Synthetic metallochaperone ZMC1 rescues mutant p53 conformation by transporting zinc into cells as an ionophore

p53 is a Zn²⁺-dependent tumor suppressor inactivated in >50% of human cancers. The most common mutation, R175H, inactivates p53 by reducing its affinity for the essential zinc ion, leaving the mutant protein unable to bind the metal in the low [Zn²⁺]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn²⁺-binding mutants by binding Zn²⁺ and buffering it to a level such that Zn²⁺ can repopulate the defective binding site, but how it accomplishes this in the context of living cells and organisms is unclear. In this study, we demonstrated that ZMC1 increases intracellular [Zn²⁺]free by functioning as a Zn²⁺ ionophore, binding Zn²⁺ in the extracellular environment, diffusing across the plasma membrane, and releasing it intracellularly. It raises intracellular [Zn²⁺]free in cancer (TOV112D) and noncancer human embryonic kidney cell line 293 to 15.8 and 18.1 nM, respectively, with half-times of 2-3 minutes. These [Zn²⁺]free levels are predicted to result in ∼90% saturation of p53-R175H, thus accounting for its observed reactivation. This mechanism is supported by the X-ray crystal structure of the [Zn(ZMC1)₂] complex, which demonstrates structural and chemical features consistent with those of known metal ionophores. These findings provide a physical mechanism linking zinc metallochaperone-1 in both in vitro and in vivo activities and define the remaining critical parameter necessary for developing synthetic metallochaperones for clinical use.