Small molecule restoration of wildtype structure and function of mutant p53 using a novel zinc-metallochaperone based mechanism

NSC319726 (ZMC1) is a small molecule that reactivates mutant p53 by restoration of WT structure/function to the most common p53 missense mutant (p53-R175H). We investigated the mechanism by which ZMC1 reactivates p53-R175H and provide evidence that ZMC1: 1) restores WT structure by functioning as a zinc-metallochaperone, providing an optimal concentration of zinc to facilitate proper folding; and 2) increases cellular reactive oxygen species that transactivate the newly conformed p53-R175H (via post-translational modifications), inducing an apoptotic program. We not only demonstrate that this zinc metallochaperone function is possessed by other zinc-binding small molecules, but that it can reactivate other p53 mutants with impaired zinc binding. This represents a novel mechanism for an anti-cancer drug and a new pathway to drug mutant p53. Significance: We have elucidated a novel mechanism to restore wild-type structure/function to mutant p53 using small molecules functioning as zinc-metallochaperones. The pharmacologic delivery of a metal ion to restore proper folding of a mutant protein is unique to medicinal chemistry and represents a new pathway to drug mutant p53.

Fluorescence measurements were taken in a 1 cm by 1 cm quartz cuvette with excitation and emission slits of 3 nm and 4 nm respectively. All experiments were conducted at 10 ˚C unless otherwise noted.

ZMC1 -Zn 2+ binding stoichiometry titration
ZnCl 2 was titrated into a solution of 10 μM ZMC1 at room temperature (RT) and the absorbance at 370 nm monitored. The absorbance values were corrected for dilution, and the rise and plateau phases of the curve were fitted with lines. The point of intersection was used to determine the saturating stoichiometry of Zn 2+ to ZMC1.

Competition binding
Freshly purified R175H (5 μM), which co-purified with 0.6 equivalents of bound Zn 2+ , was incubated with increasing concentrations of 4-(2-pyridylazo)resorcinol (PAR, Sigma-Aldrich) and left to equilibrate at 10 ˚C for 1 h. The concentration of PAR 2 Zn complex was then measured by absorbance at 500 nm. Samples were blanked against equivalent PAR solutions without protein added. No additional Zn 2+ was released from the protein when denatured in 6 M guanidine-hydrochloride (Gdn-HCl), so we concluded that all available Zn 2+ was accounted for in our competition binding curve. The K d was determined by solving the following mass action and mass conservation equations in parallel as described [3], followed by least squares minimization using a custom script written in MATLAB R2010a (MathWorks): (

Equilibrium Dialysis
Equal volumes of DBD at ~20 μM and a mixture of 20 μM/10μM ZMC1 and ZnCl 2 were loaded onto either side of an acrylic microdialysis chamber separated by a piece of 8000 MWCO dialysis tubing (BioDesignInc). The membrane was pretreated by boiling in 2% sodium bicarbonate buffer and 1 mM EDTA for 10 min to remove any excess Zn 2+ and rinsed thoroughly. Samples were shaken vigorously for 24 hrs, and the concentration of ZMC1 2 Zn complex on each side of the membrane was determined by absorbance at 370 nm using ϵ 370 = 1.4 x 10 4 M -1 cm -1 (where each concentration unit represents 1/2 of a ZMC1 2 Zn complex) as determined by our lab.

Arrested refolding
Arrested refolding experiments were performed as described previously with minor modifications [5]. DBD (25 μM) was denatured in buffer containing 5 M urea, and then rapidly diluted 50-fold into urea-free buffer with or without 2. Zn 2+ . The reaction was quenched with 10 mM β-ME for 10 min, and Zn 2+ concentration measured by PAR assay using ϵ 500 = 5.0 x 10 4 for the PAR 2 Zn complex in buffer containing 6 M Gdn-HCl as determined by our lab. Measured Zn 2+ concentration was divided by protein concentration to determine Zn 2+ content of the protein.

Synthesis of compound A6
Azetidine (85 mg, 1.5 mmol) was dissolved in 5 mL methylene chloride and diisopropylethylamine (195 mg, 1.50 mmol, 1.0 equiv.) was added followed by triphosgene (267 mg, 0.9 mmol, 0.6 equiv.) and the reaction mixture stirred at ambient temperature for 4 hrs to Where ΔA max is the absorbance change at saturating ZnCl 2 , [D] 1/2 the total concentration of A6 (as required by the 2 to 1 binding mechanism), [L] is the concentration of ZnCl 2 , and K d is the dissociation constant for drug and ligand.

Stopped-Flow Kinetics
Rapid kinetic measurements were carried out on a Bio-Logic SFM4-Q/S stopped-flow device.
The mixing dead-time was ~2 ms. Absorbance was observed at 370 nm with a 2 nm bandpass. was used to compete the binding. Reaction mixture was incubated at 4 C for 10 min prior to addition of biotin labeled probes. Binding buffer was described before [6]. DNA binding reactions were carried out at 4 C for 20 min, and resulting DNA-protein complexes were separated on a 6% polyacrylamide gel (made with 30% Acrylamide/Bis Solution (37.5:1) (Bio-Rad)) in 0.5× Tris-borate (without EDTA), running in 0.5× Tris-borate (without EDTA) buffer.

Complexes were visualized by LightShift Chemiluminescent EMSA Kit (Thermo Fisher
Scientific/Pierce). NTA and EDTA were from Sigma.

Viability assay
Viability assays were done according to the manufacture instruction of Guava ViaCount (Millipore). In brief, the cells (5x10 4 cells/well) were cultured in a 12-well plate to reach the 50-60% confluence on the second day when treated with serial dilutions of the compound. Cell growthwas measured by Guava ViaCount reagent and Guava PCA instrument after incubation for 3 days.

Immunofluorescent staining
The cells were grown on the coverslip, followed by various treatments. The coverslips were fixed with 4% paraformaldehyde for 10 min and then permealized with 0.5% Triton-X100 for 5 min. The conformation of the mutant and WT p53 proteins were recognized specifically by the antibodies PAB1620 (1:50, recognizing WT conformation) and PAB240 (1:200, recognizing mutant conformation) stained overnight, respectively. The secondary antibody, goat anti-mouse IgG was incubated for 40 min. PAB1620 and PAB240 were from EMD Chemicals.

Immunoprecipitation (IP)
The cell lysates (500 g) with various treatments were subjected to immunoprecipitation with ImmunoCruz Optima Immunoprecipitation reagent (Santa Cruz Biotechnology) using the antibody PAB240 (4 g). The pulldown was detected by western blot with p53 (FL393) (Santa Cruz Biotechnology) that recognizes all formats of the p53 protein. The density of the image relative to the Control was determined using Adobe Photoshop.

RNA extraction and quantitative RT-PCR
RNA was extracted from the cells using RNeasy kit (Qiagen) and the gene expression level was measured by quantitative RT-PCR using TaqMan gene expression assays (Life Technologies/Applied BioSystems). The gene expression level was normalized with -actin and the average was presented with standard deviation from triplicates of repeated experiments.

Western blot
The lysates (or immunoprecipitated products) were run on SDS-PAGE and transferred onto PVDF membranes. The detection of the protein level was done with the manufacture instruction (Western Lightning® Plus-ECL, Perkin-Elmer). The p21 antibody was from EMD Chemicals.
The actin antibody and the p53 (DO-1 and FL393) were from Santa Cruz Biotechnology.
The density in IP was determined using Adobe Photoshop and was expressed as the ratio of IP band to lysate band then relative to the control (no treatment).