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Zinc Metallochaperones Reactivate Mutant p53 Using an ON/OFF Switch Mechanism: A New Paradigm in Cancer Therapeutics.

ABSTRACT: Purpose: Zinc metallochaperones (ZMC) are a new class of anticancer drugs that reactivate zinc-deficient mutant p53 by raising and buffering intracellular zinc levels sufficiently to restore zinc binding. In vitro pharmacodynamics of ZMCs indicate that p53-mutant activity is ON by 4-6 hours and is OFF by 24. We sought to understand the mechanism of this regulation and to translate these findings preclinically. We further sought to innovate the formulation of ZMCs to improve efficacy.Experimental Design: We performed in vitro mechanistic studies to determine the role of cellular zinc homeostatic mechanisms in the transient pharmacodynamics of ZMCs. We conducted preclinical pharmacokinetic, pharmacodynamic, and efficacy studies using a genetically engineered murine pancreatic cancer model (KPC) to translate these mechanistic findings and investigate a novel ZMC formulation.Results:In vitro, cellular zinc homeostatic mechanisms that restore zinc to its physiologic levels function as the OFF switch in ZMC pharmacodynamics. In vivo pharmacokinetic studies indicate that ZMCs have a short half-life (< 30 minutes), which is sufficient to significantly improve survival in mice expressing a zinc-deficient allele (p53R172H) while having no effect in mice expressing a non-zinc-deficient allele (p53R270H). We synthesized a novel formulation of the drug in complex with zinc and demonstrate this significantly improves survival over ZMC1.Conclusions: Cellular zinc homeostatic mechanisms function as an OFF switch in ZMC pharmacodynamics, indicating that a brief period of p53-mutant reactivation is sufficient for on-target efficacy. ZMCs synthesized in complex with zinc are an improved formulation. Clin Cancer Res; 24(18); 4505-17. ©2018 AACR.

SUBMITTER: Yu X

PROVIDER: S-EPMC6139040 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Zinc Metallochaperones Reactivate Mutant p53 Using an ON/OFF Switch Mechanism: A New Paradigm in Cancer Therapeutics.

Yu Xin X Kogan Samuel S Chen Ying Y Tsang Ashley T AT Withers Tracy T Lin Hongxia H Gilleran John J Buckley Brian B Moore Dirk D Bertino Joseph J Chan Chang C Kimball S David SD Loh Stewart N SN Carpizo Darren R DR

Clinical cancer research : an official journal of the American Association for Cancer Research 20180618 18

<b>Purpose:</b> Zinc metallochaperones (ZMC) are a new class of anticancer drugs that reactivate zinc-deficient mutant p53 by raising and buffering intracellular zinc levels sufficiently to restore zinc binding. <i>In vitro</i> pharmacodynamics of ZMCs indicate that p53-mutant activity is ON by 4-6 hours and is OFF by 24. We sought to understand the mechanism of this regulation and to translate these findings preclinically. We further sought to innovate the formulation of ZMCs to improve efficac ...[more]

PMID: 29914895

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Project description:The tumor suppressor protein p53 induces apoptosis, cell cycle arrest, and DNA repair along with other functions in a transcription-dependent manner [Vousden, K. H. Cell 2000, 103(5), 691-694]. The selection of these functions depends on sequence-specific recognition of p53 to a target decameric sequence of gene promoters [Kitayner, M.; et al. Mol. Cell 2006, 22(6), 741-753]. Amino acid residues in p53 that directly bind to DNA were analyzed, and the replacement of A276 in p53 with selected amino acids elucidated its importance in promoter transcription. For most apoptotic and cell cycle gene promoters, position 9 of the target decameric sequence is a cytosine, while for DNA repair gene promoters, thymine is found instead. Therefore, selective binding to the cytosine at the ninth position may transcribe apoptotic gene promoters and thus can induce apoptosis and cell cycle arrest. Molecular modeling with PyMOL indicated that substitution of a hydrophilic residue, A276S, would prefer binding to cytosine at the ninth position of the target decameric sequence, whereas substitution of a hydrophobic residue (A276F) would fail to do so. Correspondingly, A276S demonstrated higher transcription of PUMA, PERP, and p21(WAF1/CIP1)gene promoters containing a cytosine at the ninth position and lower transcription of GADD45 gene promoter containing a thymine at the ninth position compared to wild-type p53. Cell cycle analysis showed that A276S maintained similar G1/G0 phase arrest as wild-type p53. Additionally, A276S induced higher apoptosis than wild-type p53 as measured by DNA segmentation and 7-AAD assay. Since the status of endogenous p53 can influence the activity of the exogenous p53, we examined the activity of A276S in HeLa cells (wild-type endogenous p53) in addition to T47D cells (mutated and mislocalized endogenous p53). The same apoptotic trend in both cell lines suggested A276S can induce cell death regardless of endogenous p53 status. Cell proliferation assay depicted that A276S efficiently reduced the viability of T47D cells more than wild-type p53 over time. We conclude that the predicted preferred binding of A276S to cytosine at the ninth position better transactivates a number of apoptotic gene promoters. Higher induction apoptosis than wild-type p53 makes A276S an attractive candidate for therapy to eradicate cancer.

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Zinc Metallochaperones Reactivate Mutant p53 Using an ON/OFF Switch Mechanism: A New Paradigm in Cancer Therapeutics.

Publications

Zinc Metallochaperones Reactivate Mutant p53 Using an ON/OFF Switch Mechanism: A New Paradigm in Cancer Therapeutics.

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