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

Abstract

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.

Figure 1.

Combination treatment of ZMC1 and cytotoxic chemotherapy or radiation does not display synergy. (A) Schematic representation of the mechanisms of ZMC1 reactivationg mutant p53^R175H. (B) TOV112D (p53R175H) cells were treated with Irinotecan, ZMC1, or a combination of both for 72 hours, after which cell viability was measured by MTS assay. (C) TOV112D cells were treated with γ-irradiation (10 Gy), ZMC1 (2 μM), or a combination of both for 72 hours, after which viability was measured by Vi-CELL Trypan Blue staining.

Figure 2.

Combination treatment of ZMC1 and cytotoxic chemotherapy or radiation in the presence of GSH displays increased synergy. (A) TOV112D cells were treated with Irinotecan, ZMC1, or a combination of both for 72 hours in the presence of 2 mM GSH, after which cell viability was measured via MTS assay. (B) TOV112D cells were treated with γ-irradiation (10 Gy), ZMC1 (2 μM), or a combination of both for 72 hours in the presence of 2 mM GSH, after which viability was measured by Vi-CELL Trypan Blue staining. * p value < 0.05. (C) Combination treatment of ZMC1 and DNA damaging reagent (Etoposide) in the presence of GSH recovers p53 transcription function as evidenced by p21 expression regulation. TOV112D cells were treated with the indicated compounds for 6 hours followed by analysis of cell lysates by Western Blot. The expression of p21 is up-regulated by ZMC1 treatment but attenuated with additional GSH. Etoposide treatment restored the p21 expression. Phospho-p53 (S15) and Phospho-p53 (S46) were also detected by Western blot after ZMC1 treatment but attenuated with additional GSH. Etoposide treatment restored the phosphorylation of the p53 protein. β-actin was used as internal control. (D) Immunoprecipitation of Acetylated p53 (K120). The p53 (K120) acetylation was up-regulated by ZMC1 treatment but attenuated with additional GSH. Concurrent Etoposide treatment increased the p53 (K120) acetylation. No Tx, no treatment; Z, ZMC1, 1 μM; G, GSH, 0.5 mM; E, Etoposide, 20 μM, ZG, ZMC1 and GSH; ZGE, ZMC1 and GSH and Etoposide; ZE, ZMC1 and Etoposide.

Figure 3.

Combination treatment of p53-reactivating reagent (NTA-derivatives) and DNA damaging reagent (Etoposide) recovers p53 transcription. ZMC1 binds Cu²⁺ with K_Cu^ZMC1 values of (7.4 ± 0.31) x 10⁻¹⁷ M and (2.0 ± 0.37) x 10⁻¹⁶ M as determined by, respectively, EGTA competition assays (A) and Zincon competition assays (B). (C) Structures of NTA, NTA-MEE, and NTA-DEE. (D) NTA (black circles), NTA-MEE (red squares), and NTA-DEE (green triangles) bind to Zn²⁺ with K_Zn^NTA = 17 nM (9), K_Zn^NTA-MEE = (327 ± 36) nM, and K_Zn^NTA-DEE = (850 ± 350) nM as determined by ZMC1 competition. (E) Immunocytochemistry fluorescent staining (IF) of the p53 protein from TOV112D cells after treatment of MEE (2.5 mM) or DEE (0.6 mM). The antibody PAB1620 recognizes WT conformation of p53. The Antibody PAB240 recognizes mutant conformation of p53. IF quantification was determined using ImageJ. *** p < 0.0001. (F) NTA (black circles), NTA-MEE (red squares), and NTA-DEE (green triangles) bind to Cu²⁺ with K_Cu^NTA = 8.2 × 10⁻¹¹ M (22), K_Cu^NTA-MEE = (2.6 ± 0.98) x 10⁻¹⁰ M, and K_Cu^NTA-DEE = (2.6 ± 0.92) x 10⁻⁹ M as determined by C12 competition assay. (G) Reactive oxygen species (ROS) was measured by Flow cytometry using CellRox-green reagent (Invitrogen) in H1299 (p53 null) cells after treatment with the indicated compounds. NTA and its derivatives did not induce ROS. The concentrations of the test are sublethal doses of 0.75 μM. (H) TOV112D cells were treated with MEE, Etoposide, or a combination at the indicated concentrations for 72 hours, followed by cell viability measurement by MTS assay. (I) TOV112D cells were treated with DEE, Etoposide, or a combination at the indicated concentrations for 72 hours, followed by cell viability measurement by MTS assay. (J) Long term effect of combination of Etoposide and MEE or DEE is evaluated by clonogenic assay. The cells were treated with vehicle control (CTL), Etoposide (Etop, 0.5 μM), MEE (0.5 mM or 0.2 mM), or DEE (0.15 mM or 0.05 mM) or combination. The quantification of colonies is shown in (K). The p value of Etoposide vs Etop + DEE (0.05 mM) is 0.0064. The p value of DEE (0.05 mM) vs. Etop + DEE (0.05 mM) is 0.0027. All other Etoposide vs combinations and MEE or DEE alone vs combinations have p values <0.001. (L) TOV112D cells are treated with ZMC1 (1 μM) or NTA (5mM) and 6 hours later, IR (0 Gy, 2 Gy, 7 Gy) and then incubated for 3 days. The cell viability was measured by Guava ViaCount. Cells treated with NTA are sensitized to ionizing radiation, while cells treated with ZMC1 are not. (M) Combination treatment of MEE or DEE and DNA damaging reagent (Etoposide) induces p53 transcription function as evidenced by p21 expression regulation, comparable to ZMC1 treatment. TOV112D cells were treated with the indicated compounds for 6 hours followed by analysis of cell lysates by Western Blot. The expression of p21 was up-regulated by ZMC1, MEE, DEE or combination with Etoposide. Phospho-p53 (S15) and Phospho-p53 (S46) were also detected by Western blot after ZMC1, MEE, DEE and combination treatment. β-actin was used as internal control. (N) Immunoprecipitation of Acetyl p53 (K120). The p53 (K120) acetylation was up-regulated by MEE, DEE and combination treatment. No Tx, no treatment; Z, ZMC1, 1 μM; E, Etoposide, 20 μM; MEE, 2.5 mM; DEE, 0.6 mM; MEE + E, MEE and Etoposide; DEE + E, DEE and Etoposide.

Figure 4.

Combination treatment of ZMC1 and other targeted therapies displays synergy. (A) Schematic Representation of ZMC mechanism and targeted therapeutics. See text for details. (B) Combination response with ZMC1 and Nultin 3a. The TOV112D cells were treated with ZMC1 (Z, 1 μM), Nutlin 3a (N, 5, 10, or 20 μM) or combination of the two compounds. The gene expression level of the p53 target genes p21, PUMA, NOXA and MDM2 were measured by qPCR. (C). In vivo efficacy of combination of ZMC1 and Nutlin 3a was assessed using xenograft assay of TOV112D cells, as shown the tumor growth curve. The tumor volumes after 17 day treatment are shown in (D). *, p < 0.05. **, p < 0.01. ***, p < 0.001. n.s., not significant. (E) Long term effect of combination of ZMC1 and ABT-199 is evaluated by clonogenic assay. The cells were treated with vehicle control (CTL), ZMC1 (Z, 1 nM) or ABT-199 (A, 5 μM), and combinations. The quantification of colonies is shown in (F). (G) In vivo efficacy of combination of ZMC1 and ABT-199 was assessed using xenograft assay of TOV112D cells, as shown the tumor growth curve. The tumor volumes after 16 day treatment are shown in (H). *, p < 0.05. **, p < 0.01. ***, p < 0.001. n.s., not significant.

Figure 5.

Schematic representation of the putative Zinc metallochaperones with mutant p53 reactivation and diminished copper binding. (A) The new ZMCs bind zinc in range of 1–500 nM which allows them to bind zinc tightly enough in the serum for zinc delivery but weak enough to allow them to donate zinc intracellularly to induce a mutant p53 conformation change. Their copper binding would need to be at least weaker than 10 pM which would lead to a significantly diminished ROS signal. (B) These compounds can combine with chemotherapy or radiation to promote the newly conformed mutant p53 to undergo PTMs that would activate it to then carry out a p53 mediated apoptotic program (C) that would synergize with the (C,R).