Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2019 Sep 15.
Published in final edited form as: Clin Cancer Res. 2018 Jun 18;24(18):4505–4517. doi: 10.1158/1078-0432.CCR-18-0822

Zinc Metallochaperones Reactivate Mutant p53 Using an ON/OFF Switch Mechanism

A New Paradigm in Cancer Therapeutics

Xin Yu 1,2, Samuel Kogan 1,2,3, Ying Chen 2, Ashley T Tsang 1,2,11, Tracy Withers 1,2, Hongxia Lin 2, John Gilleran 4, Brian Buckley 5, Dirk Moore 2,6, Joseph Bertino 2,7, Chang Chan 2,7, S David Kimball 4,8,10, Stewart N Loh 9, Darren R Carpizo 1,2,3,10,*
PMCID: PMC6139040  NIHMSID: NIHMS974549  PMID: 29914895

Abstract

Purpose

Zinc metallochaperones (ZMCs) are a new class of anti-cancer drugs that reactivate zinc deficient mutant p53 by raising and buffering intracellular zinc levels sufficiently to restore zinc binding. In vitro pharmacodynamics of ZMCs indicate that p53 mutant activity is ON by 4–6 hours and is OFF by 24. We sought to understand the mechanism of this regulation and to translate these findings pre-clinically. We further sought to innovate the formulation of ZMCs to improve efficacy.

Experimental Design

We performed in vitro mechanistic studies to determine the role of cellular zinc homeostatic mechanisms in the transient pharmacodynamics of ZMCs. We conducted pre-clinical pharmacokinetic (PK), pharmacodymanic (PD) and efficacy studies using a genetically engineered murine pancreatic cancer model (KPC) to translate these mechanistic findings and investigate a novel ZMC formulation.

Results

In vitro, cellular zinc homeostatic mechanisms that restore zinc to its physiologic levels function as the OFF switch in ZMC pharmacodynamics. In vivo PK studies indicate that ZMCs have a short half life (<30 minutes), which is sufficient to significantly improve survival in mice expressing a zinc deficient allele (p53R172H) while having no effect in mice expressing a non-zinc deficient allele (p53R270H). We synthesized a novel formulation of the drug in complex with zinc and demonstrate this significantly improves survival over ZMC1.

Conclusions

Cellular zinc homeostatic mechanisms function as an OFF switch in ZMC pharmacodynamics indicating that a brief period of p53 mutant reactivation is sufficient for on-target efficacy. ZMCs synthesized in complex with zinc are an improved formulation.

INTRODUCTION

TP53 is the most commonly mutated gene in cancer for which no effective targeted anti-cancer 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 (ZMCs) 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 (Kd) for zinc is 100–1000 -fold higher than physiological zinc concentrations (1–20 picomolar range) (10). ZMCs are zinc ionophores that raise intracellular zinc levels sufficiently above the Kd 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 anti-cancer 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 maximum tolerated dose. 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 anti-cancer 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 which 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.

MATERIALS AND METHODS

Synthesis of ZMC1 and Zn-1

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

Cell lines and Chemicals

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. 270H was a gift from Dr. Eric Collisson from University of California, San Francisco. 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

Cell growth inhibition assay was performed by MTS (Promega) or Calcein AM assay (Trevigen). Viability assays were done by Guava ViaCount (Millipore). The 24-hr 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’s t-test.

Transfection of siRNA

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.

Colony formation assays

For long-term viability, 1000 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’s t-test.

Immunofluorescent staining

Immunofluorescent staining was performed as described previously (6). The details are in Supplementary Information.

Gene expression (quantitative RT-PCR) and Western blot

The procedures were performed as described previously (6).

Intracellular free zinc concentration measurement

Intracellular free zinc concentration was measured as described previously (11). The details are in Supplementary Information.

CRISPR knockout

The CRISPR cas9 plasmid was purchased from Santa Cruz Biotechnology (Dallas, TX). Transfection of TOV112D cells was performed using Lipofectamine 2000 (Thermofisher Scientific). Twenty-four hours after transfection, cells were sorted based on 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.

RNAseq analysis

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.

Efflux analysis

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 90min, aliquots of 50 μL were collected from the receiver compartment for determination of ZMC1 concentrations by LC/MS/MS.

Pharmacokinetics and ZMC1 concentration measurement in plasma

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 in previous report (14). The details are in Supplementary Information.

Mouse experiments

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. The KPC mice (Pdx1-CRE; KRasG12D/+; p53R172H/+ and Pdx1-CRE; KRasG12D/+; p53R270H/+) were obtained from the NCI mouse repository. The nude mice NCR nu/nu are purchased from Taconic. The details of the acute toxicity and efficacy assays are in the supplementary information.

ALZET Osmotic Pumps

ALZET mini-osmotic pump, Model 2001 (1.0 microliter per hour, 7 days) was used to deliver drugs continuously (Alzet, Cupertino, CA). The details are in Supplementary Information.

Immunohistochemistry staining

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

Statistical Methods

The data were analyzed by Student’s 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.

RESULTS

The pharmacodynamics of ZMC1 in cells over 24 hours mirrors the zinc kinetics

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 panel). 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 panel).

Figure 1. The pharmacodynamics of ZMC1 in cells over 24 hours mirrors the zinc kinetics.

Figure 1

Open in a new tab

A, The p21 and p53 protein levels were examined using western blot in TOV112D cells (p53R175H) in a time course of treatment with 1 μM ZMC1for a 24 hour period. Actin is used as an internal loading control. The quantitation is shown on the right. B, Intracellular free zinc concentration was measured using FluoZin-3 fluorescent imaging. The TOV112D cells were treated with 1 μM ZMC1 over a 24 hour period. At 0, 1, 2, 4, 6, 8, 12, 16 and 24 hrs the cells were imaged with FluoZin-3. The intracellular free zinc concentration was measured according to the manufacturer's instructions. The scale bar = 25 μm. The quantitation of the zinc concentration (from Fig. 1B left panel) and the p21 protein levels (from Fig. 1A) are shown on the right. C, The expression of zinc homeostatic genes (ZnT1 and MT1A which function to decrease free zinc, and ZIP10 which increases intracellular free zinc) was measured by quantitative RT-PCR in TOV112D cells with treatment of 1 μM ZMC1 for over 24 hours. D, RNAseq analysis of TOV112D cells at baseline, 4 hours, 8 hours, and 24 hours after ZMC1 treatment. The subset of the 37 zinc homeostatic genes which were induced in response to ZMC1 treatment labeled with asterisks were shown in Supplemental Table 1.

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 fourteen importers (ZIP/SLC39) (17), nine exporters (ZnT/SLC30) (18) and the metallothionein family of eleven binding proteins. Additionally, 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 two hours, with a significant increase in zinc that peaked to 15 nM by four hours, which decreased to baseline by 8 hours (Fig. 1B, left panel). Note that the 15 nM levels would be sufficient to reactivate the p53R175H protein as it is well above the Kd of p53-R175H zinc ligation site (2 nM). 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 panel).

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 this 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 and 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).

Cellular zinc ion homeostatic mechanisms function as the OFF switch in ZMC1 pharmacodynamics

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 out 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). Due to 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 to the wild type cells (Fig. 2B). This increase in the pharmacodynamics of ZMC1 was accompanied by a three-fold increase in sensitivity in cell growth inhibition assays (EC50 69.1 nM for parental cells vs. 22.9 nM 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 to parental cells when treated for 24 hours with 1 μM 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 if 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) (21).

Figure 2. Cellular zinc ion homeostatic mechanisms function as the OFF switch in ZMC1 pharmacodynamics allowing a brief exposure to be efficacious.

Figure 2

Open in a new tab

A, Intracellular free zinc concentration was measured using FluoZin-3 fluorescent imaging in TOV112D parental cells and MT2A/MT1A KO cells. The cells were treated with 1 μM ZMC1 over an 8 hour period. Scale bar = 50 μm. The quantitation of the zinc concentration is shown on the right. B, The p21 and p53 protein levels in TOV112D parental cells and MT2A/MT1A KO cells were measured by western blot. Actin is used as an internal loading control. The quantitation of p21 levels normalized with actin is shown on the right. The data of the parental cells (left panel) is from Figure 1A. C, Cell viability measurement of TOV112D parental cells and MT2A/MT1A KO cells upon treatment of the serial dilutions of ZMC1 for 72 hours using Calcein-AM assays. Statistical analysis was performed using Student’s t-test, comparing the viability of each drug concentration between the two cell lines. *, p value < 0.05. EC50s were shown below the graph. D, Cell growth measurement of TOV112D parental cells and MT2A/MT1A KO cells upon treatment of 1 μM ZMC1 for 24 hours. The cells were fixed and stained with Crystal violet. The viable cells were counted by using automated cell counting on the Keyence BZ-X software. The average cell count per field was normalized with the vehicle control samples. 4 fields per dish and 3 dishes per treatment or control were counted. **, P value < 0.0001. E, The TOV112D cells with various time of exposure of the serial dilutions of ZMC1 and then the drug-free medium was replaced for the incubation up to 72 hours. Cell viability measurement was performed using Guava ViaCount. Statistical analysis was performed using Student’s t-test, compared to 30 min treatment with 0.01 μM. *, p value = 0.006. **, p value < 0.0001. F, Long term effect of the different exposure time (15 min, 30 min, and 60 min) of 0.01 μM ZMC1 was evaluated by clonogenic assay. The cells were incubated in drug-free medium for 14 days to form colonies followed by Crystal violet staining. The quantitation is shown on the right. The efficiency of the p53 siRNA knockdown is measured by Western blot shown on the right.

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 μM, 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.

Translating the Switch concept of ZMCs pre-clinically in pancreatic cancer

The success of the ZMC drug development program depends on demonstration that the mechanism can be translated in vivo to demonstrate pre-clinical efficacy with an acceptable therapeutic window. Important obstacles to the success of in vivo translation are: 1) can the molecules bind zinc in the plasma and deliver it intracellularly sufficiently to permit p53 mutant reactivation and 2) will there be toxicity associated with metal ion chelation.

For these pre-clinical studies, we used the genetically engineered KPC mouse model of pancreatic cancer (Pdx-1-Cre; KrasG12D/+; Tp53R172H/+ or Tp53R270H/+) (22). The Tp53R270H allele is the mouse homologue of the human hotspot p53R273H, 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 pre-clinical therapeutic studies (23–25). First, we confirmed we could detect evidence of ZMC1-induced p53R172H mutant reactivation in tumor cell lines from this model. ZMC1 treatment of the K8484 cell line (p53R172H) 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 (Fig. 3, A 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 hrs (Fig. 3B). Cell growth inhibition studies revealed that ZMC1 was selectively toxic to p53R172H cells while exhibiting resistance in both the p53R270H and p53−/− cell lines. We found this cell line to be much less sensitive to ZMC1 in comparison to the TOV112D cells (EC50 600 nM for K8484 versus 69 nM 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.

Figure 3. ZMC1 Reactivates the p53R172H in the murine KPC pancreatic cancer model.

Figure 3

Open in a new tab

A, Mutant p53 protein refolding was evaluated using conformation-specific immunofluorescent imaging with PAB240 antibody. The p53+/+ and p53R172H/+ cells were treated with 1 μM ZMC1 for 6 hours followed by immunofluorescent staining. The scale bar = 25 μm. B, The p21 protein levels were evaluated by western blot in K8484 cells upon treatment with 1 μM ZMC1 for up to 24 hours. GAPDH was used as the internal control. C, Sensitivity of K8484 (p53R172H/+), 270H (p53R270H/+) and K8483 (p53−/−) to ZMC1 was evaluated by cell growth inhibition assay (MTS assay). The cells were treated with serial dilutions of ZMC1 for 72 hours. D, Gene expression of 4 p53 regulated genes, p21, Puma, Noxa and Gdf15, after ZMC1 treatment was measured by qRT-PCR. E, Gene expression of 3 common zinc regulation genes (Znt1, Mt1, Zip10) after ZMC1 treatment was measured by qRT-PCR.

Pharmacokinetic studies of ZMC1 revealed that 2 mg/kg intravenously (IV) achieved a maximum concentration (Cmax) of 2.4 μM which was well above the concentration needed for p53 mutant reactivational activity in vitro, but the area under the curve of the last dose (AUClast) of 0.385 hr * μM and a half life of 0.59 hrs demonstrate that the compound is rapidly cleared (Table 1). This is further demonstrated after IV (2 mg/kg) and oral gavage (PO) (10 mg/kg) (Fig. 4A).

Table 1.

In vivo pharmacokinetic studies of ZMC1 in the mouse plasma

Analyte Route/Dose (mg/kg) Tmax (hr) C0/Cmax (nM) AUClast (hr*nM) AUCinf (hr*nM) T1/2 (hr) CL (mL/min/kg) Vss (L/kg) %F
ZMC1 i.v./2 - 2428 385 405 0.59 351 9.8 79
p.o./10 0.25 4556 1527 NR - - -
Open in a new tab

NR-Not reportable since AUCinf is 20% more than AUClast

Figure 4. ZMC1 improves survival in the KPC model specifically in a zinc deficient allele.

Figure 4

Open in a new tab

A, Kinetics of plasma concentrations of ZMC1 over 4 hours. B, Comparison of ZMC1 administered at 1 mg/kg IV bolus daily versus 1 mg/kg/24hr daily by continuous IV infusion (AZLET pump) in the K8484 subcutaneous model. Tumor size was measured every 2–3 days. The p-value of bolus IV vs pump IV was shown. C-D, Kaplan-Meier survival curves for KPC-172 (p53R172H) (C) and KPC-270 (p53R270H) (D) mice treated with vehicle control or ZMC1. The median survival time, number of mice in each group and the p value was shown. E-H, Relative individual tumor growth measured by ultrasound for KPC-172 and KPC-270 (Waterfall plot). The mice were treated with vehicle control or ZMC1 by IP with 5 mg/kg daily until the end point. Each bar color represents a measurement expressed as percentage change from baseline. E, KPC-172 mice treated with vehicle control. F, KPC-172 mice treated with ZMC1. G, KPC-270 mice treated with vehicle control. H KPC-270 mice treated with ZMC1. I-J, Individual tumor growth for KPC-172 (I) and KPC-270 (J).

To determine the optimal dose for efficacy experiments in this model, we performed acute toxicity studies to determine the maximum tolerated dose (MTD) and found a dose of 5 mg/kg administered intraperitoneally (IP) was well tolerated with 100% survival by seven days with no significant weight loss observed (Supplementary Fig. S6A and B). 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 B). To determine if 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 p53R172H tumor tissue compared to vehicle control while we observed almost no activity in p53R270H 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 anti-cancer activity, we hypothesized that continuous dosing (or sustained exposure) would not be necessary but rather intermittent dosing would be sufficient. We tested this pre-clinically using nude mice with subcutaneous engraftment of tumors using the K8484 cell line. We evaluated 1 μM/24 hr dosed continuously by intravenous pump to 1 μM 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-p53R172H and p53R270H 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 IP (or vehicle) daily and followed by serial ultrasound (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-p53R172H and p53R270H 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-p53R172H mice while having no such effect on the survival of the KPC-p53R270H mice (median survival 26 days, (p = 0.030) versus 10 days (p = 0.45) respectively) (Fig. 4C). Using US, we assessed response to treatment (every 3–6 days) compared to baseline in individual mice and observed regressions in 5/8 KPC-p53R172H mice while only 2/8 KPC-p53R270H mice exhibited a similar effect (Fig. 4E-H, I and J). There were 4 out of 8 KPC-p53R172H 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.

ZMCs synthesized in complex with zinc: a novel drug formulation

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). Since 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 PK study and detected a mass peak corresponding to the ZMC1 monomer as well as the Zn-1 complex (Supplementary Fig. S9). Using the KPC-p53R172H mice, we sought to determine if 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 B). 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-p53R172H 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 2/5 mice exhibited remarkably good responses with survival times of 70 and 81 days (Fig. 5C and D). We also observed regressions in 7/10 KPC-p53R172H mice (Fig. 5C). We assessed response to both ZMC1 and Zn-1 treatment using CC3 staining and found that in comparison to 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-p53R172H vs KPC-p53R270H 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-p53R172H tumor tissues but not in KPC-p53R270H controls (Supplementary Fig. S11).

Figure 5. ZMC1 complexed with zinc (Zn-1), a novel formulation, exhibits greater efficacy and less toxicity.

Figure 5

Open in a new tab

A, Synthesis of [Zn(ZMC1)2] (i.e. Zn-1). B, Kaplan-Meier survival curves for KPC-172 mice treated with Zn-1. The median survival time, number of mice in each group and the p value was shown. The data of vehicle control and ZMC1 treatment groups were from Fig. 4D. C, Relative individual tumor growth (waterfall plot) for KPC-172 mice treated with Zn-1 by IP with 5.6 mg/kg daily until the end point. Each bar color represents a measurement. D, Individual tumor growth for KPC-172 upon treatment of vehicle control, ZMC1 or Zn-1 (Spiderweb plot). The data of vehicle control and ZMC1 treatment groups were from Fig. 4J. E, Representative IHC staining with Cleaved caspase 3 for tumor tissues from KPC-172 and KPC-270 treated with vehicle, ZMC1 or Zn-1. The quantitation is shown on the right.

DISCUSSION

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. Figure 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 p53R175H, zinc levels are maintained at picomolar range (10−9 M). We have previously determined the zinc Kd of the p53R175H is approximately 2 × 10−6 M (15), thus at physiologic zinc concentrations the p53R175H is in its apo (zinc free) form and is misfolded (Fig. 6A). Upon treatment of a ZMC, cellular zinc levels rise 1000 fold (to approximately 1.5 × 10−5 M) which is high enough to allow zinc binding in the native ligation site of p53R175H causing a wild type conformational change (holo-form). This turns the switch ON and the p53R175H 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 p53R175H 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.

Figure 6. The ZMC Switch mechanism.

Figure 6

Open in a new tab

A, At physiologic zinc levels (10−9 M), p53R175H is in its Apo (zinc free) state because the mutation weakens its binding at the native ligation site (p53R175H Zn Kd 2 × 10−6 (15)). This causes the protein to misfold and lose wild type transcriptional function. B, Upon treatment with a ZMC, intracellular zinc levels increase approximately 1000 fold (1.5 × 10−5 M), and this allows zinc to bind in the p53R175H native ligation site and the protein adopts a wild type conformation (ON switch). C, Once the protein undergoes a wild type conformation change, the p53R175H exhibits wild type transcriptional activity and an apoptotic mechanism is induced. D, Within hours the cell responds to this zinc surge by activating zinc homeostatic mechanisms (increasing expression of metallothioneins, zinc exporters, decreasing expression of zinc importers) that function to lower cellular zinc levels to physiologic range. Zinc is no longer at a sufficient concentration to remain bound in the p53R175H and the protein returns to its Apo state (OFF switch).

This switch mechanism has important implications for the following 1) minimizing toxicity by requiring only brief exposure, 2) guiding the optimization of pharmacologic properties and 3) the identification of dose and scheduling. Traditionally, targeted agents in cancer drug development are optimized for pharmacologic properties that maximize drug exposure (i.e. T1/2, AUCmax) 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 (Cmax) 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 pre-clinical 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 pre-clinical 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: 1) 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. 2) 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). 3) The complex is providing supplemental zinc, which we have shown to increase the apoptotic activity of ZMC1 (6,15). 4) 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 anti-cancer drugs. The potential patient population to apply these drugs (those with zinc deficient mutant p53) is known. The frequency of the p53R175H 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.

Supplementary Material

1
2

TRANSLATIONAL RELEVANCE.

The traditional paradigm that governs the development of targeted anti-cancer drugs is to drive exposure to maximize efficacy. This is accomplished through maximizing half-life and dosing at the maximum tolerated dose. Our findings indicate that the mutant p53 reactivational activity of ZMCs is governed by a unique ON/OFF switch mechanism that allows the compounds to be effective with a brief exposure. ZMCs represent a new paradigm in cancer therapeutics defined by the idea that more is not always better and in fact can be worse, as maximizing drug exposure often increases toxicity. Metal ion chelators such as thiosemicarbazones have been plagued in the clinic by toxicity due to redox active metals such as iron or copper. These results impart an advantage to ZMCs that will allow them to be effectively dosed while avoiding these toxicities. The translation of the first in human ZMC therapeutic will be guided by these principles.

Acknowledgments

We thank Rutgers Cancer Institute of New Jersey Histology core facility to process the tumor blocks, Pharmacokinetics core facility to analyze ZMC1 content in tumor cells. We thank Dr. Kenneth Olive from Columbia University for providing KPC tumor cell lines (K8484 and K8483). We thank Dr. Eric Collisson from University of California, San Francisco for providing 270H cell line. We also thank Sai Life Sciences for performing pharamacokinetics studies and RUCDR Infinite Biologics for performing RNAseq 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 (D. Carpizo), and New Jersey Commission on Cancer Research Pre-doctoral fellowship (S. Kogan).

Footnotes

Authors’ Contributions:

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

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

Acquisition of data: X. Yu, S. Kogan, A.T. Tsang, T. Withers, H. Lin, J. Gilleran, B. Buckley Analysis and interpretation of data: X. Yu, S. Kogan, Y. Chen, B. Buckley, D. Moore, J. Bertino, C. Chan, S.D. Kimball, D.R.Carpizo

Writing, review of the manuscript: X. Yu, S. Kogan, H. Lin, J. Gilleran, S.D. Kimball, S. Loh, D.R.Carpizo

Disclosure of Potential Conflicts of Interest: Darren Carpizo and S. David Kimball are affiliated with Z53 Therapeutics, Inc. as founder and scientific board member respectively.

†

Co-first Authors

REFERENCES

  • 1.Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature 1997;387(6630):296-9. [DOI] [PubMed] [Google Scholar]
  • 2.Levine AJ, Oren M. The first 30 years of p53: growing ever more complex. Nature reviews Cancer 2009;9(10):749-58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Joerger AC, Fersht AR. Structure-function-rescue: the diverse nature of common p53 cancer mutants. Oncogene 2007;26(15):2226-42. [DOI] [PubMed] [Google Scholar]
  • 4.Freed-Pastor WA, Prives C. Mutant p53: one name, many proteins. Genes & development 2012;26(12):1268-86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Blanden AR, Yu X, Loh SN, Levine AJ, Carpizo DR. Reactivating mutant p53 using small molecules as zinc metallochaperones: awakening a sleeping giant in cancer. Drug Discov Today 2015;20(11):1391-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Yu X, Vazquez A, Levine AJ, Carpizo DR. Allele-specific p53 mutant reactivation. Cancer Cell 2012;21(5):614-25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Meplan C, Richard MJ, Hainaut P. Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells. Oncogene 2000;19(46):5227-36. [DOI] [PubMed] [Google Scholar]
  • 8.Butler JS, Loh SN. Structure, function, and aggregation of the zinc-free form of the p53 DNA binding domain. Biochemistry 2003;42(8):2396-403. [DOI] [PubMed] [Google Scholar]
  • 9.Hainaut P, Milner J. A structural role for metal ions in the "wild-type" conformation of the tumor suppressor protein p53. Cancer Res 1993;53(8):1739-42. [PubMed] [Google Scholar]
  • 10.Bozym RA, Thompson RB, Stoddard AK, Fierke CA. Measuring picomolar intracellular exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor. ACS chemical biology 2006;1(2):103-11. [DOI] [PubMed] [Google Scholar]
  • 11.Blanden AR, Yu X, Wolfe AJ, Gilleran JA, Augeri DJ, O'Dell RS, et al. Synthetic Metallochaperone ZMC1 Rescues Mutant p53 Conformation by Transporting Zinc into Cells as an Ionophore. Molecular pharmacology 2015;87(5):825-31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Yu Y, Kalinowski DS, Kovacevic Z, Siafakas AR, Jansson PJ, Stefani C, et al. Thiosemicarbazones from the old to new: iron chelators that are more than just ribonucleotide reductase inhibitors. Journal of medicinal chemistry 2009;52(17):5271-94. [DOI] [PubMed] [Google Scholar]
  • 13.Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc 2006;1(5):2315-9. [DOI] [PubMed] [Google Scholar]
  • 14.Lin H, Yu X, Eng OS, Buckley B, Kong AN, Bertino JR, et al. A sensitive liquid chromatography-mass spectrometry bioanalytical assay for a novel anticancer candidate--ZMC1. Biomed Chromatogr 2015;29(11):1708-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Yu X, Blanden AR, Narayanan S, Jayakumar L, Lubin D, Augeri D, et al. Small molecule restoration of wildtype structure and function of mutant p53 using a novel zinc-metallochaperone based mechanism. Oncotarget 2014;5(19):8879-92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Colvin RA, Holmes WR, Fontaine CP, Maret W. Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasis. Metallomics : integrated biometal science 2010;2(5):306-17. [DOI] [PubMed] [Google Scholar]
  • 17.Eide DJ. Zinc transporters and the cellular trafficking of zinc. Biochimica et biophysica acta 2006;1763(7):711-22. [DOI] [PubMed] [Google Scholar]
  • 18.Palmiter RD, Huang L. Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pflugers Archiv : European journal of physiology 2004;447(5):744-51. [DOI] [PubMed] [Google Scholar]
  • 19.Andrews GK. Cellular zinc sensors: MTF-1 regulation of gene expression. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 2001;14(3–4):223-37. [DOI] [PubMed] [Google Scholar]
  • 20.Kimura T, Kambe T. The Functions of Metallothionein and ZIP and ZnT Transporters: An Overview and Perspective. Int J Mol Sci 2016;17(3):336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kerns EH, Di L, Petusky S, Farris M, Ley R, Jupp P. Combined application of parallel artificial membrane permeability assay and Caco-2 permeability assays in drug discovery. J Pharm Sci 2004;93(6):1440-53. [DOI] [PubMed] [Google Scholar]
  • 22.Hingorani SR, Wang L, Multani AS, Combs C, Deramaudt TB, Hruban RH, et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer cell 2005;7(5):469-83. [DOI] [PubMed] [Google Scholar]
  • 23.Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 2009;324(5933):1457-61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Frese KK, Neesse A, Cook N, Bapiro TE, Lolkema MP, Jodrell DI, et al. nab-Paclitaxel potentiates gemcitabine activity by reducing cytidine deaminase levels in a mouse model of pancreatic cancer. Cancer Discov 2012;2(3):260-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Provenzano PP, Cuevas C, Chang AE, Goel VK, Von Hoff DD, Hingorani SR. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer cell 2012;21(3):418-29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Cook N, Frese KK, Bapiro TE, Jacobetz MA, Gopinathan A, Miller JL, et al. Gamma secretase inhibition promotes hypoxic necrosis in mouse pancreatic ductal adenocarcinoma. J Exp Med 2012;209(3):437-44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004;303(5659):844-8. [DOI] [PubMed] [Google Scholar]
  • 28.Bykov VJ, Issaeva N, Shilov A, Hultcrantz M, Pugacheva E, Chumakov P, et al. Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nature medicine 2002;8(3):282-8. [DOI] [PubMed] [Google Scholar]
  • 29.Xue W, Zender L, Miething C, Dickins RA, Hernando E, Krizhanovsky V, et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 2007;445(7128):656-60. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS974549-supplement-1.docx (32.3KB, docx)
2
NIHMS974549-supplement-2.pdf (769.4KB, pdf)

RESOURCES