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

  • ️Wed Jan 01 2014

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

Xin Yu et al. Oncotarget. 2014.

Abstract

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.

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Figures

Figure 1
Figure 1. Binding interactions of ZMC1, DBD, and zinc

A, titration of ZMC1 and ZnCl2. Extrapolation of the linear phases shows 0.51 ± 0.01 equivalents of Zn2+ at saturation. Inset shows normalized absorbance spectra for ZMC1 alone (black) and ZMC1-Zinc complex (red). B, measurement of ZMC1-zinc complex dissociation rate. Solutions of ZnCl2 and ZMC1 were mixed 1:1 with 2 mM EDTA to the final concentrations indicated (written ZnCl2/ZMC1). The traces are an average of 3-4 injections and when fit to a single exponential, yield nearly identical rates, which we combine to report a koff of 2.6 ± 0.1 s−1. C, structures of ZMC1 and A6 and their Kd's for Zn2+. D, determination of the Kd of R175H DBD for Zn2+.R175H DBD (5 μM, 0.6 equivalents Zn2+ co-purified) was incubated with increasing concentrations of PAR to compete for the available Zn2+. The concentration of PAR2Zn2+ complex was determined by absorbance, and the parallel mass action and mass conservation equations solved to determine Kd. Kd's from the 2-site model are 2.1 ± 0.8 nM for the tighter site (Kd1) and below the detectable limit for the weaker site (Kd2), respectively.

Figure 2
Figure 2. ZMC1 functions as a Zn-metallochaperone

A, Trp fluorescence used to measure the fraction of R175H and WT DBD saved from Zn2+-arrested refolding. Values are normalized to no treatment and no ZnCl2 controls. Protein was unfolded in 5 M urea, refolded by rapid dilution in the presence of 2.5 μM ZnCl2, then rescued by the indicated treatment. Representative traces available in Supplementary Fig. S2. Error bars are ± SD. B, remetallationof WT and R175H DBD by Zn2+binding compounds. Apo DBD was incubated with ZnCl2 and either EDTA, ZMC1, NTA. The Zn2+ content of the resultant proteins was measured by PAR assay. ZMC1 and NTA can restore Zn2+ to pre-apoization levels. Error bars are ± SD. C, R175H and WT DBD remetallated by ZMC1 and NTA are native by Trp fluorescence. All spectra are of 0.5 μM protein desalted into buffer after the indicated treatment to remove any excess drug or zinc. Gray dashed lines are basis spectra from 0.5 μM WT DBD either freshly purified (native), unfolded in 8 M urea (unfolded), or misfolded with 5 μM ZnCl2 (misfolded). All spectra are similar to native DBD, with the exception of R175H remetallated with NTA, which is suggestive of a small amount of misfolding. D, quantification of Zn2+ transferred from ZMC1 to apo DBDs. The stoichiometries are 1.0 ± 0.1 equivalents (WT) and 0.5 ± 0.1 (R175H). Error bars are ± SD. E, electrophoretic mobility shift assay (EMSA) using WT, R175H and R248W DBD. The WT DBD is used as a positive control (Lane 9). Only the combination of ZMC1 (20μM) and ZnCl2 (10μM) restores site-specific DNA binding to the R175H DBD (Lane 5). The protein-DNA complex is specific because the unlabeled DNA competes for binding (Lane 10). F, EMSA demonstrating the importance of the stoichiometetry of ZMC1:Zn2+for the restoration of DNA binding. G, cell viability assay using serial dilutions of ZMC1. At 10 μM ZMC1, 25 μM ZnCl2 was supplemented. The ZMC1:Zn2+stoichiometric relationship applies to the pharmacodynamics of ZMC1 in p53-R175H cells. H, EMSA demonstrating that remetallation of the apo R175H DBD can occur in the absence of ZMC1. Free zinc of 2 nM to 10 μM and 20 μM ZMC1 were added to the indicated reactions.

Figure 3
Figure 3. The zinc metallochaperone function is not unique to ZMC1

A, structure of NTA and its Kd for Zn2+. B, EMSA indicating R175H DBD binds to DNA in the presence of NTA and zinc with the same trend as ZMC1, but not with NTA alone. R175H DBD with ZMC1, zinc and WT DBD are used as positive controls. C, NTA arrested refolding traces. Experiments were run as in Figure S3 but with the indicated concentrations of NTA. NTA was effective at rescuing zinc induced misfolding, but less so than EDTA or ZMC1. D, immunoprecipitation (IP) of p53 protein from R175H cells after treatment of ZMC1 (1 μM) or NTA (5 mM) with the mutant specific PAB240 antibody. The density of western blot bands from IP and lysates were calculated and normalized to no treatment control. E, immunocytochemistry fluorescent staining (IF) of p53 protein from R175H cells after treatment of ZMC1 (1 μM), NTA (5 mM) or EDTA (500 μM or 1 mM). The antibody PAB1620 recognizes WT conformation of p53. Scale bar = 25 μm.

Figure 4
Figure 4. ZMC1 reactivates additional p53 missense mutants with impaired zinc binding

A, ZMC1 inhibits cell growth in zinc-binding p53 mutants. Mutations are labeled in the legend. B, ZMC1 induces a WT like conformational change in zinc binding mutants, shown by IF. The antibodies, PAB240 and PAB1620 are same as Figure 3E. Scale bar = 25 μm. C, ZMC1 induces expression of p53 regulated genes (p21 and PUMA) in zinc binding mutants, shown by quantitative RT-PCR. Relative gene expression level is normalized to β-actin. D, ZMC1 reduces mutant p53 stability in zinc binding mutants shown by western blot. The p53 protein level is detected by p53 antibody. β-actin is an internal control.

Figure 5
Figure 5. ZMC1 transactivates mutant p53 through increasing ROS levels

A, exogenous GSH (2 mM) abrogates ZMC1 mediated cell growth inhibition. B, ZMC1 induces ROS levels in cells as detected by 8-oxy-dGUO staining. The right panel is quantification of staining. C, expression of Nrf2 and downstream targets is elevated after ZMC1 (1 μM) or H2O2 (300μM) but not NAC (5 mM) treatment. D, NAC (5 mM) does not inhibit ZMC1 mediated conformation change shown in IF. Scale bar = 25 μm. E, NAC (5 mM) abrogates ZMC1 mediated p53 transactivation in p53R175H cells and reduces expression of p21 and PUMA genes. F, ZMC1 transcriptionally activates p53-R175H through ROS mediated post-translational modifications. NAC attenuates p53 post-translational modification induced by ZMC1. With addition of NAC, ATM, p53-Ser15 and Ser46 phosphorylation are reduced and p21 induction is eliminated. G, ZMC1 induces p53-K120 acetylation in p53-R175H cells.

Figure 6
Figure 6. Dual mechanism of ZMC1

A1, in the absence of zinc both WT and R175H DBD are in the their zinc free (apo) forms. The Kd1 of WT p53 is << Kd1 of R175H p53 (≈2 nM). The Kd2 of both WT and R175H p53 are ≥ 1 μM. A2, at physiologic concentrations of zinc, WT p53 is zinc bound (holo) (Kd1<[Zn2+]<Kd2). The R175H p53 is in its apo form ([Zn2+]<Kd1<Kd2). A3, in high concentrations of zinc, both WT p53 and R175H p53 are misfolded (Kd1<Kd2<[Zn2+]). A4, in the presence of ZMC1 both WT and R175H p53 are in their holo form despite (Kd1<Kd2<[Zn2+]). B, once the WT conformation change has occurred the increase in ROS by ZMC1 induces N-terminal post-translational modifications of mutant p53 (ser-15, 46, lys-120) that transactivate mutant p53 and induce an apoptotic program.

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