Methylated Nucleotide-Based Proteolysis-Targeting Chimera Enables Targeted Degradation of Methyl-CpG-Binding Protein 2

Abstract

Methyl-CpG-binding protein 2 (MeCP2), a reader of DNA methylation, has been extensively investigated for its function in neurological and neurodevelopmental disorders. Emerging evidence indicates that MeCP2 exerts an oncogenic function in cancer; however, the endeavor to develop a MeCP2-targeted therapy remains a challenge. This work attempts to address it by introducing a methylated nucleotide-based targeting chimera termed methyl-proteolysis-targeting chimera (methyl-PROTAC). The methyl-PROTAC incorporates a methylated cytosine into an oligodeoxynucleotide moiety to recruit MeCP2 for targeted degradation in a von Hippel-Lindau- and proteasome-dependent manner, thus displaying antiproliferative effects in cancer cells reliant on MeCP2 overexpression. This selective cytotoxicity endows methyl-PROTAC with the capacity to selectively eliminate cancer cells that are addicted to the overexpression of the MeCP2 oncoprotein. Furthermore, methyl-PROTAC-mediated MeCP2 degradation induces apoptosis in cancer cells. These findings underscore the therapeutic potential of methyl-PROTAC to degrade undruggable epigenetic regulatory proteins. In summary, the development of methyl-PROTAC introduces an innovative strategy by designing a modified nucleotide-based degradation approach for manipulating epigenetic factors, thereby representing a promising avenue for the advancement of PROTAC-based therapeutics.

Graphical Abstract

INTRODUCTION

Epigenetic modification refers to the heritable alteration of deoxyribonucleic acid (DNA) without influencing the DNA sequence,^1,2 including DNA modifications and histone posttranslational modifications.^1–4 DNA methylation refers to the covalent modification of the C-5 position of the cytosine ring (5-methylcytosine, 5 mC), which is catalyzed by DNA methyltransferases (DNMTs),^5,6 and this modification could be removed by a chain of reactions catalyzed by ten-eleven translocation (TET), thymine DNA glycosylase (TDG), and base excision repair enzyme (BER).^5,6 On the other hand, the methylated DNA could be specifically recognized by a pivotal reader protein, methyl-CpG-binding protein 2 (MeCP2), which has been implicated in the pathogenesis of neurological and neurodevelopmental disorders.^7–17 Intriguingly, MeCP2 has also been established as an oncogene and is upregulated in various types of human cancers.^18–20 However, due to the lack of the MeCP2 inhibitor, it remains a challenge to target MeCP2 for cancer treatment.^19–26 We and others have demonstrated that nucleotide-based proteolysis-targeting chimera (PROTAC) platforms are effective in degrading undruggable targets such as ribonucleic acid (RNA)-binding proteins,²⁷ transcription factors,^28–30 G-quadruplex binding proteins,³¹ cellular Myc (c-Myc),³² and telomeric repeat-binding factor proteins (TRFs).³³ Thus, we aim to test whether the nucleotide-based PROTAC design can be adopted for targeted degradation of epigenetic readers such as MeCP2 in cancer cells.

Here, we propose the development of a novel methodology termed methylated nucleotide-based targeting chimera (methyl-PROTAC) as a proof of concept to expand the application of DNA-based PROTACs for targeting epigenetic regulators (Figure 1). To accomplish this goal, an oligodeoxynucleotide (ODN)-containing methylated cytosine, which is used as a ligand to recruit MeCP2, was incorporated with the von Hippel-Lindau (VHL) ligand to recruit VHL E3 ligase, thus facilitating the ubiquitination and subsequent degradation of MeCP2. The construction of the methyl-PROTAC was achieved through a copper-free strain-promoted azide—alkyne cycloaddition (SPAAC) reaction, as we previously described.^29,33,34 Upon the cellular introduction of the methyl-PROTAC, the methylated oligonucleotide moiety directly engages with the MeCP2 protein, leveraging its specific binding affinity. Simultaneously, the VHL ligand moiety recruits the VHL E3 ligase, enabling the subsequent ubiquitination of MeCP2 (Figure 1).

Figure 1.

Methyl-PROTAC strategy hijacking the VHL E3 ligase for targeted degradation of MeCP2 in cells.

RESULTS AND DISCUSSION

Design of Methylated-ODN as a Ligand for MeCP2.

MeCP2 specifically binds to methylated cytosine in DNA, providing an opportunity to target the MeCP2 protein.^35–40 MeCP2 recognizes a DNA sequence containing a GTATC_mCGGATAC consus through multiple hydrogen bonds, where the methyl group on the cytosine enhances the hydrophobic interactions (Figure S1).³⁶ However, it has been observed that nonmethylated cytosine within specific sequences can also interact with MeCP2, albeit with weaker binding affinity.^35,37,40 To investigate the indispensability of methylated cytosine for MeCP2 binding, we designed two biotin-modified ligands: Biotin-GTATCCGGATACTTTGTATCCGGATAC with either nonmethylated or methylated cytosine at the C-position, referred to as biotin-C ODN and biotin-_mC ODN, respectively. The biotinylated DNA oligomers were synthesized by incorporating a TTT linker (Figures 2A and S2) and annealed (Figure 2B) as previously described.²⁹ To assess the interaction between MeCP2 and the biotinylated ODNs, streptavidin–biotin pull-down assays were performed using the biotinylated ODNs as baits (Figure 2C,D). As shown in Figure 2C,D, biotin-_mC ODN, but not biotin-C ODN, could precipitate MeCP2 protein from the Flag-MeCP2 transfected HEK293 cell lysate, indicating that the methylation of cytosine within the ODN sequence is likely essential for the MeCP2 interaction. Consequently, the _mC ODN was selected for subsequent assays.

Figure 2.

MeCP2 protein binds with methylated ODN (_mC ODN). (A) Biotin-ODN sequences with or without methylation on the cytosine. (B) Monitoring of annealed ODNs by PAGE analysis. (C,D) Binding of _mC ODN to Flag-MeCP2 extracted from HEK293 cells, monitored with immunoblotting using anti-MeCP2 or anti-Flag antibodies as indicated.

Methyl-PROTAC-Induced MeCP2 Degradation in Cancer Cells.

To evaluate the ability of methyl-PROTAC to trigger MeCP2 degradation in cancer cells, we utilized the A375 cell line as a cellular model for evaluation. We designed 18 PROTACs, collectively referred to as methyl-PROTACs, by employing azide-modified _mC ODN as a ligand for MeCP2 and bicyclooctyne-containing compounds 1b–18b as VHL E3 ligands conjugated through the SPAAC reaction^29,33 (Figure 3A). To ensure the optimal positioning of the E3 ligase and substrate protein in the PROTAC ternary structure,^41–43 a series of VHL ligands 1b—18b²⁹ were synthesized by coupling a bicyclooctyne group onto VHL1⁴⁴ with diverse linkers (Figure S3). To obtain the methyl-PROTAC degraders 1c–18c, a click reaction was performed for 16 h at room temperature, and the efficiency was monitored using the polyacrylamide gel electrophoresis (PAGE) analysis. As demonstrated previously, most of the compounds achieved yields exceeding 80%. We further optimized the efficiency for compounds 7c—10c, which showed decent yields of around 80% (Figure 3B). Upon successful synthesis of the methyl-PROTACs, A375 cells were treated with compounds 1c—18c (5 μg/mL, 24 h), in which 9c, 10c, 12c, 13c, and 15c significantly degraded MeCP2, while other compounds could not do so in this experimental setting (Figure 3C). Among these methyl-PROTACs, 9c and 10c exhibited particularly remarkable and stable degradation efficiency (Figure S6) and were used as lead compounds in the following assays.

Figure 3.

Methyl-PROTAC targets MeCP2 for degradation. (A) Schematic illustration of the SPAAC reaction between VHL ligands 1–18 with two types of linkers (alkyl and PEG) and azide-modified mC ODN for the synthesis of methyl-PROTAC 1c–18c. (B) PAGE analysis of the click reaction efficiency. The click efficiency was determined and is indicated below the corresponding bands. (C) Screening of methyl-PROTAC 1c–18c for targeted degradation of MeCP2. A375 cells were treated with 5 μg/mL of methyl-PROTAC 1c–18c for 24 h, and the resulting cells were harvested for western blotting analysis to assess the degradation of MeCP2. (D) Induction of cell death by methyl-PROTAC 9c and 10c in A375 cells. A375 cells expressing mCherry were treated with mC ODN, 9c, and 10c, at 5 μg/mL for 24 h, and then cell imaging was acquired for analyzing the cell number. Data are presented as mean ± SD (n = 3) and are analyzed by one-way ANOVA with Tukey’s correction. ***p < 0.001, ***p < 0.0001. NC: negative control. (E) Dose-dependent analysis of methyl-PROTAC 10c with different concentrations (0, 2.5, 5, 7.5, and 10 μg/mL) on A375 cells for 24 h. (F) Competition experiments of VH032, MG132, and free mC ODN with 10c in MeCP2 degradation. Data are presented as the mean ± SD from three independent experiments.

To further investigate the efficacy of 9c and 10c, A375 cancer cells expressing mCherry were treated with 9c, 10c, and _mC ODN as a negative control, at a concentration of 5 μg/mL for 24 h, followed by the analysis of cell numbers (Figure 3D). Methyl-PROTACs 9c and 10c demonstrated potent inhibition of A375 cell proliferation, indicating their promising anticancer effects. Moreover, a dose-dependent analysis of methyl-PROTAC 10c was performed at different concentrations (0, 2.5, 5, 7.5, and 10 μg/mL) on A375 cells for 24 h. The results indicated that 5 and 7.5 μg/mL 10c exhibited the most efficient inhibition of cell proliferation (Figures 3E and S7). The degradation of MeCP2 by 10c was blocked by the VHL ligand VH-032 (5 μM), the proteasome inhibitor MG132 (2.5 μM), and free _mC ODN (5 μg/mL), suggesting that methyl-PROTAC specifically mediated MeCP2 degradation in VHL- and proteasome-dependent manners (Figures 3F, S8, S9, and S10).

Methyl-PROTAC-Mediated MeCP2 Degradation Elicits Anticancer Activity.

To further investigate the antiproliferative activity of methyl-PROTAC, we conducted cell growth curves, colony formation assays, and MTT assays in multiple cancer and noncancerous normal cell lines (Figure 4A). Transfection with 2.5 μg/mL _mC ODN, 9c, and 10c exhibited a robust inhibitory effect on cancer cells compared to normal cells by suppressing cancer cell proliferation and inducing cancer cell death within 24 h, while normal cell lines human foreskin fibroblasts 1 (HFF1) and human fetal lung 1 (LF1) were relatively resistant (Figure 4B,C). Subsequently, we performed a colony formation assay to further evaluate the impact of methyl-PROTAC on cancer cell transformation in vitro. As anticipated, methyl-PROTAC 9c and 10c significantly inhibited the potential for colony formation in various cancer cell lines, whereas _mC ODN exhibited a relatively lower efficiency (Figures 4D and S11). These results suggest that the methyl-PROTAC is relatively more potent than _mC ODN in targeting MeCP2 as the degradative effect of methyl-PROTAC surpasses the suppressive effect of _mC ODN, acting as a DNA decoy (Figure 4D).

Figure 4.

MeCP₂ degradation by methyl-PROTAC efficiently induces cell death in tumor cells compared to normal cells. (A) Scheme of the effects of methyl-PROTAC-mediated MeCP₂ degradation on the growth curve, colony formation, and IC₅₀ of cancer cells. (B) Methyl-PROTAC 9c or 10c suppressed the proliferation of noncancerous normal cells after 24 h. Cells were treated in 5 μg/mL of 9c or 10c for 3 days, and the cell number was counted daily. Data are presented as the mean ± SD from three independent experiments. (C) Methyl-PROTAC 9c or 10c suppressed the proliferation of cancer cells. Cells were treated with 5 μg/mL of 9c or 10c, and the cell number was counted daily. Data were presented as the mean ± SD from three independent experiments. (D) Methyl-PROTAC 9c or 10c inhibits the colony formation of cancer cells. HeLa cells were treated with 2.5 μg/mL of methyl-PROTAC 9c or 10c, and the colony number was calculated 2 weeks after the treatment. (E) IC₅₀ of methyl-PROTAC 10c on normal and cancer cells. Cells were treated with the indicated dose of 10c for 72 h, and the cell viability was measured. Data were presented as mean ± SD and analyzed by using GraphPad Prism software. (F) Western blot analysis of whole-cell lysates obtained from the indicated cell lines, and (G) Kaplan–Meier analysis of the mRNA expression level of MeCP2. N, T: Normal, Tumor.

Next, we determined the half-maximal inhibitory concentration (IC₅₀) of methyl-PROTAC in both normal and cancer cells. Methyl-PROTAC 10c exhibited an IC₅₀ of approximately 18 ± 3, 39 ± 6, and 38 ± 3 nM on MDA-MB-468, MCF-7), and A375 cancer cells, respectively (Figure 4E). In contrast, its anticancer activity was relatively moderate against HCT116 and HeLa) cell lines, with an IC₅₀ of around 120 and 400 nM. Interestingly, the IC₅₀ of 10c on noncancerous normal cells was approximately 10-fold or even higher compared to those of cancer cells (Figures 4E and S12). Therefore, we hypothesized that methyl-PROTAC leads to anticancer activity that is likely proportional to the abundance of MeCP2. To test the hypothesis regarding the relationship between MeCP2 abundance and the cytotoxic response of different cell lines, we assessed MeCP2 mRNA) and protein levels in multiple cancerous and noncancerous normal cells. Our findings revealed variable levels of MeCP2 among different cancer cell lines (Figure 4F,G) and relatively low MeCP2 abundances in normal cells. This observation may explain why certain cancer cell lines, such as MDA-MB-231, characterized by low MeCP2 expression levels, were unresponsive to the methyl-PROTAC, resembling the behavior of normal cells. It was previously shown that tumor cells are addicted to the expression of driving oncoproteins, which can be exploited as a possible cancer cell vulnerability for targeted anticancer therapies.^45,46 Notably, the methyl-PROTAC exhibited its most potent activity in cell lines with relatively high MeCP2 levels, including MDA-MB-468, MCF7, and A375, while displaying moderate effects in HCT116 and HeLa cells, which exhibited intermediate protein levels as that of MeCP2 (Figure 4E). This observation suggests a cell-line-dependent response to the methyl-PROTAC.

Methyl-PROTAC Modulates the Apoptosis Signaling Pathway by Downregulating Bcl-2 and Bcl-xL.

Subsequently, the mechanism underlying methyl-PROTAC-induced cell death was analyzed using cancer cell lines (A375 and MDA-MB-468) and noncancerous normal lines (LF1 and HFF1). We found that methyl-PROTAC down-regulated antiapoptotic proteins [B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL)] in the cancer cell lines, accompanied by cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP1), leading to activation of the apoptosis pathway (Figures 5A and S13). In contrast, the apoptosis pathway remained largely unaffected in the normal cell line (Figures 5A and S14). Our data suggest that methyl-PROTAC-triggered MeCP2 degradation rewires the apoptosis pathway to suppress the proliferation of cancer cells in a MeCP2-dependent manner while exerting minimal effects on normal cells (as summarized in Figure 5B).

Figure 5.

Analysis of the impact of _mC ODN, 9c, and 10c on cell signaling in cancer cells compared to noncancerous normal cells. (A) Assessment of the apoptosis pathway in cancer cells (A375 and MDA-MB-468) and normal cells (LF1 and HFF1) upon treatment by methyl-PROTAC. (B) Scheme representation illustrating the differential expression levels of MeCP2 in normal and cancer cells (left), and the inhibition of the apoptosis pathway in cancer cells through upregulation of antiapoptosis genes Bcl-2 and Bcl-xL, along with downregulation of proapoptotic genes such as cleaved caspase-3 (Right). Proposed mechanism underlying the action of methyl-PROTAC in inducing cancer cell death via MeCP2 modulation.

CONCLUSIONS

Considering the lack of MeCP2 inhibitors to date, this work represents the initial instance of a therapeutic approach, specifically targeting MeCP2, presenting a potential anticancer strategy. The prototype methyl-PROTAC 10c demonstrated effective degradation of MeCP2, leading to cytotoxicity, specifically in cancer cells, while sparing normal cells. This significant outcome underscores the potential value of methyl-PROTAC as an efficient cancer-targeting PROTAC strategy. Our study provides a compelling proof-of-concept by harnessing the methyl-PROTAC to effectively degrade MeCP2, thereby forging a novel path toward targeting CpG reader proteins in cancer cells. The ingenious utilization of methylation-modified cytosine in the methyl-PROTAC strategy augments the binding affinity and specificity toward the MeCP2 protein while presenting an opportunity to expand this approach to develop diverse PROTACs targeting other epigenetic DNA or RNA modifications, such as DNA-6mA and RNA-m6A, that are specifically recognized by reader proteins. However, it is essential to consider that the methylation binding domain in cells encompasses not only MeCP2 but also other homologues, including methyl-CpG-binding domain protein 1 (MBD1).³⁶ While the present study primarily focuses on MeCP2, it is imperative to expand the investigation to encompass other homologues, which warrants future exploration.

Additionally, the clinical translation of methyl-PROTAC poses several challenges, notably regarding permeability, stability, and the limited available delivery approaches. Transfection, a complex process involving the introduction of foreign DNA or RNA into cells, is beset by intricate hurdles encompassing factors such as limited transfection efficiency, potential cell toxicity, and immune responses, which needs further optimization for clinical application.⁴⁷ In parallel, DNA-based PROTAC molecules encounter analogous limitations shared with their counterparts that modulate mRNA and/or protein levels, exemplified by antisense oligonucleotides, small interfering RNA (siRNA), and short hairpin RNA (shRNA).⁴⁸ However, DNA-based PROTAC molecules continue to exhibit distinct advantages.⁴⁹ These advantages encompass multifaceted target engagement, augmented specificity, mitigation of off-target effects, pliability in modular design, and the potential for posttranslational modifications of target proteins, transcending the confines of mere downregulation. Lastly, despite substantial efforts dedicated to unraveling MeCP2-related signaling pathways, the downstream mechanisms induced by MeCP2 exhibit remarkable diversity.^20,50,51 The apoptosis pathway related to Bcl-2 and Bcl-xL has been investigated in the current work, but more detailed mechanisms demand further scrutiny and investigation. Nevertheless, methyl-PROTAC represents a novel concept in which modified nucleotides are successfully integrated into the PROTAC system, paving the way for nucleotide-based PROTACs to become a versatile platform in the future. In this study, MeCP2, the well-known oncogenic CpG reader protein, is first explored for anticancer drug discovery through PROTAC. The MeCP2-dependent cytotoxicity observed in this study suggests cell line-specific effects of this strategy, indicating its potential for broad application and development.