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

ABSTRACT: p53 is a Zn(2+)-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 [Zn(2+)]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn(2+)-binding mutants by binding Zn(2+) and buffering it to a level such that Zn(2+) 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 [Zn(2+)]free by functioning as a Zn(2+) ionophore, binding Zn(2+) in the extracellular environment, diffusing across the plasma membrane, and releasing it intracellularly. It raises intracellular [Zn(2+)]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 [Zn(2+)]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.

SUBMITTER: Blanden AR

PROVIDER: S-EPMC4407733 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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

Blanden Adam R AR Yu Xin X Wolfe Aaron J AJ Gilleran John A JA Augeri David J DJ O'Dell Ryan S RS Olson Eric C EC Kimball S David SD Emge Thomas J TJ Movileanu Liviu L Carpizo Darren R DR Loh Stewart N SN

Molecular pharmacology 20150220 5

p53 is a Zn(2+)-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 [Zn(2+)]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn(2+)-binding mutants by binding Zn(2+) and buffering it to a level such that Zn(2+) can repopulate ...[more]

PMID: 25710967

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Project description:Globally, more antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance (AMR). The development of novel ionophores, a class of antimicrobials used exclusively in animals, holds promise as a strategy to replace or reduce essential human antimicrobials in veterinary practice. PBT2 is a zinc ionophore with recently demonstrated antibacterial activity against several Gram-positive pathogens, although the underlying mechanism of action is unknown. Here, we investigated the bactericidal mechanism of PBT2 in the bovine mastitis-causing pathogen, Streptococcus uberis In this work, we show that PBT2 functions as a Zn2+/H+ ionophore, exchanging extracellular zinc for intracellular protons in an electroneutral process that leads to cellular zinc accumulation. Zinc accumulation occurs concomitantly with manganese depletion and the production of reactive oxygen species (ROS). PBT2 inhibits the activity of the manganese-dependent superoxide dismutase, SodA, thereby impairing oxidative stress protection. We propose that PBT2-mediated intracellular zinc toxicity in S. uberis leads to lethality through multiple bactericidal mechanisms: the production of toxic ROS and the impairment of manganese-dependent antioxidant functions. Collectively, these data show that PBT2 represents a new class of antibacterial ionophores capable of targeting bacterial metal ion homeostasis and cellular redox balance. We propose that this novel and multitarget mechanism of PBT2 makes the development of cross-resistance to medically important antimicrobials unlikely.IMPORTANCE More antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance. Therefore, the elimination of antimicrobial crossover between human and veterinary medicine is of great interest. Unfortunately, the development of new antimicrobials is an expensive high-risk process fraught with difficulties. The repurposing of chemical agents provides a solution to this problem, and while many have not been originally developed as antimicrobials, they have been proven safe in clinical trials. PBT2, a zinc ionophore, is an experimental therapeutic that met safety criteria but failed efficacy checkpoints against both Alzheimer's and Huntington's diseases. It was recently found that PBT2 possessed potent antimicrobial activity, although the mechanism of bacterial cell death is unresolved. In this body of work, we show that PBT2 has multiple mechanisms of antimicrobial action, making the development of PBT2 resistance unlikely.

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Dataset Information

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

Publications

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

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