A cancer-selective zinc ionophore inspired by the natural product

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A cancer-selective zinc ionophore inspired by the natural product naamidine A Rachel M Vaden, Katrin P. Guillen, Justin M. Salvant, Celine B. Santiago, Joseph B. Gibbons, Satya S Pathi, Sasi Arunachalam, Matthew S Sigman, Ryan E. Looper, and Bryan E. Welm ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00977 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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Title: A cancer-selective zinc ionophore inspired by the natural product naamidine A Authors: Rachel M. Vaden1, Katrin P. Guillen2,3, Justin M. Salvant1, Celine B. Santiago1, Joseph B. Gibbons1, Satya S. Pathi2,3, Sasi Arunachalam2,3, Matthew S. Sigman1, Ryan E. Looper*1, Bryan E. Welm*3,4 Author affiliations: 1Department of Chemistry, University of Utah, 315 S 1400 E, Salt Lake City, Utah, 84112, USA. 2Department

of Oncological Sciences, 3Huntsman Cancer Institute, 4Department of Surgery, University of Utah, 2000

Circle of Hope, Salt Lake City, UT 84112, USA *Corresponding authors: E-mail: [email protected], [email protected] Keywords: zinc trafficking, zinc dyshomeostasis, cancer therapeutics, small molecule design, natural products ABSTRACT

We present data demonstrating the natural product mimic, zinaamidole A (ZNA), is a modulator of metal ion homeostasis causing cancer-selective cell death by specifically inducing cellular Zn2+-uptake in transformed cells.

ZNA’s cancer

selectivity was evaluated using metastatic, patient-derived breast cancer cells, established human breast cancer cell lines, and 3-dimensional organoid models derived from normal and transformed mouse mammary glands. Structural analysis of ZNA demonstrated that the compound interacts with zinc through the N2-acyl-2-aminoimidazole core.

Combination

treatment with ZnSO4 strongly potentiated ZNA’s cancer-specific cell death mechanism, an effect that was not observed with other transition metals. We show that Zn2+-dyshomeostasis induced by ZNA is unique and markedly more selective than other known Zn2+-interacting compounds such as clioquinol. The in vivo bioactivity of ZNA was also assessed and revealed that tumor-bearing mice treated with ZNA had improved survival outcomes. Collectively, these data demonstrate that the N2-acyl-2-aminoimidazole core of ZNA represents a powerful chemotype to induce cell death in cancer cells concurrently with a disruption in zinc homeostasis. INTRODUCTION The marine natural product naamidine A (NA) was isolated in 1987

HO

HO

from the marine sponge Leucetta chagosensis (Fig. 1).1 During the

N N

initial biological evaluation of NA, it was reported that the natural product exhibited sub-micromolar inhibition of Epidermal Growth Factor (EGF)-stimulated DNA synthesis, a response that was not observed with other mitogenic stimuli.2 Although NA inhibited EGFmediated signaling in A-431 cells, it was demonstrated that this

Me N

MeO Me O

O naamidine A

Me N

NH

N

MeO

O N Me

N

N

O

ZnII N N

O

N

N Me O

N OMe

N Me

Zn( naamidine A)2 OH

Figure 1. Structure of naamidine A and its Zn2+ coordination complex.

effect occurred independently of EGF-EGFR binding. Subsequent in vitro studies focused on the MAPK/ERK signaling pathway found that NA generated robust and sustained ERK1/2 signal leading to cell cycle arrest in the G0-G1 phase. ACS Paragon Plus Environment

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However, treatment of cells with the known MEK inhibitor, U0126, which decreases ERK-phosphorylation levels, did not rescue cell death. Subsequent in vivo tumor xenograft studies revealed that treatment with NA induced apoptosis via activation of caspase 3.3 JC-1 staining showed that NA causes mitochondrial membrane depolarization and this was correlated with the induction of apoptosis, providing an alternative explanation for the cytotoxicity of NA in A-431 cells. Since the isolation of NA, numerous studies have described the cytotoxicity of related Leucetta alkaloids against cancer cells, but have failed to discern the relative toxicity between transformed and untransformed cell lines. 4-6 We were intrigued that NA and related natural products had been isolated as Zn2+ dimers from the marine environment (e.g. 2:1 Zn:naamidine A) (Fig. 1).7,8 An ecological hypothesis followed that these compounds may have evolved to serve as zinc siderophores as free Zn2+ is functionally limited in shallow marine environments, analogous to free Fe2+ pools in terrestrial environments.9 Unlike iron siderophores, which typically display three chelating groups to accommodate iron’s octahedral ligand arrangement, these natural products have perhaps evolved to address the problem of selectively coordinating a tetrahedral zinc ion through the formation of mixed dimeric complexes. Thus, these natural products may serve as the marine equivalent of terrestrial bacterial siderophores by mobilizing free Zn2+ for uptake into host cells. Mitochondrial membrane potential has been linked to the availability of Zn2+ pools; if these natural products function to increase intracellular Zn2+, it follows that they could trigger mitochondrial membrane depolarization, thereby offering a mechanistic hypothesis for the apoptotic phenotype observed with NA.10 Taken together, we aimed to dissect the potential role of these natural products as Zn2+ binders versus agonists of the MAPK pathway. Herein, we report the discovery of zinaamidole A (ZNA), a naamidine A-inspired N2-acyl-2-aminoimidazole whose biological activity is highly selective for transformed cells.

Decoupling the activity of NA from ERK activation by

replacement of the N-Me-dehydrohydantoin allowed for the development of a class of compounds capable of delivering superstoichiometric payloads of zinc selectively into cancer cells. In all, the work presented applies multiple in vitro and in vivo model systems to extensively characterize ZNA’s effects in different biological settings; the data demonstrate that ZNA targets a unique and vulnerable pathway in transformed cells that regulates zinc homeostasis. RESULTS AND DISCUSSION Identification of an NA analog capable of selectively inhibiting cancer cell proliferation Given the two previously reported broad characteristics of NA (MAPK pathway perturbation and direct Zn2+ binding), we employed a synthetic chemistry strategy to parse the architectures in the natural product that associate with each phenotype.

Five-membered heterocycles with multiple heteroatoms (e.g., the N-Me-dehydrohydantoin) are

frequently regarded as promiscuous binders.11 Our first attempt to decouple the complex activity of NA focused on removal of the N-Me-dehydrohydantoin.

To this end, we developed new chemistry to succinctly access N2-acyl-2-

aminoimidazoles, thus maintaining a Lewis-base that could coordinate Zn2+, but not the plethora of heteroatoms as in the hydantoin that could lead to promiscuous binding.12,13 A subset of these newly synthesized molecules was first evaluated in a pre-clinical cancer model previously developed in our laboratories, utilizing primary metastatic tumor cells derived from malignant pleural effusions (PEs) of breast cancer patients.14

The use of these cells afforded the opportunity to model metastatic disease and

chemoresistance as established during patient treatment from physiologically-relevant exposure to clinical therapies. Utilizing these cells, we conducted a small molecule screen with the overall goal of identifying compounds that target the growth and survival of cancer cells that are refractory to standard clinical treatments. In a parallel screen, immortalized human mammary epithelial cells (hTERT-HMEC) were used to discriminate molecules with toxicity towards normal cells. ACS Paragon Plus Environment


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From this screen, two key discoveries were found: 1) NA was not selective for transformed cells and induced 100% decrease in cell viability at 10 M in both the PE and hTERT-HMEC cells and 2) a novel N2-acyl-2-aminoimidazole (compound 1) was initially identified as selectively inhibiting the PE cells (84% inhibition at 10 M) versus hTERT-HMEC (18% inhibition at 10 M).14 Compound 1 was the only compound in the initial screen possessing the C5-aryl substitution pattern. Ten other NA analogs all possessed the C4-benzyl substituent as in the parent natural product and were either not active or not selective. To begin to understand the unique activity of these N2-acyl-C5-aryl-2-aminoimidazoles, we prepared a small, focused library of structurally related compounds drawing on synthetic methods developed previously by our group.13

To elucidate whether the C5-aryl group or the N2-acyl group might dominate potency, two sets of

compounds were generated with the first being varied in the N2-acyl group (compounds 1-7) and the second set (compounds 8-11) varied in the C5-aryl substituent (Fig. 2A). MCF-10A and MCF-7 cells, untransformed mammary epithelial cells and a breast cancer cell line respectively, were used to evaluate this set of compounds

(Fig. 2B). When assessing analog activity, we evaluated both the EC50 in MCF-7 cells and the difference in area under the curve (AUC) between MCF-7 and MCF-10A to allow for comparison of analogs that induced only partial reductions in cell viability. Incorporation of aryl groups containing different substitutions (compounds 2-6), demonstrated that 2-FPh substitution (compound 4) displayed a significant increase in potency and selectivity.

Figure 2. Evaluation of a focused library of NA analogs. (A) Focused library of N2-acyl-C5-aryl-2-aminoimidazoles alongside NA. (B) EC50 values for MCF-7 cells represent the mean of three replicates. AUC represents the difference in AUC between MCF-7 and MCF-10A cells. Structure of the novel small molecule zinaamidole A (compound 4, ZNA). (C) Dose response measurements following ZNA treatment (4 days) in immortalized primary human mammary epithelial cells (hTERT-HMEC) and primary metastatic chemoresistant breast cancer cells (PE1005339). Values plotted represent the mean and standard deviation of three replicates. (D) Dose response measurements following ZNA treatment (5 days) in an untransformed mammary epithelial cell line (MCF-10A) and three breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). Values plotted represent the mean and standard deviation of three replicates. (E) Following 24 hours of treatment with ZNA, cells were cultured in ZNA-free media for 21 days; colonies were then stained with crystal violet and quantified. Values plotted represent the mean and standard deviation of four replicates. (F) Real-time PCR (RT-PCR) measurements of metal trafficking gene induction following either 3 or 24 hours of treatment with ZNA (30 M, in quadruplicate). All data was internally normalized to GAPDH expression.

Transposition of the fluorine to the meta (compound 5) and para (compound 6) positions and the 2,6-diflouro-substitution (compound 8), confirmed that the 2-FPh substitution impacted potency. Alteration of the C5 aryl substitution proved less ACS Paragon Plus Environment

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consequential for the potency of analogs; however, more electron-deficient substitution appeared to be less selective between cell lines (e.g. compounds 9 and 11). Compounds 4 and 10, which differ only in a 4-OMePh versus 3-OMePh at the C5-substitution, have similar activity. From this evaluation, compound 4 was identified, which was named zinaamidole A (ZNA), as having the greatest balance of potency and selectivity between transformed and untransformed breast cancer cells. Thus, ZNA served as our tool compound to further probe the mechanism by which these novel heterocyclic cores, inspired by NA, were selectively inducing tumor cell death. Validating ZNA’s selective anti-proliferative activity ZNA’s initial screening profile in MCF-7 / MCF-10A cell lines was validated with a 12-point dose response assay using malignant pleural effusion cells (PE1005339) obtained from a second breast cancer patient with chemoresistant disease. The data revealed that ZNA was effective at low micromolar concentrations against tumor cells (EC50 = 4.12 M) and maintained a relatively large therapeutic treatment window even at high concentrations (IC50 >100 M) (Fig. 2C). To further assess the generality of ZNA’s breast cancer selectivity and cytotoxicity, dose response assays were performed using three breast cancer cell lines (MCF-7, T47D, and MDA-MB-231) in comparison to the untransformed mammary epithelial cell line (MCF-10A) (Fig. 2D). As with the immortalized human breast epithelial cells and primary patient-derived cancer cells, treatment with ZNA resulted in decreased cell viability in the malignant cell lines, whereas the untransformed breast epithelial cell line exhibited reduced sensitivity to the compound. Further experiments were conducted to assess the effects of short-term ZNA treatment on long-term cell survival and proliferation. Cells were treated for 24 hours with ZNA then cultured in the absence of the small molecule for three weeks and surviving colonies were stained with crystal violet and quantified (Fig. 2E). From this assay, it was discovered that short-term exposure, even at normally sub-lethal concentrations, significantly affected long-term proliferative potential in cancer cells; again, as with the dose response assays, the effect on untransformed MCF-10A cells was greatly attenuated compared to the malignant cell types tested. The small molecule ZNA activates metal response genes The activity of ZNA against patient-derived cancer cells and established cancer cell lines, but not untransformed cells, suggests the small molecule targets a vulnerable pathway common to transformed cells. To help identify this pathway, MCF-7 breast cancer cells were treated with ZNA for 3 and 12 hours, followed by RNAseq profiling to determine transcriptome changes induced by the compound.

Genes associated with cell cycle regulation were significantly

downregulated following treatment while the most upregulated genes encoded metallothionein proteins MT1F, MT1X, and MT2A (Supplemental Fig. 1 and 2). Members of the metallothionein family are cysteine-rich, redox-active proteins known to directly bind transition metals such as copper and zinc and are involved in intracellular metal distribution and sequestration.15 Transcriptome profiling also revealed increased expression of two transcripts encoding Zn2+ transport proteins, SLC30A1 and SLC30A2, following treatment with ZNA. To validate the RNAseq data, real-time PCR (RT-PCR) gene expression studies were conducted for the five metal trafficking genes identified as differentially upregulated (Fig. 2F). Following ZNA treatment, metal trafficking genes were highly expressed in MCF-7 cells (75-fold), confirming the RNAseq data, but only moderately induced in untransformed MCF-10A cells (5-fold). Collectively, transcriptome profiling and RT-PCR results suggest that ZNA treatment affects intracellular metal trafficking; furthermore, the transcriptional changes associated with SLC30A1 and SLC30A2 more specifically suggest that Zn2+ dyshomeostasis may occur following exposure to the small molecule, consistent with our initial siderophore hypothesis for the natural product NA.



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ZNA induces intracellular Zn2+ accumulation in cancer cells Zinc is a biologically important transition metal that is critical for transcriptional, metabolic, and enzymatic processes within cells.

Dysregulation of zinc homeostasis is critical in many cancers and may contribute to the

proliferative and metabolic properties of tumors.16-21 We were interested in assessing whether ZNA affected zinc uptake and localization in cancer cells and whether zinc dysregulation mediated the compound’s cancer-selective mechanism of action. To determine whether ZNA affected Zn2+ uptake, we used the fluorescent Zn2+ indicator FluoZin-3 and flow cytometry to quantify intracellular Zn2+ levels following ZNA treatment (Fig. 3A). Cells were treated with ZNA for 1, 3, 24 or 48 hours, followed by staining with FluoZin-3. We found that ZNA treatment of MCF-7 cells resulted in a small but measurable increase in FluoZin-3 fluorescence as early as 1 hour following treatment and a 13-fold increase after 48 hours. In contrast, MCF-10A cells showed only a 2-fold increase in intracellular Zn2+ at 48 hours. FluoZin-3 staining was also assessed by fluorescence microscopy of MCF-10A and MCF-7 cells treated with ZNA or DMSO for 48 hours. Similar to the flow cytometry analysis, strong FluoZin-3 fluorescence was observed in MCF-7, but not MCF-10A cells following ZNA treatment (Fig. 3B). Considering that increased metallothionein gene expression might participate in attenuating a rise in intracellular Zn2+, cells were exposed to ZNA in conjunction with an inhibitor of transcription (Actinomycin D, ACTD) for 24 hours and intracellular Zn2+ was subsequently measured by flow cytometry analysis of FluoZin-3 (Supplemental Fig. 3). Co-treatment of cells with ZNA and ACTD resulted in a statistically significant increase in intracellular Zn2+ compared to ZNA-alone in MCF-7 cells. intracellular

Zn2+

These data suggest gene transcription in MCF-7 cells plays a role in attenuating

levels, which is consistent with the increased expression of metallothionein and Zn2 transporter genes in

cells treated with ZNA. In contrast, ACTD did not increase FluoZin-3 staining in MCF-10A cells, suggesting that either ZNA induces differential uptake of Zn2+ between the two cell types or MCF-10As cells have a greater ability than MCF-7s to buffer or efflux intracellular Zn2+ without requiring transcriptional activity. Given these results, we next sought to determine whether a direct interaction between ZNA and Zn2+ was required for ZNA’s cytotoxic effects. The addition of ZnSO4 to a solution of ZNA generated an isolable Zn2+-ZNA complex. 1H

NMR analysis of the resulting species revealed that the benzylic methylene singlet was transformed into an AB quartet,

suggesting the presence of a diastereotopic methylene group and thus a tetrahedral 2:1 (ZNA)2:Zn2+ complex. This was ultimately confirmed by X-ray crystallography, which revealed a tetrahedral Zn2+ center with ZNA bound in a bidentate fashion through N3- of the 2-aminoimidazole and the pendant N2-acyl group (Fig. 3C). To further define the role of the ZNA-Zn2+ complex in a biological context, an N2-methylated derivative of ZNA (N2-Me-ZNA) that would be incapable of binding Zn2+ as an anionic ligand was prepared (Fig. 3D). Evaluation of the activity of N2-Me-ZNA on cell viability after a 96-hour exposure indicated that N2-methylation significantly abrogated the compound’s cytotoxic effect. These data strongly suggest that ZNA’s cytotoxicity is directly coupled to its ability to complex Zn2+ ions. We next assessed whether complexation with other physiologically relevant cations might also impact activity. The complexation of ZNA with Cu2+ or Fe2+ provided colored complexes (max = 510 and 650 nm respectively) that were amenable for UV absorbance-based assays but attempts to quantify the ZNA-Zn2+ interaction proved problematic due to the colorless nature of the complex. However, a qualitative assessment based on competitive titration of Zn2+ with the Cu2+-ZNA or Fe2+-ZNA complexes resulted in a significant decrease in the absorbance for the ZNA:Fe2+ complex at 650 nM, suggesting that Zn2+ readily competes for ZNA binding with iron binding. In contrast, the addition of Zn2+ to the ZNA:Cu2+ complex resulted in a persistence of the absorbance at 510 nm suggesting that Zn2+ does not compete for ZNA binding and thus complexes preferentially with Cu > Zn > Fe (Fig. 3E).


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Figure 3. ZNA promotes increases in intracellular Zn2+. (A) Quantification of intracellular Zn2+ using the fluorescent indicator FluoZin-3; values represent the mean and SEM of three replicates and statistical significance indicates comparison with the control sample. (B) FluoZin-3 (green), LysoTracker (red), and Hoechst (blue) staining of MCF-10A and MCF-7 cells treated with ZNA (30 M) for 48 hours. Scale bar = 50 m. (C) X-ray structure of the Zn(ZNA)2 complex. (D) Structure of the N2-methylated derivative of ZNA, N2-Me-ZNA and measurement of cell viability (ATP quantification) following 48 hours of treatment. Values plotted represent the mean and SEM of three replicates. (E) UV-Vis Competition experiments of Zn2+ with the preformed complexes of ZNA with Cu2+ and Fe2+.

Co-treatment with Zn2+ strongly potentiates the cytotoxicity and cancer selectivity of ZNA Considering the observed binding interactions of ZNA with Zn2+, Cu2+ and Fe2+, we next evaluated their effects on ZNA’s cytotoxic phenotype (Fig. 4A).

The combination of ZNA and Fe2+ had relatively little effect on either the

untransformed or transformed cells lines, consistent with the observed weak binding of ZNA with Fe2+. Strong synergy was discovered between ZNA and Cu2+, and between ZNA and Zn2+; co-treatment with ZNA and Cu2+ resulted in nonselective cell death, with cytotoxicity observed in both MCF-10 and MCF-7 cell lines. treatment with ZNA and

Zn2+

In contrast, the combination

was both highly synergistic and cancer selective. Evaluation of a broader series of metals

including Ni, Co and Mn resulted in minimal changes in cell viability compared to treatment with ZNA alone (Supplemental Fig. 4).

To assess whether this potentiation of ZNA’s activity and selectivity was related to an increased cellular

accumulation of Zn2+ we again quantified Zn2+ cellular uptake. As noted in the absence of exogenous Zn2+, a 13-fold increase in cellular concentration is observed in MCF-7 cells after 48 hrs (Fig. 3A). In the presence of 30 M Zn2+, which is thought to reflect the average exchangeable pool of Zn2+ in human plasma,22 MCF-7 cells were found to have a 170 times higher intracellular Zn2+ concentration after just 3 hours, as measured by FluoZin-3 staining (Fig. 4B). This increase in total cellular Zn2+ in MCF-7 cells following a three-hour treatment with ZNA or ZNA/ZnSO4 was also confirmed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Supplemental Fig 5). While untransformed MCF10A cells also experienced an increase in intracellular Zn2+ with co-treatment, the level was less than 10-fold higher than the vehicle control and was below detectable levels by ICP-AES analysis. In all, the combination of ZNA and ZnSO4 was strongly synergistic (Supplemental Fig. 6) in cancer cells as measured by intracellular zinc uptake and cell death assays. Subsequent dose response experiments revealed that the maximal inhibition of cell viability occurred at a ~1:1 concentration of ZNA:Zn2+ (Fig. 4C). Again, this demonstrates the critical nature of ZNA:Zn2+ binding as related to activity, with higher ZNA concentrations relative to Zn2+ resulted in partially diminished activity. These drastically different rates and magnitude of Zn2+ accumulation, based both on concentration of exogenous metal and metal-ligand stoichiometry led ACS Paragon Plus Environment


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us to distinguish cytostatic and cytotoxic phenotypes observed in the absence and presence of exogenous Zn2+ respectively. Co-treatment of cells with ZNA and ZnSO4 resulted in cell death of >80% of MCF-7 cells within 24 hours, a synergistic increase compared to ZNA alone (Fig. 4D).

Figure 4. Cu2+ and Zn2+ potentiate ZNA’s cytotoxic phenotype in malignant cells. (A) Effect on cell viability of ZNA treatment in combination with exogenously added transition metals. Cell viability was quantified by measuring cellular ATP content following 24 hours of treatment. Values plotted represent the mean and SEM of three replicates normalized to respective controls. (B) Zn2+ uptake in MCF-7 and MCF-10A cell lines as measured by relative fluorescence with FluoZin-3. (C) Titration of ZNA in the presence of 30 M ZnSO4. MCF-7 cell viability was measured after 24 hours of treatment by quantifying cellular ATP content. (D) Measurement of cell viability following treatment with ZNA in combination with ZnSO4. Cells were treated for 24 hours, stained with propidium iodide, and subsequently analyzed by flow cytometry. The values plotted represent the mean and SEM of three replicates normalized to the control sample. (E) Measurement of cellular proliferation following treatment with ZNA (30 M) and/or ZnSO4 (30 M) for 6 hours. Values represent the mean percent EdU incorporation and SEM of three experimental replicates. Statistical significance annotated for conditions as compared to control.

Most notably, the untransformed MCF-10A line was unaffected by either ZNA alone or the combination treatment. These data demonstrate that the ZNA/ZnSO4 combination induces potent cancer-selective cellular effects within a short time period and suggest that treatment with ZNA alone is largely cytostatic. Similarly, a 6-hour treatment with ZNA/ZnSO4 was sufficient to induce a complete block in EdU incorporation in MCF-7 cells (Fig. 4E), while only partially inhibiting EdU incorporation in MCF-10A cells. Treatment of either cell type with ZNA alone resulted in only a partial reduction in EdU incorporation, again consistent with the induction of a cytostatic phenotype. Importantly, ZNA/ZnSO4 was selective for cancer cells- nearly all MCF-7 cells exhibited either cell death or cell cycle arrest within 24 hours after treatment, in contrast to the minimal effects observed in MCF-10A cells. ZNA induces caspase independent cell death

Having noted a strong effect on PI uptake, we assessed several cell death pathways to better understand the mechanism by which ZNA/ZnSO4 kills cancer cells (Fig. 5). Given NA’s reported induction of apoptosis via the activation of caspase 3 we first examined this pathway. Since MCF-7 cells lack functional caspase 3,23 we examined T47D, MDAMB-231, and MCF-10A cell to establish whether ZNA-treated cells undergo apoptosis (Fig. 5A). Surprisingly, caspase 3/7, 8, or 9 activity was either minimal or not observed in ZNA or ZNA/ZnSO4 treated cells in the three cell lines examined ACS Paragon Plus Environment

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(even after 72 hours). These data demonstrate that ZNA alone and in combination with ZnSO4 do not promote classic caspase-mediated apoptotic cell death, suggesting NA and ZNA induce different mechanisms of cell death. We examined whether ZNA induced necroptosis, a caspase-independent mechanism of cell death that occurs through the activation of receptor interaction protein kinase 1 (RIP1).24 Cell viability was not affected by the RIP1 inhibitor Necrostatin, which suggests ZNA and ZNA/ZnSO4 induce a non-necroptotic mechanism of cell death (Supplemental Fig 7).25

Figure 5. ZNA induces caspase independent cell death. (A) Quantification of caspase activity following treatment with ZNA and ZnSO4 for either 6 or 72 hours. Staurosporine (STS) was used as a positive control. The plotted values represent the mean and standard deviation of three replicates. (B) Assessment of FluoZin-3 (green), LysoTracker (red), and Hoechst (blue) staining of MCF-10A and MCF-7 cells treated with ZNA (30 M) and/or ZnSO4 (30 M) for 2 hours. Scale bar = 10 m. (C) Quantification of FluoZin-3 and LysoTracker co-localization. (D) Cytoplasmic cathepsin activity as measured with an enzymatic assay following 6 hours of treatment. Values represent the mean activity compared to the control and SEM of three experimental replicates. (E) Cytoplasmic cathepsin activity as measured with cytoplasmic fractionation followed by cellular fractionation.

Vesicles such as lysosomes have been reported to function as reservoirs for zinc when intracellular levels of the metal ion exceed a cell’s ability to buffer concentrations through metallothioneins or efflux mechanisms.26,27

We

hypothesized that lysosomes may sequester excess Zn2+ following ZNA/ZnSO4 co-treatment, resulting in lysosomal dysfunction. To evaluate this hypothesis, we first determined whether Zn2+ accumulated in lysosomes by co-staining ZNA-treated cells with FluoZin-3 and LysoTracker (Fig. 5B). Quantification of the co-localization revealed a significant increase in FluoZin-3 and LysoTracker overlap in MCF-7 cells upon combination treatment with ZNA and ZnSO4, suggesting that intracellular zinc accumulates preferentially in the lysosome (Fig. 5C). Excessive storage of intracellular zinc in the lysosome has been reported to induce lysosome membrane permeabilization (LMP), resulting in the release of cathepsins into the cytoplasm followed by the induction of cell death.28.29 Indeed, treatment with the combination of ZNA ACS Paragon Plus Environment


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and ZnSO4 resulted in a significant increase in cytosolic cathepsin activity after 6 hours in MCF-7 cells but not MCF-10As (Fig. 5D). Treatment of MCF-7s with ZNA/ZnSO4 also results in an increase in cytosolic cathepsins B, D and L relative to untreated cells (Fig. 5E). Taken together, these data suggest that synergism of ZNA and ZnSO4 induces cancer specific cell death through a caspase-independent mechanism and may be mediated, at least in part, by LMP. ZNA’s cancer-specific cytotoxicity is maintained in three-dimensional organoid culture models Having demonstrated that ZNA is highly synergistic with Zn2+ and selective for killing transformed versus untransformed cell lines, we further evaluated the compound’s activity in more complex tissue models. The activity of ZNA and ZNA/Zn2+ was assessed in a three-dimensional (3D) organoid model of normal mammary growth and morphogenesis and compared with primary tumor organoids derived from Polyomavirus Middle T-antigen (PyMT) mammary tumors (Fig. 6A/B).30,31 When grown in laminin-rich 3D gels (Matrigel), primary mouse mammary epithelial cells (MECs) establish organoids that undergo branching morphogenesis and replicate the normal cellular architecture of mammary ducts.22 Upon exposure to toxic compounds, dying cells are actively extruded from the organoid, resulting in a visual cue that can be used to evaluate a compound’s cytotoxicity (Fig. 6C). Mouse primary MECs embedded in Matrigel were treated with ZnSO4, ZNA, or a combination of ZNA/ZnSO4, and after 144 hours, Calcein AM and SYTOX Orange were used to identify live and dead cells, respectively (Fig. 6A and Supplemental Fig. 8A). Qualitatively, treatment with Zn2+, ZNA or ZNA/Zn2+ permitted healthy organoid development in these untransformed primary cells. Quantification of the branching phenotypes revealed that mammary organoids treated with ZnSO4 or ZNA exhibited a 24-hour delay in branching morphogenesis (compare with 72-hour time point images in Supplemental Fig. 8B), and at the assay’s endpoint, a minor reduction in healthy, fully branched organoids was observed (Fig. 6D). Overall, normal mammary organoids tolerated both ZNA- and ZNA/ZnSO4-treatment and exhibited minimal SYTOX Orange uptake or extrusion of dying cells.

Greater than 60% of combination-treated organoids were healthy with no visible extruding cells, and

approximately 90% of organoids exhibited either full or partial branching. These effects mirrored the partial cytostatic effects observed with untransformed MCF-10A cells in the two-dimensional cell culture assays (Fig. 6E). Evaluation of ZNA’s activity in 3D culture using PyMT primary tumor organoids demonstrated marked cancer-specific cytotoxicity akin to the cell culture assays (Fig. 6B and Supplemental Fig. 8B). In contrast to normal mammary organoids, PyMT tumor organoids exhibited a strong cell extrusion phenotype in about 70% of organoids treated with ZNA-alone, while ZNA/ZnSO4 co-treatment was cytotoxic to over 80% of the organoids. These data demonstrate that ZNA has broad anticancer activity against primary tumor cells and established cancer cell lines of mouse or human origin and maintains its cancer-selective activity in complex tissue models.


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Figure 6. Three-dimensional culture models recapitulate ZNA’s phenotype. (A) Matrigel-embedded organoids of mouse mammary epithelial cells were stimulated to branch with fibroblast growth factor-2 (FGF2). Cells were treated with ZNA and/or ZnSO4 or a vehicle control starting at 48 hours to assess the effects of the small molecule on branching morphogenesis. Following 144 hours of growth, organoids were stained with Hoechst nuclear stain (blue), calcein AM live cell stain (green), and SYTOX Orange dead cell stain (red). Scale bar = 20 m. (B) Mouse Polyomavirus Middle T-Antigen (PyMT) tumor cells were embedded as organoids into Matrigel and cultured for 144 hours. Starting at 48 hours, cells were treated with ZNA and/or ZnSO4 or a vehicle control for the duration of the assay. Following 144 hours of growth, organoids were stained with Hoechst nuclear stain (blue), calcein AM live cell stain (green), and SYTOX Orange dead cell stain (red). Scale bar = 20 mm. (C) Representative examples of the phenotypes observed in the three-dimensional assay. Scale bar = 20 mm. (D) Quantification of the phenotypes observed in panels A and B.

ZNA suppresses tumor growth in an in vivo mouse mammary tumor model Having characterized the relationship between ZNA and Zn2+ in multiple different in vitro models, we next sought to establish the relevance of ZNA in a mouse tumor model. To determine bioactivity, we first evaluated whether ZNA would elicit a response in normal tissue. Non-tumor bearing FVB/NJ mice were treated with ZNA (at 100 mg/kg administered via intraperitoneal injection) or vehicle control for either 3 or 24 hours. RT-PCR was used to assess mouse metallothionein expression (MT1 and MT2) in renal and hepatic tissue from the treated mice. The results revealed that MT1 and MT2 expression were increased following 3 hours of ZNA treatment as compared to control treated mice but were decreased following 24 hours (Fig. 7A). This trend is consistent with the results of the analogous in vitro RT-PCR experiments conducted with breast cancer cell lines suggesting that ZNA has activity in vivo. To evaluate the activity of ZNA in a cancer model we generated PyMT-expressing mouse mammary cells and transplanted the cells into the fat pads of 3week old female recipient mice.31 Tumor-bearing mice were placed in four treatment cohorts: ZNA (100 mg/kg administered via intraperitoneal injection), ZnSO4 (25 mM administered continuously via drinking water), a combination of ZNA and ZnSO4, and a control group (PBS administered via intraperitoneal injection). Mice were treated once a day for 21 days and were euthanized when tumors reached an endpoint diameter of 2 cm. We found that treatment with either ZNA alone or ZNA/ZnSO4 resulted in a significant survival advantage compared to the control-treated or ZnSO4-only groups (Fig. 7B). Additionally, no general toxicity, adverse reactions, or decreases in body weight were observed in the mice treated with ZNA or with the combination of ZNA and ZnSO4. Taken together, these data provide evidence that ZNA represents a novel class of Zn2+ modulating compounds with in vivo anticancer activity and low general toxicity. ACS Paragon Plus Environment


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Figure 7. ZNA suppresses tumor growth in an in vivo mouse mammary tumor model. (A) Real-time PCR (RT-PCR) measurements of metallothionein gene induction following either 3 or 24 hours of treatment with ZNA in non-tumor bearing FVB mice (100 mg/kg, intraperitoneal administration). All data was internally normalized to b-actin gene expression. Statistical significance indicates comparison with the control sample to which each condition was normalized. (B) Kaplan-Meier survival analysis. FVB mice bearing transplanted PyMT mammary tumors were treated according to the following conditions for 21 days: Control (intraperitoneal PBS injection once per day), ZNA (100 mg/kg, intraperitoneal injection once per day), ZnSO4 (25 mM administered continuously via drinking water), or combinatorial treatment (100 mg/kg ZNA, intraperitoneal injection once per day and 25 mM ZnSO4, administered continuously via drinking water). Statistical significance: Control versus ZNA, ** (p=0.01); ZnSO4 versus combinatorial treatment, ** (p=0.007).

The effect of ZNA on transformed cells is distinct from other known zinc interactors including NA Clinically used as an antiparasitic, clioquinol serves as the most well-studied Zn2+ ionophore. Its clinical use was discontinued due to the emergence of subacute myleo-optic neuropathy after high, cumulative doses of the drug in a subset of patients.32 The recent discovery of clioquinol’s anticancer properties, as a result of its ionophore activity, are well documented and have led to clinical trials to evaluate the potential of the drug to treat hematological malignancies.29,33-36 Clioquinol’s ionophore activity has also been exploited to decrease the pathogenesis of Huntington’s disease and the Aplaques associated with Alzheimer’s disease (AD), ultimately progressing to phase II clinical trials for targeting amyloid deposition in AD.34,37-39 Clioquinol’s activity, with IC50s in the 5-50 M range against a variety of cancer cell lines, has been reported to reduce tumor volume in vivo and has been shown to induce apoptosis by caspase activation via lysosomal membrane depolarization (LMP).29 In comparison, ZNA induces LMP but does not trigger caspase activation, suggesting that while clioquinol and ZNA share Zn2+ ionophore activity, their cytotoxic effects diverge.

In an effort to better

understand this reported divergence of these phenotypes, we directly compared the cellular and cytotoxic effects induced by ZNA, clioquinol, and NA in the presence and absence of exogenous Zn2+ (Fig. 8). We first measured the effects of the three molecules alone on intracellular Zn2+ levels in untransformed mouse MECs and MCF-10A cells, in comparison to MCF-7 cells (Fig. 8A).


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Figure 8. The effect of ZNA on transformed cells is distinct from other known zinc modulators. (A) Quantification of intracellular Zn2+ using the fluorescent indicator FluoZin-3 following 3 hours of treatment; values represent the mean and SEM of three replicates and statistical significance indicates comparison with the control sample. (B) Measurement of cell viability (ATP quantification) following 48 hours of treatment. Values plotted represent the mean and SEM of three replicates. (C) Measurement of cell viability following treatment with ZnSO4 in combination with ZNA (30 M), clioquinol (30 M), or NA (15 M). Cells were treated for 48 hours, stained with propidium iodide, and subsequently analyzed by flow cytometry. The values plotted represent the mean and SEM of three replicates normalized to the control sample. (D) Comparative dose response curves with both ZNA and clioquinol and +/- 30 M ZnSO4 in MCF-10 and MCF-7 cells. (E) Matrigel-embedded organoids of mouse mammary epithelial cells were stimulated to branch with fibroblast growth factor-2 (FGF2). Cells were treated with Clioquinol, NA, and/or ZnSO4 or a vehicle control starting at 48 hours to assess the effects of the small molecule on branching morphogenesis. Following 144 hours of growth, organoids were stained with Hoechst nuclear stain (blue), calcein AM live cell stain (green), and SYTOX Orange dead cell stain (red). Scale bar = 20 mm. (F) Quantification of the phenotypes observed in panel E.

Clioquinol induced an increase in intracellular Zn2+ in each of the cell types tested even in the absence of exogenous Zn2+; NA treatment, however, resulted in decreased or unchanged intracellular Zn2+ levels, suggesting that the natural product chelates and sequesters Zn2+ instead of acting as an ionophore. Examination of clioquinol and NA’s effects on cell viability revealed that co-treatment with exogenous ZnSO4 induced opposite effects: ZnSO4 potentiated clioquinol’s activity, but antagonized NA’s cytotoxic effect (Fig. 8B). The combination of clioquinol and ZnSO4 was toxic to both ACS Paragon Plus Environment


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transformed and untransformed cells, in contrast to the cancer selective effects of ZNA. A time-course experiment was designed to fully evaluate the kinetics of these effects and again, clioquinol/ZnSO4 was found to be cytotoxic towards all cell types tested while ZNA/ZnSO4’s cytotoxic effect was only observed in the transformed cell line (Fig. 8C).

A

comparative dose-response experiment in the presence of 30 M ZnSO4 showed that even at low concentrations, clioquinol killed both transformed and untransformed cell lines with an EC50 = 0.29 M and 0.37 M against the MCF-7 and MCF-10A cell lines, respectively (Fig. 8D). In contrast, ZNA remained highly selective with an EC50 = 0.43 M against MCF-7s and an EC50 > 100 M against MCF-10As. Finally, we tested clioquinol and NA in the 3D branching assay to assess effects on normal mammary organoids relative to ZNA (Figs. 8E, 8F, and Supplemental Fig. 8C). We found that clioquinol-, clioquinol/ZnSO4, and NA-treatment killed 100% of the organoids in the experiment while exogenous ZnSO4 rescued the cytotoxic effects of NA (Fig. 8F). In all, these data illustrate that ZNA’s effects on cells are unique from other known Zn2+ modulators. These results demonstrate that ZNA is a unique structural scaffold that can differentially affect Zn2+ trafficking between transformed and untransformed cell types with high selectivity. Further studies will evaluate the structural components and cancer intrinsic mechanisms that mediate ZNA’s selective cytotoxicity. DISCUSSION AND CONCLUSIONS Zinc is an essential trace metal and it is estimated that 10% of the proteome binds the ion, with 40% of these proteins functioning as transcription factors and the remaining 60% operating in enzymatic or ion transport capacities.40 Proteins require zinc for a variety of functions, including coordination of inter- and intramolecular interactions, stabilization of tertiary and quaternary structures, and catalytic activity. Zinc also participates in biological signaling cascades as a secondary messenger by propagating signals produced by extracellular stimuli.41 Many critical biological processes such as proliferation, immune signaling, development, autophagy, and apoptosis depend upon ion homeostasis.42-48 Serum zinc levels are maintained at micromolar levels and at the cellular level, metal-binding proteins of the metallothionein family suppress excess free ion levels by direct binding and sequestration.49,50 Intracellular localization of zinc within vesicles also provides a dynamic mechanism for buffering exchangeable ion concentrations.51,52 Overall, zinc is essential for biological function and an inability to properly buffer intracellular levels can affect critical cellular processes. Two families of zinc transport proteins, the ZnT (SLC30A) and ZIP (SLC39A) families, have been identified as key participants in intracellular regulation of the ion.53 Ten ZnT and fourteen ZIP protein family members have been identified to date. Through this network of transporters, zinc levels can be tuned to meet cellular demands and maintain ion homeostasis. In breast cancer, differential zinc levels have been observed between malignant and nonmalignant breast tissue and aberrant ZnT and ZIP gene expression has been proposed to support this observed phenotype.17,18 Elevated expression of SLC39A10 (ZIP10) has been associated with invasive behaviors in breast cancer while increased SLC39A7 (ZIP7) expression has been reported in the case of a tamoxifen-resistant breast cancer cell line.19,20 Altered zinc levels in prostate cancer have also been characterized, and lower levels of the ion are clinically associated with more aggressive disease states.21,54 These studies collectively indicate that malignant cells manage zinc differently than untransformed cells, although the degree and direction of demand for the ion are cancer type-specific. While correlations between cancer and zinc dyshomeostasis are well documented, the molecular mechanisms that lead to these observed phenotypes in transformed cells have not been fully elucidated. Zinaamidole A (ZNA), a small molecule ionophore inspired by the natural product naamidine A, delivers superstoichiometric payloads of zinc specifically into transformed cell types leading to cytostatic and cytotoxic effects through a non-apoptotic pathway. This effect was consistent in four different experimental models: 2D breast cancer cell ACS Paragon Plus Environment

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line assays, 2D assays using primary, patient-derived breast cancer cells, 3D mouse tumor organoid assays, and an in vivo mouse model of breast cancer. When benchmarked against the known zinc modulator clioquinol, ZNA appears to have unique cancer-selectivity, particularly in the presence of exogenous zinc. We found that the addition of exogenous zinc potentiated the effects of both clioquinol and ZNA, but the clioquinol/ZnSO4 combination induced cell death in both transformed and untransformed cells. The premise of this investigation, that NA’s reported selective anti-cancer effects are predicated on its ability to chelate zinc, is not supported by our study. Rather, as evidenced by the rescue of cellular toxicity upon the addition of exogenous zinc, NA behaves as a zinc chelator not an ionophore. Our investigation into the chemical characteristics that underpin ZNA’s zinc-modulating activity led us to identify the N2-acyl-2-aminoimidazole as a key architectural component. Additionally, the structure-activity relationships explored suggest that modulation of the pKa of N3 on the 2-aminoimidazole core may play a role in tuning the compound’s ability to act as a ligand for zinc. Methylation at the N2-position, as with N2-Me-ZNA, abolishes the activity of ZNA, presumably through interference of metal complex formation via inhibition of the anionic binding mechanism displayed in the crystal structure of ZNA. Studies to further illuminate the structural and electronic components responsible for this unique chemotype as related to ZNA’s selectivity are ongoing. This work demonstrates not only that zinc management differs between transformed and untransformed breast cells, but that this difference can be exploited for therapeutic benefit. This new zinc ionophore chemotype represents the most selective tool to dissect the differences in zinc regulation between normal and cancer cells (both in vitro and in vivo). These studies will facilitate the identification of new, therapeutically accessible vulnerabilities in cancer. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications websiteat DOI: Additional data and materials and methods for RNAseq experiments, additional cell viability experiments with exogenous transition metals, three-dimensional organoid comparisons for ZNA, clioquinol and naamidine A, zinc synergy experiments and characterization of all organic compounds synthesized.

ACKNOWLEDGEMENTS This study was supported by the National Institutes of Health (R01CA140296, M. S. Sigman and B. E. Welm; R01CA143815, B. E. Welm; and 2R01RGM090082-A1, R. E. Looper). Shared resources used in this project were supported in part by P30CA042014 awarded to the Huntsman Cancer Institute. REFERENCES 1. Carmely, S.; Kashman, Y. (1987) Naamines and naamidines, novel imidazole alkaloids from the calcareous sponge Leucetta chagosensis. Tetrahedron Lett. 28, 3003-3006. 2. Copp, B. R.; Fairchild, C. R.; Cornell, L.; Casazza, A. M.; Robinson, S.; Ireland, C. M. (1998) Naamidine A Is an Antagonist of the Epidermal Growth Factor Receptor and an in Vivo Active Antitumor Agent. J. Med. Chem. 41, 39093911. 3. LaBarbera, D. V.; Modzelewska, K.; Glazar, A. I.; Gray, P. D.; Kaur, M.; Liu, T.; Grossman, D.; Harper, M. K.; Kuwada, S. K.; Moghal, N.; Ireland, C. M. (2009) The marine alkaloid naamidine A promotes caspase-dependent apoptosis in tumor cells. Colloq. Inse. 20, 425-436. 4. Gibbons, J. B.; Gligorich, K. M.; Welm, B. E.; Looper, R. E. (2012) Synthesis of the Reported Structures for Kealiinines B and C. Org. Lett. 14, 4734-4737. ACS Paragon Plus Environment


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A cancer-selective zinc ionophore inspired by the natural product

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