homeostatic mechanisms (increasing expression of metallothioneins, zinc exporters, decreasing the expression of zinc importers) that function to lower cellular zinc levels to physiologic range. Zinc is no longer at a sufficient concentration to remain bound in the p53R175H and the protein returns to its Apo state (off switch).