Development of ergosterol peroxide probes for cellular localisation studies

Abstract

Ergosterol peroxide selectively displays biological activity against a wide range of diseases, however, its mode of action remains unknown. Here we present an efficient synthesis of ergosterol peroxide chemical probes for in vitro anticancer evaluation, live cell studies and proteomic profiling. The ergosterol peroxide analogues show promising anti-proliferation activity against triple negative breast cancer cellular models, revealing information on the structure-activity-relationship of this natural product in order to develop superior analogues. The combined cellular studies demonstrate that ergosterol peroxide is distributed across the cytosol with significant accumulation in the endoplasmic reticulum (ER). These chemical probes support our efforts towards uncovering the potential target (s) of ergosterol peroxide against triple negative breast cancer cell lines.

Graphical Abstract

Cell-permeable ergosterol peroxide probes were synthesized to advance its biological understanding and therapeutic potential.

Introduction

The ergosterol peroxide natural product (2, Figure 1) is a compound isolated from plants,¹ algae,² lichens,³ anemonae,⁴ corals,⁵ and mushrooms⁶ such as Ganoderma lucidum among others.⁷ Ergosterol peroxide has been reported as an anti-tumour agent,⁸ and displays proapoptotic,⁹ anti-inflammatory/immunosuppressive effects,¹⁰ anti-mycobacterial, and anti-proliferative activity against cancer cell lines.^{6, 11–13} Ergosterol peroxide triggers apoptosis, and modulates the cell cycle of cancer cell models in a dose-dependent manner.^8,13 We have shown that ergosterol peroxide displays anti-proliferative effects through G1 phase cell cycle arrest, apoptosis induction via caspase 3/7 activation, and PARP cleavage. It decreases migratory and invasive effects of triple negative cancer cells while inhibiting the expression of total AKT1, AKT2, BCL-XL, Cyclin D1 and c-Myc in the tested breast cancer cells.¹¹

Figure 1.

Synthesis of ergosterol peroxide (EP) and its analogues.

We and others have studied the biological activity of the extract of Ganoderma lucidum and shown that this extract induces tumour reduction in vivo breast cancer models.^11a Ergosterol peroxide belongs to the steroidal family of natural products, sharing a cholesterol core and an unexpected endoperoxide bridge as the reactive center of the molecule (Figure 1). Derivatives lacking a peroxidic bond have no significant activity (EC₅₀>50µM). Although the apoptosis induction presumably arises from this motif and several studies have reported the affected potential pathways, the underlying molecular mechanism of ergosterol peroxide in cancer (in vitro and in vivo murine cancer models) is not fully understood.^14–15

We are particularly interested in studying ergosterol peroxide’s properties against triple-negative breast cancer cellular models. Triple-negative breast cancer is diagnosed when breast cancer cells lack the three common types of protein receptors: oestrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER-2). Triple-negative breast cancer is associated with advanced disease stage, higher-grade tumours at diagnosis, an increased recurrence risk, and a poor five-year survival rate (relative to other breast cancer subtypes).¹⁶

Most cancer-related deaths are a result of metastasis, and metastatic tumour cells are genetically unstable, with no single dominant pathway likely to control metastasis in most cancers. Thus, there is a growing interest in developing new pharmacological treatments from natural products, which might induce their effects via primary and secondary molecules that cross-talk in cancer-dependent signaling pathways rather than affecting a single biological target. There is a unanimous agreement that ergosterol peroxide induces cells death in cancer cell models, but the specific biological target(s) remain elusive. Several reports indicate that ergosterol peroxide might influence various pathways across cancer subtypes.¹⁷ Wu and our group have shown that ergosterol peroxide induces reactive oxygen species (ROS), which are radicals with a sole unpaired electron in the outermost shell of electrons.^11,17 Accordingly, the expression of ROS-detoxifying antioxidant proteins is altered in cancer cells. However, ROS display anticancer properties by decreasing cell proliferation, damaging DNA, and inducing apoptosis, among other mechanisms.¹⁸ Endoperoxides are known for generating radicals. It is hypothesized that oxyl radicals are generated by homolytic cleavage of the peroxide, which ultimately lead to a stable carbon-carbon radical and presumably bind to specific proteins (Fig. 1). Although such reactivity is expected to be promoted by Iron (II),¹⁹ we did not detect improvement in cytotoxicity by combination treatments of iron chelators and ergosterol peroxide (data not shown).

The objectives of our study are: a) to generate a focus library of ergosterol peroxide probes to evaluate their cytotoxic/apoptotic potential via CellTiterGlo and propidium iodide viability assays against breast cancer cell models [epithelial cell model: MDA-MB-231 (ER-/PR-/HER2-), luminal A cell model: T-47D (ER+/PR+/HER2-) both of which are P53 mutant, SUM149 (invasive ductal carcinoma, ER-/PR-/HER2-deactivated), and the non-cancerous cell models: BJ (fibroblast) and HMEC (primary mammary epithelial, b) to assess the subcellular accumulation of ergosterol peroxide probes, and c) its potential biological target (s) in triple negative breast cancer cell models.

Results and Discussion

First, we generated ergosterol peroxide by treatment of the natural product ergosterol with catalytic radical initiator (phloxine B) in the presence of oxygen and visible light, to undergo a hetero Diels-Alder reaction in excellent yields (93% isolated yield, Figure 1). The hydroxyl functional group at C3 represents a feasible site for functionalization strategies, while maintaining the rest of the molecule intact. Thus, we generated several ergosterol peroxide analogues (3a–3k, Figure 2) to investigate the biological effects of a diverse group of linkers at the C3-hydroxyl group. We have previously shown that benzyl carbamates of ergosterol peroxide displayed superior biological activity to ergosterol peroxide, presumably due to improved aqueous solubility.¹¹

Figure 2.

Ergosterol peroxide analogues (3a–3k)

Oxidation of compound 3 with TPAP/NMO at RT provided the enone system 3a. The synthesis of 3b–3c, 3h, 3i, 3j, and 3k involved esterification reactions of ergosterol peroxide with the corresponding carboxylic acid, acyl chloride, or anhydride (see SI for detailed information). Synthesis of 3d–3g involved S_N2-type reactions to provide the desired compounds in good yields. For the synthesis of the biotinylated compound, ergosterol peroxide was subjected to the mixed anhydride of biotin to afford 3k in good yield (Figure 2). The synthesized compounds were characterized by NMR (¹H and ¹³C) and mass spectrometry (see SI). Next, the ergosterol peroxide fluorescent probes (3l–3n) were synthesized for the anticipated live-cell analysis, including organelle accumulation and proteomic profiling studies (Scheme 1). We envisioned ergosterol peroxide conjugates of fluorescent dyes would enable live-cell imaging to explore their subcellular interactions. Furthermore, the fluorescent probes were rationally designed to be spectrally orthogonal to organelle-fluorescent trackers for co-localisation studies using fluorescence microscopy. The syntheses of ergosterol peroxide probes with the corresponding fluorescent reagents: tetramethylrhodamine (TAMRA) and boron-dipyrromethene (Bodipy)²⁰ were evaluated. Recently, Bu et al. generated a coumarin fluorescent probe of ergosterol peroxide that accumulates in the mitochondria.¹⁰ However, our unbiased fluorescent ergosterol peroxide probes were intended to uncover the specific subcellular accumulation sites of ergosterol peroxide with minimum influence from the fluorescent dye to provide insight into ergosterol peroxide mode of action. The chemical nature of the fluorescent dyes can affect the cellular permeability of the compound and the site of accumulation, thus we minimized the size of the linker to avoid excessive changes to the properties of ergosterol peroxide. First, the tetramethylrhodamine probe 3l (Scheme 1) was synthesized by treatment of commercially available TAMRA acid, propargyl amine (2 equiv.) with PyBOP and Hünig’s base to afford the corresponding fluorescein-propargyl amide (90% yield). Next, synthesis of the corresponding coupling partner was achieved via (a) acylation of ergosterol peroxide with acyl chloride, followed by (b) azide displacement to provide the desired intermediate (see SI). The copper‐catalysed azide–alkyne cycloaddition (CuAAC)²¹ was mediated by CuSO₄ and sodium ascorbate in a solvent mixture of t-BuOH and water (1:1) to provide 3I in 68% yield. Next, we generated the desired probe 3m (with a short linker between the fluorescent core and the ergosterol peroxide) via acylation of Bodipy propionic acid with 2,4,6-trichlorobenzoyl chloride in the presence of Et₃N, followed by ergosterol peroxide addition, to provide 3m in 70% yield. The synthesis of compound 3n consisted of a single-step condensation of ergosterol peroxide with Bodipy N-hydroxysuccinimide ester (BDY 630-X-NHS, Scheme 1) and Et₃N to afford the expected compound 3n in 60% yield.

Scheme 1.

Reagents and conditions for syntheses of 3l–3n probes: A. 1. (a) PyBOP, DMSO, Hünig's base, 25°C, (b) sodium ascorbate (0.2eq), CuSO₄.7H₂O (0.1eq), t-BuOH:H₂O =1:1 (v/v). B. (a) 2,4,6-trichloro-benzoyl chloride, Et₃N, 25°C, CH₂Cl₂, 1hr. 2. (b) ergosterol peroxide, DMAP, CH₂Cl₂, 25°C, 10hr. C. (a) BDY 630-X-NHS, EP, Et₃N, 25°C, CH₂Cl₂.

Previous biological studies of ergosterol peroxide¹¹ suggested that the endoperoxide bridge is required for biological activity against cancer cell models. To gather further information on the structure-activity-relationship of ergosterol peroxide, modifications were conducted at the C3 position and their cytotoxicity was assessed by an established cell viability assay previously described.²² A summary of the cytotoxicity findings is shown in Table 1, and cell death was validated by propidium iodide apoptosis assay (SI). The triple-negative breast cancer cell lines MDA-MB-231 and SUM149 and the ER-positive cell line T-47D were evaluated along with the non-cancerous cell lines BJ and HMEC to determine therapeutic index.²² Our study shows that the triple-negative breast cancer cell model, SUM149 was the most sensitive cell line towards these compounds, while the estrogen receptor positive cell line T-47D was the least responsive under the evaluated conditions.

Table 1.

Cytotoxicity of ergosterol peroxide probes via CTG assay. EC₅₀ value expressed as the mean of three independent experiments.

Number	MDA-MB-231 EC50 (µM)	SUM149 EC50 (µM)	T-47D EC50 (µM)	HMEC EC50 (µM)	TI(HMEC/SUM149)
Phosphoramide	>11	>12	ND	>10	>1
Doxorubicin	1	1	ND	>10	>11
Taxol	2	0.01	ND	>10	>800
Capecitabine	5	5	ND	10	>2
3	18	9	19	>20	>3
3a	7	5	10	>13	>3
3b	16	3	>20	>20	>4
3c	>20	5	>20	>20	>4
3d	>20	10	>20	>20	>2
3e	16	10	>20	>20	>2
3f	9	5	>20	>20	>4
3g	37	5	>20	>20	>4
3h	25	21	17	>20	>1
3i	33	5	18	>20	>4
3j	8	4	11	>13	>4
3k	10	4	>10	>14	>6
3l	20	18	>20	>20	>1
3m	18	2	18	>15	>6
3n	7	2	16	>15	>6

Ergosterol peroxide has been reported to have variable potency across cancer cell lines, which is attributed to modest aqueous solubility (<50 µM), limiting its bioavailability to the cell. In addition, at 5–10 mM concentrations in DMSO, precipitation occurs after 168 hr at RT. Suitable formulation strategies are currently under development. Some of the generated compounds also displayed poor aqueous solubility, indicating that liposomal delivery (formulation/prodrug approaches) will be necessary to overcome this potential liability. In terms of stability, ergosterol peroxide is surprisingly stable as we did not observe any degradation by MS/MS in PBS over 72 hr at 37 °C, however there is a report on the conversion of ergosterol peroxide to ergosterol^6b, presumably via retro-Diels-Alder reaction. Noteworthy, the oxidized compound 3a was homogeneously soluble at 100 µM in PBS, this compound displayed improved activity, which was partially attributed to the improved aqueous solubility of this compound. We used Caco-2 cell model permeability assay (derived from a human colon adenocarcinoma)22 for examining the permeability property ergosterol peroxide, and found that it displays good cellular permeability (Avg Papp, A/B, nm/s= 342.21, and efflux ratio B2A/A2B= 0.93) as compared to control drug carbamazepine (Avg Papp, A/B, nm/s= 375.73, and efflux ratio B2A/A2B= 0.74).

Next, we explored the bioactivity of the ergosterol peroxide analogues. Compound 3b, chloride 3c, and azide 3d showed promising activity against SUM149, with little or no activity observed at the tested concentrations against MDA-MB-231 and T-47D. The corresponding alkynyl ether 3e showed moderate bioactivity, and compound 3j which containing the longer side chain showed superior bioactivity against the cancer cell lines tested. The phosphonate ester 3f showed improved activity; however, the corresponding phosphonic acid (3g), the phosphocholine (3h), and glutaric ester (3i) displayed poor activity, presumably due to the carboxylic acid/charged moiety of these compounds that may limit passive diffusion across the membrane. The biotinylated compound 3k demonstrated cytotoxicity comparable to that of ergosterol peroxide. Cellular evaluation of compound 3l revealed the compound did not accumulate in the cell and showed poor cytotoxicity. Further studies indicate 3l display poor solubility in both organic and inorganic solvents (as it readily precipitates in DMSO or PBS at 1mM). Lastly, the fluorescent probe 3m disclosed cytotoxicity comparable to that of ergosterol peroxide. However, 3n showed superior bioactivity activity across cancer cell lines. The combined cytotoxicity studies indicate the introduction of lipophilic substituents at the C3 hydroxyl group can improve the potency of this natural product. The fluorescent tag significantly improves the potency of ergosterol peroxide, presumably due to its ability to accumulate in the cell.

To validate the anti-proliferative effects of these compounds (3k, 3m–3n), their activity was validated via propidium iodide assay (SI, Figure S1–3). Significantly, ergosterol peroxide and its analogues, particularly compounds 3k, 3m–3n, displayed moderate therapeutic index comparable to capecitabine, a chemotherapy approved drug.

Next, we evaluated co-localisation of ergosterol peroxide with orthogonal probes for cellular organelles. When possible, we used commercially available chemical probes for live cell staining organelles (endoplasmic reticulum, mitochondria, and lysosomes). However, to study the Golgi apparatus or peroxisome no orthogonal dye was available for our chemical probe 3m–3n. Therefore, transiently transfected with Red Fluorescent Protein (RFP) or Green Fluorescent Protein (GFP) organelle-labelled cellular models were generated as shown in Figure 3 (MDA-MB-231, Figure 3. A–B and SUM149, see SI) cells.^23a Also, stably transformed cancer cell lines transfected with mRuby-peroxisomes-2 plasmid^23b were generated (Figure 3C).

Figure 3.

Transient & stable organelle–labelled MDA-MB-231. A. Transiently transfected GFP fused to the peroxisomal C-terminal targeting sequence cells with nuclear blue stain. B. Transiently transfected RFP fused to Golgi apparatus resident enzyme N-acetyl galactosaminyl transferase cells with nuclear blue stain. C. Stable mRuby-peroxisomal targeting signal 1 cells.

We hypothesized that ergosterol peroxide would accumulate in the peroxisome due to its endoperoxide chemical nature. Probe 3l was the first evaluated; as previously mentioned, no cell staining was observed. Either the compound did not penetrate the cell or it was pumped out of the cell too rapidly to measure. As shown in washout experiments with probe 3m and the corresponding BodipyFL methyl ester (Figure S1–4.1), 3m distributes across the cytosol and appears to accumulate in cellular substructures. Then, compound 3m was evaluated in co-localisation studies utilizing an array of organelle markers. A pattern opposite to our hypothesis was captured, no co-localisation was detected with RFP-labelled peroxisome cellular model (Figure 4A). Similarly, no co-localisation was observed with the Golgi apparatus (Figure 4B), lysosomes, (Figure 4C) or mitochondria (MitoTracker Deep Red, Figure 4D). However, substantial co-localisation was detected with the endoplasmic reticulum using blue/white endoplasmic reticulum tracker along with 3m (Figure 4E). Consistent observations were recorded for both MDA-MB-231 and SUM149 cancer cell lines (see SI). The antiproliferative activity of 3m was comparable to that of ergosterol peroxide, thus the combined results suggest that ergosterol peroxide is likely to accumulate in the cytosol and partially co-localise to the endoplasmic reticulum.

Figure 4.

Co-localisation studies of 3m in MDA-MB-231 (see SI for SUM149 cells). A. Peroxisome. B. Golgi. C. Lysosome. D. Mitochondria. E. Endoplasmic reticulum.

After generating the probe 3n (MW= 1030 g/mol), which carries a longer linker between the core of ergosterol peroxide and the fluorescent tag than 3m (MW=702 g/mol), we tested the hypothesis that such chemical change would affect the accumulation of the compound. Washout experiments with probe 3n and the corresponding BDY 630-X ester (Figure S1–4.2) were conducted, 3n permeated the cell and accumulated in cellular substructures. Interestingly, a different subcellular accumulation pattern was observed for 3n in the GFP-peroxisome cell lines (Figure 5A) as previously recorded for probe 3m. Then to validate the findings, the orthogonal MitoTracker Green was utilized to investigate the compound distribution in the cell. To our surprise, 3n demonstrated selective accumulation in the mitochondria. This finding explained our cytotoxicity data, showing 3n as the most potent compound of the evaluated ergosterol peroxide analogues across cancer cell lines, as accumulation in the mitochondria presumably directly influences the apoptotic program.

Figure 5.

Evaluation of probe 3n. A. cell light peroxisome-GFP/MDA-MB-231. B. MitoTracker Green and 3n.

Target identification of natural products and small molecules remains one of the biggest challenges in chemical biology,²⁴ and it is possible that ergosterol peroxide affects several biological pathways. Affinity-based proteomic target identification methods are still the most promising methods to elucidate biological targets in drug discovery. Thus, a pull-down experiment with the biotinylated probe 3k, was designed. The compound displayed biological activity analogous to ergosterol peroxide. Incubation of the biotinylated probe 3k bound to Dynabeads M-280 with cell lysates for 2 hr at 4 °C overcomes some of the concerns related to compound solubility/bioavailability and potential secondary effects of the compound during cell death. Also, we generated a biotinylated cholesterol probe to identify nonspecific protein binders as a control. The bound protein species were identified by mass spectrometry using the spectral counting method and the complete data set is provided (SI).

The distribution of potential protein targets interacting with 3k in SUM149 and MDA-MB-231 cell models is shown in Figure 6 respectively. They are represented in scatter plots displaying the ratios of Spectral Counts probe/ Spectral Counts Control against the corresponding p-values. Excitingly, two potential biological targets, Rab interacting lysosomal protein like 1 (RIPL1) and E3 ubiquitin-protein ligase (UBR4) were identified in both SUM149 and MDA-MB-231 cell lines. These proteins are known to be distributed across the cytosol and the plasma membrane, which agrees with our co-localisation results. RIPL1 is involved in regulating cell shape and polarity via cellular protein transport,²⁵ while UBR4 is involved in ubiquitination and subsequent degradation of certain proteins, interacts with the retinoblastoma-associated protein in the nucleus, calcium-bound calmodulin in the cytoplasm and regulates integrin-mediated signaling.²⁶ Other relevant targets for these cell lines are shown in blue (PRKDC, FAS, EZRI, and GDIB for SUM149, and eIF4B and PP1A for MDA-MB-231) and they will be evaluated in future studies.

Figure 6.

–Scatter plots displaying potential protein targets of 3k, Spectral Counts (SC) against p-values. A. SUM149. B. MDA-MB-231.

Conclusions

In summary, our study describes the synthesis of ergosterol peroxide probes from the readily accessible natural product ergosterol and evaluates their biological potential against breast cancer cell models. These chemical tools can be used for live cell imaging studies to delineate the mechanism of action of ergosterol peroxide in cancer cells. We disclose a short synthesis route to ergosterol peroxide probes and demonstrate these probes accumulate in the cytosol with subcellular specificity. Our ergosterol peroxide probes show promising anti-proliferation activity against cancer cell models in alignment with reported studies,^9,10–12,17 providing information on the role of substituent groups at the hydroxyl group and potential biological targets of this natural product. Furthermore, these chemical probes indicate that ergosterol peroxide is a promising molecular scaffold as an early lead compound for further therapeutic development against breast cancer. Live cell imaging reveals significant co-localisation of 3m with endoplasmic reticulum fluorescent markers, while the probe 3n with a longer linker between the borane core and ergosterol peroxide showed accumulation in the mitochondria. We demonstrate that chemical modifications can lead to superior potency by causing accumulation in specific organelles to induce greater cellular damage. The identified potential targets warrant future validation studies via molecular biology/chemical biology approaches.