Intrinsically Disordered N-terminal Domain (NTD) of p53 Interacts with Mitochondrial PTP Regulator Cyclophilin D.

Mitochondrial permeability transition pore (mPTP) plays crucial roles in cell death in a variety of diseases, including ischemia/reperfusion injury in heart attack and stroke, neurodegenerative conditions, and cancer. To date, cyclophilin D is the only confirmed component of mPTP. Under stress, p53 can translocate into mitochondria and interact with CypD, triggering necrosis and cell growth arrest. However, the molecular details of p53/CypD interaction are still poorly understood. Previously, several studies reported that p53 interacts with CypD through its DNA-binding domain (DBD). However, using surface plasmon resonance (SPR), we found that both NTD-DBD, NTD and NTD (1-70) bind to CypD at ∼µM KD. In solution NMR, NTD binds CypD with µM affinity and mimics the pattern of FLp53 binding in chemical shift perturbation. In contrast, neither solution NMR nor fluorescence anisotropy detected DBD binding to CypD. Thus, instead of DBD, NTD is the major CypD binding site on p53. NMR titration and MD simulation revealed that NTD binds CypD with broad and dynamic interfaces dominated by electrostatic interactions. NTD 20-70 was further identified as the minimal binding region for CypD interaction, and two NTD fragments, D1 (residues 22-44) and D2 (58-70), can each bind CypD with mM affinity. Our detailed biophysical characterization of the dynamic interface between NTD and CypD provides novel insights on the p53-dependent mPTP opening and drug discovery targeting NTD/CypD interface in diseases.

Benzothiazolyl and Benzoxazolyl Hydrazones Function as Zinc Metallochaperones to Reactivate Mutant p53.

We identified a set of thiosemicarbazone (TSC) metal ion chelators that reactivate specific zinc-deficient p53 mutants using a mechanism called zinc metallochaperones (ZMCs) that restore zinc binding by shuttling zinc into cells. We defined biophysical and cellular assays necessary for structure-activity relationship studies using this mechanism. We investigated an alternative class of zinc scaffolds that differ from TSCs by substitution of the thiocarbamoyl moiety with benzothiazolyl, benzoxazolyl, and benzimidazolyl hydrazones. Members of this series bound zinc with similar affinity and functioned to reactivate mutant p53 comparable to the TSCs. Acute toxicity and efficacy assays in rodents demonstrated C1 to be significantly less toxic than the TSCs while demonstrating equivalent growth inhibition. We identified C85 as a ZMC with diminished copper binding that functions as a chemotherapy and radiation sensitizer. We conclude that the benzothiazolyl, benzoxazolyl, and benzimidazolyl hydrazones can function as ZMCs to reactivate mutant p53 in vitro and in vivo.

EGCG binds intrinsically disordered N-terminal domain of p53 and disrupts p53-MDM2 interaction.

Epigallocatechin gallate (EGCG) from green tea can induce apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Using SPR and NMR, here we report a direct, µM interaction between EGCG and the tumor suppressor p53 (KD = 1.6 ± 1.4 µM), with the disordered N-terminal domain (NTD) identified as the major binding site (KD = 4 ± 2 µM). Large scale atomistic simulations (>100 µs), SAXS and AUC demonstrate that EGCG-NTD interaction is dynamic and EGCG causes the emergence of a subpopulation of compact bound conformations. The EGCG-p53 interaction disrupts p53 interaction with its regulatory E3 ligase MDM2 and inhibits ubiquitination of p53 by MDM2 in an in vitro ubiquitination assay, likely stabilizing p53 for anti-tumor activity. Our work provides insights into the mechanisms for EGCG's anticancer activity and identifies p53 NTD as a target for cancer drug discovery through dynamic interactions with small molecules.

Zinc shapes the folding landscape of p53 and establishes a pathway for reactivating structurally diverse cancer mutants.

Missense mutations in the p53 DNA-binding domain (DBD) contribute to half of new cancer cases annually. Here we present a thermodynamic model that quantifies and links the major pathways by which mutations inactivate p53. We find that DBD possesses two unusual properties-one of the highest zinc affinities of any eukaryotic protein and extreme instability in the absence of zinc-which are predicted to poise p53 on the cusp of folding/unfolding in the cell, with a major determinant being available zinc concentration. We analyze the 20 most common tumorigenic p53 mutations and find that 80% impair zinc affinity, thermodynamic stability, or both. Biophysical, cell-based, and murine xenograft experiments demonstrate that a synthetic zinc metallochaperone rescues not only mutations that decrease zinc affinity, but also mutations that destabilize DBD without impairing zinc binding. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients annually in the U.S.

Combinatorial Therapy of Zinc Metallochaperones with Mutant p53 Reactivation and Diminished Copper Binding.

Chemotherapy and radiation are more effective in wild-type (WT) p53 tumors due to p53 activation. This is one rationale for developing drugs that reactivate mutant p53 to synergize with chemotherapy and radiation. Zinc metallochaperones (ZMC) are a new class of mutant p53 reactivators that restore WT structure and function to zinc-deficient p53 mutants. We hypothesized that the thiosemicarbazone, ZMC1, would synergize with chemotherapy and radiation. Surprisingly, this was not found. We explored the mechanism of this and found the reactive oxygen species (ROS) activity of ZMC1 negates the signal on p53 that is generated with chemotherapy and radiation. We hypothesized that a zinc scaffold generating less ROS would synergize with chemotherapy and radiation. The ROS effect of ZMC1 is generated by its chelation of redox active copper. ZMC1 copper binding (K Cu) studies reveal its affinity for copper is approximately 108 greater than Zn2+ We identified an alternative zinc scaffold (nitrilotriacetic acid) and synthesized derivatives to improve cell permeability. These compounds bind zinc in the same range as ZMC1 but bound copper much less avidly (106- to 107-fold lower) and induced less ROS. These compounds were synergistic with chemotherapy and radiation by inducing p53 signaling events on mutant p53. We explored other combinations with ZMC1 based on its mechanism of action and demonstrate that ZMC1 is synergistic with MDM2 antagonists, BCL2 antagonists, and molecules that deplete cellular reducing agents. We have identified an optimal Cu2+:Zn2+ binding ratio to facilitate development of ZMCs as chemotherapy and radiation sensitizers. Although ZMC1 is not synergistic with chemotherapy and radiation, it is synergistic with a number of other targeted agents.