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

TP53 is the most commonly mutated gene in cancer for which no effective targeted anticancer drug exists. The majority of mutations (>70%) are missense that generate a defective protein found at high levels in cancer cells due to loss of MDM2-mediated negative feedback (1, 2). Restoring wild-type structure/function of mutant p53 (henceforth “reactivation”) using small molecules is a highly sought after goal in cancer therapeutics.

There are three major classes of mutant p53′s: destabilizing, DNA contact, and zinc-binding mutants. The differences among the categories partly explain why mutant p53 has been difficult to target for drug development. Destabilizing mutations are often found in the beta-sandwich core of the DNA-binding domain (DBD) and act by lowering the melting temperature of p53 to where it partially unfolds at 37°C. Zinc-binding mutants are classified by their proximity to the four amino acids involved in coordinating the single zinc ion and by impairing zinc binding they cause the protein to misfold (3). The most well-characterized zinc-binding mutant is the R175H, which is also the most frequently found missense mutation in cancer (4). In contrast, DNA contact mutations such as R248W and R273H typically diminish DNA affinity while having little effect on stability or zinc-binding affinity and hence resemble the WT structure.

We recently discovered a new class of mutant p53 reactivators called zinc metallochaperones (ZMC) that represent a new pathway to pharmacologically target the class of zinc-deficient mutant p53′s by restoring zinc binding (5, 6). The ZMC mechanism is predicated on a number of important concepts based on the relationship of zinc to p53; chiefly that the structure of p53 can become flexible by manipulating zinc (7–9). Mutants like the R175H are in the apo (zinc-free) form in the cell because their binding affinity (K_d) for zinc is 100–1,000-fold higher than physiologic zinc concentrations (1–20 picomolar range; ref. 10). ZMCs are zinc ionophores that raise intracellular zinc levels sufficiently above the K_d of the R175H DBD to allow zinc to ligate in the native site and refold the protein (11). ZMCs do not reactivate the DNA contact mutants as these mutations do not have impaired zinc binding.

The traditional paradigm in targeted anticancer drug development is to select a small molecule that binds its target with high affinity and demonstrates a pharmacokinetic profile that maximizes exposure. Furthermore, dosing is often determined by driving exposure to the MTD. ZMCs are a very different drug development program in that they do not directly bind the ultimate target (p53) but rather affect its structure/function indirectly by raising and buffering intracellular zinc levels to trigger a WT p53 program. Metal ion chelators have been investigated as anticancer drugs and have been plagued by toxicity pertaining to the chelation of redox active metals (Fe²⁺, Cu²⁺) which is dose limiting (12). Demonstrating efficacy through the ZMC mechanism with minimal exposure would be an advantage to the development of ZMCs.

Here, we have extended the understanding of the ZMC mechanism by demonstrating that cellular zinc homeostatic mechanisms regulate the mutant p53 reactivational activity functioning as an OFF switch by restoring physiologic zinc levels in cells. In addition, this switch can be accomplished with a very brief exposure of drug both in vitro and in vivo indicating that only a burst of mutant p53 reactivation is necessary to induce complete cancer cell death. This switch indicates that a ZMC with a short half life in vivo is sufficient that imparts an advantage of minimizing potential problems with metal ion toxicity. This represents a significant departure from the traditional paradigm in targeted cancer therapeutics and will impact the future translation of ZMCs to the clinic.

ZMC1 and Zn-1 were synthesized by the Rutgers Translational Sciences group. The synthesis of Zn-1 is detailed in Supplementary Information.

TOV112D and KPC tumor cell lines (K8484, 270H, and K8483) were cultured in DMEM with 10% FBS. K8484 and K8483 were gifts from Dr. Kenneth Olive from Columbia University (New York, NY). 270H was a gift from Dr. Eric Collisson from University of California, San Francisco (San Francisco, CA). TOV112D was purchased from ATCC. Cell lines were authenticated by examination of morphology and growth characteristics. All chemicals were purchased from Sigma-Aldrich or otherwise indicated.

Cell growth inhibition assay was performed by MTS (Promega) or Calcein AM assay (Trevigen). Viability assays were done by Guava ViaCount (Millipore). The 24-hour cell growth assays were performed by Crystal Violet staining (13). The procedures are described in Supplementary Information. Statistical significance of the data, obtained from three independent experiments, each with triplicates, was calculated with Student t test.

The siRNA (Santa Cruz Biotechnology or Dharmacon) transfection were performed using RNAiMAX (Invitrogen), following the manufacturer's instructions. The efficiency of the knockdown was measured by quantitative PCR or Western blot analysis.

For long-term viability, 1,000 cells per well on 6-well plates were treated with various concentrations for varying lengths of time, then, cells were cultured with drug-free complete medium for 14 days without interference. Cells were fixed with 10% formalin and stained with 0.05% crystal violet at the end of 14-day period of cell culture (13). Statistical significance of the data, obtained from three independent experiments, each with triplicates, was calculated with Student t test.

Immunofluorescent staining was performed as described previously (6). For details, see Supplementary Information.

Gene expression (quantitative RT-PCR) and Western blot analysis were performed as described previously (6).

Intracellular free zinc concentration was measured as described previously (11). For details, see Supplementary Information.

The CRISPR cas9 plasmid was purchased from Santa Cruz Biotechnology. Transfection of TOV112D cells was performed using Lipofectamine 2000 (Thermo Fisher Scientific). Twenty-four hours after transfection, cells were sorted on the basis of GFP expression, and single cells were sorted directly into 96-well plates containing DMEM and 10% FBS. The plates were incubated until single cells developed into colonies.

RNA was extracted using Qiagen RNeasy kit and quantified by Agilent RNA Nano. The library preparation was performed with the Illumina TruSeq RNA Library Preparation Kit V2. The library was sequenced on the Illumina NextSeq (high output kit) and 2 × 150bp paired-end sequencing.

Caco-2 cells were seeded in 24-well millcell plates and incubated for 21 days. For A–B transport, ZMC1 and loperamide were added to the Apical side. For B–A transport, ZMC1 and loperamide were added to the basal side. At 0, 15, 30, 60, and 90 minutes, aliquots of 50 μL were collected from the receiver compartment for determination of ZMC1 concentrations by LC/MS-MS.

ZMC1 was administered to male BALB/c mice and plasma was harvested at various time followed by LC/MS-MS analysis for pharmacokinetics. Quantitation of ZMC1 in plasma samples was analyzed by a validated LC/MS-MS assay as mentioned in a previous report (14). For details, see Supplementary Information.

Mice are housed and treated according to guidelines and all the mouse experiments are done with the approval of Institutional Animal Care and Use Committee (IACUC) of Rutgers University (Piscataway, NJ). The KPC mice (Pdx1-CRE; KRas^G12D/+; p53^R172H/+ and Pdx1-CRE; KRas^G12D/+; p53^R270H/+) were obtained from the NCI mouse repository. The nude mice NCR nu/nu were purchased from Taconic. The details of the acute toxicity and efficacy assays are given in the Supplementary Information.

ALZET mini-osmotic pump, Model 2001 (1.0 μL/hour, 7 days) was used to deliver drugs continuously (Alzet). For details, see Supplementary Information.

The mouse tissues were harvested and were subjected to IHC staining with cleaved caspase-3 (CC3, 1:100; Cell Signaling Technology). The staining was quantified using ImageJ software.

The data were analyzed by Student t test with an overall significance level of P < 0.05. P values were described in each figure and the text. The survival times for KPC mice were analyzed with log-rank test. The pairwise comparison was done between groups using a Cox proportional hazards test.

The pharmacodynamics of ZMC1 in tumor cells can be reflected by mutant p53 and p21 levels over time (6, 15). Mutant p53 protein levels decrease after ZMC1 treatment [due to restoration of MDM2-mediated negative feedback (6)] and p21 levels (a p53 downstream target) increase (Fig. 1A, left). Notably, this activity is transient over a 24-hour time period with activation maximally seen at 6–8 hours followed by loss of activity by 24 hours (Fig. 1A, right).

Raising intracellular [Zn²⁺]_free can become toxic in the absence of zinc muffling mechanisms that eukaryotic cells have evolved to lower excessively high free zinc levels (16). Intracellular [Zn²⁺]_free is tightly controlled by zinc transporters, binders, and sensors. Zinc transporters include 14 importers (ZIP/SLC39; ref. 17), nine exporters (ZnT/SLC30; ref. 18), and the metallothionein family of 11 binding proteins. In addition, metal-responsive element-binding transcription factor-1 (MTF-1) is a Zn sensor that regulates the expression of zinc homeostatic genes (19). We hypothesized that ZMC1 treatment would induce these cellular mechanisms to normalize zinc concentrations.

We studied the ZMC1-induced zinc kinetics using the FluoZin3 (FZ3)-AM fluorescent zinc indicator in TOV112D cells (Fig. 1B). Zinc levels rapidly rose at 2 hours, with a significant increase in zinc that peaked to 15 nmol/L by 4 hours, which decreased to baseline by 8 hours (Fig. 1B, left). Note that the 15 nmol/L levels would be sufficient to reactivate the p53^R175H protein as it is well above the K_d of p53-R175H zinc ligation site (2 nmol/L). Importantly, when we plot these zinc kinetics with those of p21 (from Fig. 1A), we found that this peak in zinc levels precedes the peak in p21 levels temporally (Fig. 1B, right).

We measured the expression of three of the most ubiquitously expressed zinc homeostatic genes (exporter ZnT1, importer ZIP10, and binding protein MT1A) in TOV112D cells after ZMC1 treatment. ZnT1 exports and MT1A buffers cellular free zinc, and both function to lower zinc levels, so we expected their expression to increase. ZIP10 increases zinc concentrations, thus we hypothesized a decrease in its expression. We found that ZnT1, MT1A, and ZIP10 expression changed accordingly in response to ZMC1 treatment (Fig. 1C). While these data matched our hypothesis, we next sought to investigate the impact of ZMC1 on the totality of cellular zinc homeostatic genes using RNAseq.

Analysis of the RNAseq dataset revealed that 16 of the 37 zinc homeostatic genes displayed significant expression changes in response to ZMC1 treatment (Fig. 1D; Supplementary Table S1). ZnT1, ZnT2, ZnT3, ZIP1, ZIP9, MT1G, MT1X, MT2A were markedly induced in TOV112D cells in response to ZMC1 treatment with kinetics that correlated with the ZMC1-induced zinc influx. ZnT4, ZnT7, ZIP3, ZIP6, ZIP7, ZIP10, ZIP11, and ZIP14 had decreased expression in response to ZMC1-induced zinc influx. We validated the gene expression induction of ZnT1, MT1A, and MT2A and found that while MT1A was induced to a greater degree than in the RNAseq data, MT2A was more highly induced consistent with the RNAseq data (Supplementary Fig. S1).

To determine the functional relevance of cellular zinc homeostatic mechanisms to ZMC1 pharmacodynamics, we manipulated several zinc homeostatic genes using both a knockdown and knockout approach. We performed a knockout of the MT1A and MT2A genes using CRISPR-cas9 technology. These metallothioneins function as cytosolic zinc buffers (20) and both were significantly induced by ZMC1 in TOV112D cells. Thus, we reasoned they would likely be important in lowering zinc levels induced by ZMC1. Indeed their expression was highly induced by ZMC1 by qPCR and their knockout was confirmed by qPCR (Supplementary Fig. S2). Knockout of these genes proved to be important for cell survival as only one of 192 clones survived the selection. One possible explanation for the survival of this clone was the upregulation of the zinc exporter, ZnT1 as a compensatory mechanism (Supplementary Fig. S2). We determined the effect of this knockout on ZMC1 induced zinc kinetics and found that zinc levels increased earlier, peaked higher, and sustained longer in the knockout cells versus the wild-type controls (Fig. 2A). Because of the compensation in the knockout cells, we also examined these kinetics upon acute knockdown of MT1A or MT2A by siRNA and found similar results (Supplementary Fig. S3). In the knockout cells, we found a similar effect on the pharmacodynamics of ZMC1 where p21 induction was not only greater, but also longer in comparison with the wild-type cells (Fig. 2B). This increase in the pharmacodynamics of ZMC1 was accompanied by a 3-fold increase in sensitivity in cell growth inhibition assays (EC₅₀ 69.1 nmol/L for parental cells vs. 22.9 nmol/L for knockout; Fig. 2C). A similar increase in sensitivity was observed with siRNA knockdown of either MT2A or MT1X (Supplementary Fig. S4). In a crystal violet staining assay, the MT1A/MT2A KO cells showed a 45% (19% vs 42%) reduction in cell growth compared with parental cells when treated for 24 hours with 1 μmol/L ZMC1 (Fig. 2D). Together, these results support the hypothesis that the cellular zinc homeostatic mechanisms function as an OFF switch to the ZMC mechanism. We investigated whether ZMC1 is being actively effluxed out of the cell as an alternative hypothesis for its transient activity. Using the Caco-2 permeability assay, we found that the efflux ratio (B–A/A–B) was 2.2, a low score not consistent with a molecule being actively effluxed (Supplementary Table S2; ref. 21).

Given the transient pharmacodynamics of the ZMC1, we hypothesized that maximum or continuous drug exposure would not be necessary for effective reactivation of mutant p53. In vitro cell growth inhibition experiments in cancer drug development are typically performed by continuously exposing cells to a compound for 72 hours followed by an assay for cell viability. We performed a series of “washout” experiments using both cell viability and colony formation assays in which we varied the drug exposure time. We exposed TOV112D cells to ZMC1 at serial dilutions for 30 minutes, 1, 2, 4, 6, 24, 48, and 72 hours. For time points less than 72 hours, we removed the media containing ZMC1 and replaced with media without ZMC1. Cells were harvested for viability at 72 hours. We found there to be a similar magnitude of cell growth inhibition between exposure times of 24, 48, and 72 hours (Supplementary Fig. S5). Interestingly, we found that even 4 and 6 hours were as effective as 24 hours. Thirty minutes appears to be the time point where there was a significant reduction in efficacy (Fig. 2E). We validated this result using the colony formation assay in which cells were exposed to ZMC1 for a specified time period and then allowed to grow in media without drug for two weeks. This method allows us to assess the durability of the ZMC1 response. Like the cell growth inhibition assay, we found that using a dose 0.01 μmol/L, an exposure of less than 30 minutes was sufficient to significantly reduce the number of colonies by day 14. We even found in this assay that at least 15 minutes was sufficient. This effect was “on target” as it could be abrogated by mutant p53 knockdown using an siRNA to p53 (Fig. 2F). These data demonstrate that the OFF switch exerted by the cellular zinc homeostatic mechanisms has important implications for the in vivo translation of ZMCs, specifically that the duration of exposure needed for activity is brief.

The success of the ZMC drug development program depends on demonstration that the mechanism can be translated in vivo to demonstrate preclinical efficacy with an acceptable therapeutic window. Important obstacles to the success of in vivo translation are: (i) can the molecules bind zinc in the plasma and deliver it intracellularly sufficiently to permit p53-mutant reactivation and (ii) will there be toxicity associated with metal ion chelation.

For these preclinical studies, we used the genetically engineered KPC mouse model of pancreatic cancer (Pdx-1-Cre; Kras^G12D/+; Tp53^R172H/+ or Tp53^R270H/+; ref.22). The Tp53^R270H allele is the mouse homolog of the human hotspot p53^R273H, a non–zinc-deficient negative control. Pancreatic cancer is a highly lethal malignancy that suffers from a lack of effective systemic chemotherapy, thus novel agents showing efficacy in this model are in high demand. The model faithfully recapitulates many aspects of pancreatic cancer biology and has been used extensively in preclinical therapeutic studies (23–25). First, we confirmed we could detect evidence of ZMC1-induced p53^R172H-mutant reactivation in tumor cell lines from this model. ZMC1 treatment of the K8484 cell line (p53^R172H) induced a conformation change in the mutant protein as evidenced by loss of the mutant-specific antibody (PAB240) on immunoflourescent staining as well as an induction of p21 by Western blot analysis (Fig. 3A and B). This cell line also exhibited similar pharmacodynamics of ZMC1 over 24 hours to those seen in the TOV112D cells with a peak in p21 at 8 hours that returned to baseline by 24 hours (Fig. 3B). Cell growth inhibition studies revealed that ZMC1 was selectively toxic to p53^R172H cells while exhibiting resistance in both the p53^R270H and p53^−/− cell lines. We found this cell line to be much less sensitive to ZMC1 in comparison with the TOV112D cells (EC₅₀ 600 nmol/L for K8484 vs. 69 nmol/L for TOV112D) while the others could not reach an EC₅₀; Fig. 3C). Gene expression of the p53-regulated genes (p21, Puma, Noxa, and Gdf15) increased with ZMC1 treatment (Fig. 3D). Moreover, gene expression of zinc regulators (Znt1, Mt1, and Zip10) in K8484 cells was similar to TOV112D cells (Fig. 3E). Specifically, the genes that function to decrease intracellular zinc levels are upregulated (Znt1, Mt1) and the genes that function to increase cellular zinc levels decrease in response to ZMC1. These studies confirmed ZMC1 p53-mutant reactivation in this model in vitro as well as an induction of zinc homeostatic mechanisms.

Pharmacokinetic studies of ZMC1 revealed that 2 mg/kg intravenously achieved a maximum concentration (C_max) of 2.4 μmol/L, which was well above the concentration needed for p53-mutant reactivational activity in vitro, but the area under the curve of the last dose (AUC_last) of 0.385 hour * μmol/L and a half life of 0.59 hours demonstrate that the compound is rapidly cleared (Table 1). This is further demonstrated after intravenous (2 mg/kg) and oral gavage (10 mg/kg; Fig. 4A).

To determine the optimal dose for efficacy experiments in this model, we performed acute toxicity studies to determine the MTD and found a dose of 5 mg/kg administered intraperitoneally was well tolerated with 100% survival by 7 days with no significant weight loss observed (Supplementary Fig. S6A and S6B). At a dose of 10 mg/kg, we observed some toxicity including seizure-like activity and hypothermia with approximately 60% survival and up to 20% weight loss (Supplementary Fig. S6A and S6B). To determine whether the 5 mg/kg dose was sufficient to induce the ZMC mechanism, we performed pharmacodynamics experiments in KPC mice administered four doses of drug prior to immunostaining tumor tissue for the apoptotic marker cleaved caspase-3 (CC3). We detected a significantly greater level of staining in the p53^R172H tumor tissue compared with vehicle control while we observed almost no activity in p53^R270H tissue indicating an “on-target” effect (Supplementary Fig. S7). This revealed CC3 staining to be a reliable marker of pharmacodynamic activity in vivo and that the 5 mg/kg dose daily was sufficient for activity.

Given that a brief exposure of a ZMC is sufficient for anticancer activity, we hypothesized that continuous dosing (or sustained exposure) would not be necessary but rather intermittent dosing would be sufficient. We tested this preclinically using nude mice with subcutaneous engraftment of tumors using the K8484 cell line. We evaluated 1 μmol/L/24 hours dosed continuously by intravenous pump to 1 μmol/L by intravenous bolus daily and found no significant difference (P = 0.35; Fig. 4B). This is consistent with our in vitro results in which short exposures were as efficacious as continuous exposures.

Given the unique switch mechanism of ZMC1, we hypothesized that ZMC1 should exhibit efficacy with limited drug exposure in vivo. This contrasts with more traditional targeted therapeutics for which it is necessary to select a compound with a longer half life and maximal exposure. We then evaluated ZMC1 in the KPC model using both the KPC-p53^R172H and p53^R270H alleles using overall survival as the primary endpoint. In this model, mice are enrolled when they develop a dominant pancreatic tumor that is 4–5 mm in size by ultrasound. Mice received ZMC1 5 mg/kg i.p. (or vehicle) daily and followed by serial US every 3 days until moribund. The published median survival in this model from that point is approximately 14 days (23). We found similar results with our control (vehicle treated) mice in both of the KPC-p53^R172H and p53^R270H groups (median survival 15.5 and 14 days, respectively; Fig. 4C and D). However, we found that ZMC1 significantly increased the survival of the KPC-p53^R172H mice while having no such effect on the survival of the KPC-p53^R270H mice [median survival 26 days, (P = 0.030) vs. 10 days (P = 0.45) respectively; Fig. 4C]. Using US, we assessed response to treatment (every 3–6 days) compared with baseline in individual mice and observed regressions in 5/8 KPC-p53^R172H mice while only 2/8 KPC-p53^R270H mice exhibited a similar effect (Fig. 4E–J). There were 4 out of 8 KPC-p53^R172H mice with survival more than 26 days. Overall ZMC1 was well tolerated with no mice requiring treatment breaks and no significant weight loss observed. These results demonstrate that ZMC1 can improve survival in mice specifically with tumors expressing a zinc-deficient p53 mutation.

We previously showed that administering ZMC1 with supplemental zinc increases its apoptotic function (6). We have also determined using X-ray crystallography that binding of ZMC1 to zinc is 2:1 (11). Because ZMCs pass through cell membranes as a charge neutral complex with a 2:1 stoichiometry, we hypothesized that synthesizing the ZMC complexed with zinc in a 2:1 ratio will improve potency. The chemistry to produce the [Zn(ZMC1)₂] (named Zn-1) is illustrated (Fig. 5A). We evaluated Zn-1 in cell growth inhibition assays with the TOV112D cell line using the monomer as a control. We found that Zn-1 was indeed more potent than the monomer alone with the complex resulting in nearly 100% cell kill (Supplementary Fig. S8). Using LC/MS-MS, we examined the serum of mice administered Zn-1 in a formal pharmacokinetic study and detected a mass peak corresponding to the ZMC1 monomer as well as the Zn-1 complex (Supplementary Fig. S9). Using the KPC-p53^R172H mice, we sought to determine whether Zn-1 was more potent in vivo. We first determined the MTD using acute single-dose exposure toxicity assays and found that the complexes were well tolerated up to 15 mg/kg without significant weight loss indicating the complexes are less toxic than the monomer (MTD 10 mg/kg Supplementary Fig. S6A; Supplementary Fig. S10A and S10B). To compare the efficacy of Zn-1 to ZMC1 at 5 mg/kg, we dosed the molar equivalent of Zn-1 (5.6 mg/kg). We found that like ZMC1, Zn-1 treatment also significantly increased the survival of the KPC-p53^R172H mice (median survival 35 days) versus vehicle control (P = 0.00065). Furthermore, Zn-1 treatment also performed better than the monomer (P = 0.023; Fig. 5B). When we assessed the response of individual mice by tumor volume to Zn-1, we found that 2 of 5 mice exhibited remarkably good responses with survival times of 70 and 81 days (Fig. 5C and D). We also observed regressions in 7 of 10 KPC-p53^R172H mice (Fig. 5C). We assessed response to both ZMC1 and Zn-1 treatment using CC3 staining and found that in comparison with controls, staining was significantly increased in both the ZMC1 and Zn-1–treated tumors (P = 0.0015 and 0.019 for ZMC1 and Zn-1 respectively; Fig. 5E). In support of this, we conducted a separate pharmacodynamic experiment in which mice bearing subcutaneous tumors from cell lines KPC-p53^R172H versus KPC-p53^R270H were treated with one dose of ZMC1. Gene expression of three p53 target genes Puma, Noxa, and p21 increased after ZMC1 treatment in the KPC-p53^R172H tumor tissues but not in KPC-p53^R270H controls (Supplementary Fig. S11).

In this study, we have used ZMC1 to illuminate another key aspect of the mechanism of ZMCs, which is the cellular response to zinc through zinc homeostatic mechanisms. Fig. 6 illustrates how our concept of the mechanism of ZMC's has evolved to resemble an ON/OFF switch. In a cancer cell expressing a zinc-deficient mutant like p53^R175H, zinc levels are maintained at the picomolar range (10⁻⁹ mol/L). We have previously determined the zinc K_d of the p53^R175H is approximately 2 × 10⁻⁶ mol/L (15), thus at physiologic zinc concentrations the p53^R175H is in its apo (zinc free) form and is misfolded (Fig. 6A). Upon treatment of a ZMC, cellular zinc levels rise 1,000-fold (to approximately 1.5 × 10⁻⁵ mol/L), which is high enough to allow zinc binding in the native ligation site of p53^R175H causing a wild-type conformational change (holo-form). This turns the switch ON and the p53^R175H functions like wild-type p53 to induce apoptosis (Fig. 6B and C). In response to this zinc surge, cellular zinc homeostatic mechanisms are activated to lower zinc back to physiologic levels. As a result, zinc can no longer bind to the p53^R175H and it returns to its apo form turning the switch OFF, (Fig. 6D). It is possible that different cancer cells express different levels of zinc modulators and this might affect the sensitivity or resistance to a ZMC. This is the subject of future investigations.

This switch mechanism has important implications for the following (i) minimizing toxicity by requiring only brief exposure, (ii) guiding the optimization of pharmacologic properties, and (iii) the identification of dose and scheduling. Traditionally, targeted agents in cancer drug development are optimized for pharmacologic properties that maximize drug exposure (i.e., t_1/2, AUC_max) as the goal is to achieve a relatively constant level that exceeds the binding affinity of the target (IC₅₀) and/or the cell growth–inhibitory/killing properties (EC₅₀) in the cancer cell. In contrast, ZMCs will need to be optimized for maximum concentration in the serum (C_max) where the concentration needs to exceed the EC₅₀ in the cancer cell expressing a zinc-deficient mutant p53 for a relatively brief period of time (less than 30 minutes based upon our data).

Our data demonstrate as proof of principle that the ZMC mechanism can be translated in vivo to establish efficacy in a complex preclinical model of cancer with an acceptable therapeutic window. In fact, we were able to show single-agent efficacy of both ZMC1 and Zn-1 in a cancer model that has been notoriously resistant to many targeted therapies as single agents (23, 24, 26). This indicates that ZMCs may have the potential for efficacy in pancreatic adenocarcinoma, one of the most chemotherapy-insensitive cancers.

The findings regarding the relationship between the duration of exposure and efficacy for ZMCs are particularly impactful to their translation in humans and may have a broader relevance to pharmacologic strategies to turn on or restore p53 signaling in wild-type or mutant tumors (27, 28). Specifically with respect to p53, longer durations of activity are not necessary. In other words, less is more. Precedence for this concept exists from experiments using mouse models of cancer where short duration of p53 signaling are as effective as long ones (29).

The [Zn(ZMC1)₂] (Zn-1) complex represents an innovation in drug design and our data in the KPC model provides robust preclinical evidence that this formulation is superior to the monomer in both efficacy and toxicity. There are several reasons why the complexes are more potent than the monomers: (i) delivering the active complex (Zn-1) does not require the monomers to self assemble with zinc in vivo. The 2:1 complex will reversibly dissociate, and the resulting monomers will become diluted, eliminating their ability to form active complex. The second-order kinetics of complex formation effectively terminates the ZMC activity as the complex dissociates. (ii) They deliver the optimal ratio of chelator to zinc for ZMC function. We have shown that when the molar ratio of the monomer exceeds zinc by a ratio of 4:1, the activity is lost because all of the available zinc is monomer bound (15). (iii) The complex is providing supplemental zinc, which we have shown to increase the apoptotic activity of ZMC1 (6, 15). (iv) There is less monomer available to bind alternative divalent metal ions such as iron or copper. The latter reason might also explain why the Zn-1 complex is less toxic then the monomer.

Our efficacy data in the KPC model demonstrating mutant p53 allele specificity highlights one of the most attractive aspects of developing ZMCs as potential anticancer drugs. The potential patient population to apply these drugs (those with zinc-deficient mutant p53) is known. The frequency of the p53^R175H mutation in pancreatic cancer is consistent what is reported across all cancers (approximately 4%–5%). In an era of precision medicine, sequencing a patient's TP53 gene is quite common and the first proof-of-concept clinical trial for a ZMC should require a zinc-deficient p53 mutation as an inclusion criteria.

S.D. Kimball is listed as a co-inventor on a series of patents involving zinc complexes and compounds that form zinc complexes for the treatment of cancer, owned by Rutgers University and licensed to Z53 Therapeutics. He is a consultant/advisory board member for Z53 Therapeutics. D.R. Carpizo is an employee of and holds ownership interest (including patents) in Z53 Therapeutics. No potential conflicts of interest were disclosed by the other authors.

Conception and design: X. Yu, S. Kogan, S.D. Kimball, D.R. Carpizo

Development of methodology: X. Yu, S. Kogan, D.R. Carpizo

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Yu, S. Kogan, A.T. Tsang, T. Withers, H. Lin, J. Gilleran, D.R. Carpizo

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Yu, S. Kogan, Y. Chen, D. Moore, J. Bertino, C. Chan, S.N. Loh, D.R. Carpizo

Writing, review, and/or revision of the manuscript: X. Yu, S. Kogan, H. Lin, J. Gilleran, B. Buckley, D. Moore, S.D. Kimball, S.N. Loh, D.R. Carpizo

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Yu, S. Kogan, T. Withers, B. Buckley, D.R. Carpizo

Study supervision: X. Yu, D.R. Carpizo

The authors thank Rutgers Cancer Institute of New Jersey Histology core facility for processing the tumor blocks and Pharmacokinetics core facility for analyzing ZMC1 content in tumor cells. They thank Dr. Kenneth Olive from Columbia University for providing KPC tumor cell lines (K8484 and K8483). They thank Dr. Eric Collisson from University of California, San Francisco, for providing 270H cell line. They also thank Sai Life Sciences for performing pharamacokinetics studies and RUCDR Infinite Biologics for performing RNA-seq studies. This study was supported by grants from the National Cancer Institute (K08 CA172676, R01 CA200800), the Sidney Kimmel Foundation for Cancer Research, Pancreatic Cancer Action Network-AACR Innovative Grant, Breast Cancer Research Foundation (to D.R. Carpizo), and New Jersey Commission on Cancer Research Pre-doctoral fellowship (to S. Kogan).

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