A platinated prodrug leveraging PROTAC technology for targeted protein degradation and enhanced antitumor efficacy

Abstract

Proteolysis targeting chimeras (PROTACs), which catalytically degrade disease-related proteins, can overcome the limitations of traditional small-molecule inhibitors and thus have revolutionized the field of targeted therapy. Building on this advancement, we present platinated PROTAC [PROTAC–Pt(IV)], a new class of “dual-action” prodrug that leverages the ubiquitin–proteasome system-mediated degradation capabilities of PROTAC and exhibits the advantages of Pt-based anticancer prodrugs. PROTAC–Pt(IV) exhibits exceptional cytotoxicity, with half-maximal inhibitory concentration values in the nanomolar range. It outperformed conventional inhibitor-based Pt(IV) prodrugs by up to three orders of magnitude by efficiently degrading the target protein BRD4 in a range of human cancer cells. PROTAC–Pt(IV) induces cancer cell death through mechanisms including augmented apoptosis, p21-mediated cell cycle arrest, and immune activation via PD–L1 downregulation. Compared with PROTAC alone, PROTAC–Pt(IV) more effectively suppressed the growth of tumor xenografts in a mouse model via its altered pharmacokinetic properties. Collectively, the development of PROTAC–Pt(IV) marks a revolution in dual-action Pt(IV) anticancer prodrugs and offers a promising avenue for enhanced and targeted cancer therapies.

---

Introduction

Proteolysis targeting chimeras (PROTACs) represent a groundbreaking approach to targeted therapy.¹ In contrast to traditional inhibitors, PROTACs rely on the recruitment of an E3 ubiquitin ligase to the target protein, which induces ubiquitination and subsequent proteasomal degradation.² This unique mechanism of action of PROTACs not only inhibits the target protein's function but also eliminates it from the cellular milieu, offering a robust strategy for targeting disease-related proteins. Crucially, PROTACs offer catalytic, irreversible, and enduring deactivation of proteins of interest and can effectively counter tumor drug resistance in tumors stemming from protein reactivation and mutations.³ Additionally, PROTACs can target “undruggable” proteins without necessitating attachment to their specific “binding sites”, thus broadening the scope of potential therapies.⁴ Nonetheless, the large sizes and complex structures of PROTACs often impede their pharmacokinetic properties and maximal therapeutic efficacy.⁵

Platinum-based chemotherapeutics, including cisplatin, carboplatin, and oxaliplatin, remain pivotal components of oncological regimens, yet their clinical utility is often compromised by severe toxicity and the development of acquired resistance.⁶ In recent years, the emergence of Pt(IV) prodrugs has garnered significant interest; these prodrugs provide a promising path forward, given their enhanced stability and the capacity to integrate additional functional groups to enable dual-action capabilities and synergistic effects.^7–12 Despite these advancements, current dual-action Pt(IV) prodrugs, which typically incorporate inhibitors targeting specific proteins, face several limitations. Inhibitors can bind their targets reversibly, reducing the efficacy of Pt(IV) prodrugs and necessitating sustained drug levels for optimal performance. In addition, the lack of isoform specificity among inhibitors may give rise to off-target effects.¹³

Herein, we introduce a new class of Pt(IV) prodrug, platinated PROTAC [PROTAC–Pt(IV)], which integrates a PROTAC molecule to a Pt(IV) center. Compared with a conventional inhibitor-based “dual-action” Pt(IV) prodrug, PROTAC–Pt(IV) features remarkable increases in cytotoxicity (up to 1754-fold) against a spectrum of human cancer cells, which is achieved through targeted protein degradation (Fig. 1). PROTAC–Pt(IV) also demonstrates superior in vivo tumor inhibition relative to standalone PROTAC molecules, which is attributed to altered pharmacokinetics resulting from strategic modification. Our findings not only represent a step forward in the field of dual-action Pt(IV) prodrugs but also provide a viable strategy for enhancing the pharmacokinetic profiles of PROTAC molecules, offering a promising avenue for more effective and targeted cancer therapies.

Fig. 1 Scheme illustration of the action mechanism of PROTAC–Pt(IV) that allows for protein degradation and conventional inhibitor-based dual-action Pt(IV) prodrugs.

Results and discussion

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) family, is critical for cancer cell growth and survival, rendering it an appealing target in cancer treatment.¹⁴ The inhibition of BRD4 has been shown to sensitize cancer cells to platinum-based chemotherapy.^15,16 ARV771, an extensively studied von Hippel-Lindau (VHL)-based BRD4 PROTAC,¹⁷ is a standard model for mechanistic studies and methodological development, making it an optimal choice for constructing the PROTAC–Pt(IV) in this study. To avoid hindering VHL E3 ligase recruitment, which can occur when the hydroxyl group in the VHL ligand is modified,¹⁸ we selected a carbonate linker for conjugation; this ensures the efficient release of the intact PROTAC molecule upon intracellular reduction of the Pt(IV) prodrug (Scheme 1).¹⁹ Following this design, we first synthesized complex 1via the oxidation of carboplatin with H₂O₂ in acetic acid, and then coupled the complex with ARV771 using a carbonate linker, resulting in the creation of the first platinated PROTAC (Scheme S1†). We also synthesized a conventional inhibitor-based Pt(IV) prodrug, JQ1–Pt(IV), for comparison (Scheme S2†). PROTAC–Pt(IV) and JQ1–Pt(IV) were fully characterized using ¹H, ¹³C, and ¹⁹⁵Pt nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS), and their purity was determined by high-performance liquid chromatography (HPLC) prior to application in the subsequent experiments (Fig. S1–S9†).

Scheme 1 Reduction of PROTAC–Pt(IV) to release carboplatin and intact ARV771.

The design of prodrugs aims to ensure their stability in the physiological environment and activation specifically at the target site, thereby reducing side effects.²⁰ After synthesizing PROTAC–Pt(IV), we first assessed its stability using HPLC. PROTAC–Pt(IV) remained stable in PBS and complete medium for 24 h, with over 98% and 83% of the complex persisting unchanged in buffer and medium, respectively (Fig. S10A, B, and D†). Next, we examined the reduction properties of PROTAC–Pt(IV) in the presence of sodium ascorbate. After 24 h, over 99% of the prodrug was reduced (Fig. S10C and D†). LC-HRMS analysis confirmed an emerging peak at the 13 min mark, representing the intact ARV771 ligand as the reduced product (Fig. S10E†). Additionally, the formation of carboplatin was verified by LC-HRMS (Fig. S10F†). Collectively, these results demonstrate the effectiveness of the carbonate strategy for releasing intact ARV771 ligand to enable subsequent protein degradation upon Pt(IV) reduction, while simultaneously avoiding premature release within the buffer solution (Scheme 1).

We next examined the cytotoxicity of PROTAC–Pt(IV). A panel of cell lines, namely A2780 and A2780cisR (human ovarian carcinoma), MCF-7 (human breast carcinoma), HeLa (human cervical carcinoma), MRC5 (human normal lung), A549 and A549cisR (human lung adenocarcinoma), and 4T1 (murine breast carcinoma), were selected to evaluate the anticancer activity of the prodrug in vitro. The half-maximal inhibitory concentration (IC₅₀) values are presented in Table S1.† Carboplatin exhibited IC₅₀ values in the high micromolar range, while JQ1–Pt(IV), the conventional inhibitor-based Pt(IV) prodrug, yielded IC₅₀ values consistently exceeding 25 μM. In contrast, PROTAC–Pt(IV) exerted significantly more potent cytotoxicity than carboplatin and JQ1–Pt(IV) across various cancer cell lines. Notably, PROTAC–Pt(IV) demonstrated potencies up to 1754-fold greater than that of carboplatin, with effects observed in the nanomolar range; these results position PROTAC–Pt(IV) among the most potent Pt(IV) prodrugs (Fig. 2A). For example, in A2780 cells, the IC₅₀ value of PROTAC–Pt(IV) is 11 nM, significantly outperforming carboplatin (19 300 nM) and JQ1–Pt(IV) (>25 000 nM). A comparative analysis of PROTAC–Pt(IV) and carboplatin across various cell lines demonstrated distinct anticancer profiles for each compound (Fig. S11†), suggesting that PROTAC–Pt(IV) could effectively treat cancer types that do not respond to traditional Pt(II) anticancer drugs. To further assess the inhibitory effects of the prodrugs on cell proliferation and colony formation from a single cell, we conducted a colony formation assay. PROTAC–Pt(IV) exhibited significant inhibition of A2780 cell colony formation compared with carboplatin and JQ1–Pt(IV) at the same concentration, indicating an enhanced ability of PROTAC–Pt(IV) to inhibit cancer cell proliferation (Fig. 2B).

Fig. 2 Cytotoxicity of PROTAC–Pt(IV). (A) IC₅₀ values of different complexes in various cancer cell lines. IC₅₀ value of JQ1–Pt(IV) is greater than 25 000 nM in various cancer cell lines. (B) Images from cell colony formation assay with A2780 cells treated with 10 nM different complexes for 14 days. (C) The volume of A2780cisR cell spheroids treated with 100 nM different complexes for 7 days.

To better mimic the natural tumor microenvironment, we conducted cytotoxicity assessments of the complexes in 3D multicellular tumor spheroids (MCTs) composed of A2780cisR cells and monitored the spheroids’ morphological changes.²¹ The MCTs treated with DMF, carboplatin, and JQ1–Pt(IV) exhibited continuous growth over time, suggesting active cell proliferation within the spheroid (Fig. 2C and S12†). In contrast, the sizes of MCTs treated with PROTAC–Pt(IV) were maintained or even slightly reduced, similar to the effects observed with ARV771 alone or in combination with carboplatin, indicating the cessation of cell proliferation (Fig. 2C). Furthermore, the edges of these MCTs appeared unclear and blurry, suggesting a loss of structural integrity or detachment of cells. Furthermore, the PROTAC–Pt(IV)-treated spheroids became progressively less transparent and darker over time, indicating cell necrosis within the interior regions (Fig. S12†). These findings collectively suggest that PROTAC–Pt(IV) not only inhibits cell proliferation but also effectively penetrates the tumor to induce cell death.

To gain more insight into the action mechanism, we next investigated the accumulation of carboplatin, JQ1–Pt(IV), and PROTAC–Pt(IV) within A2780 cells. The level of Pt in cells treated with PROTAC–Pt(IV) was measured to be 4.1 ng Pt/10⁶ cells, significantly higher than the level measured in cells treated with carboplatin and JQ1–Pt(IV) (Fig. 3A). This increased cellular accumulation of PROTAC–Pt(IV) may have been due to the large lipophilic structure of the PROTAC molecule, indicated by its high octanol–water partition coefficient (log Po/w, Fig. S13†), which could enhance its cellular penetration through passive diffusion. As DNA is the primary target of platinum drugs and an essential factor in their cytotoxic mechanism,²² we subsequently evaluated the level of Pt binding to genomic DNA in A2780 cells. PROTAC–Pt(IV) exhibited the highest level of Pt binding to DNA (Fig. 3B), confirming the efficient release of Pt after cellular entry. Compared with JQ1–Pt(IV), PROTAC–Pt(IV) displayed 6.8-fold higher cellular accumulation and a 2.4-fold increase in the level of DNA binding in A2780 cells; however, PROTAC–Pt(IV) was shown to be 1754 times more cytotoxic than JQ1–Pt(IV) in this cell line, indicating that the catalytic protein degradation capability of PROTAC–Pt(IV) contributes to its cytotoxicity.

Fig. 3 (A) Cellular accumulation of platinum from A2780 cells upon treatment with 10 μM of different complexes for 6 h. (B) Levels of platinum binding to genomic DNA from A2780 cells upon treatment with 10 μM of different complexes for 24 h. (C) Western blotting images and quantification of BRD4 in A2780 cells upon treatment with 1 μM of different complexes for 24 h. (D) Confocal images of immunofluorescence of BRD4 in A2780 cells upon treatment with 1 μM of different complexes for 12 h. Scale bars, 20 μm.

To further investigate the action mechanism and intracellular release of ARV771, a PROTAC molecule that specifically targets BRD4 for degradation,¹⁷ we treated A2780 cells with PROTAC–Pt(IV) and assessed the expression level of BRD4 using western blotting and immunofluorescence. Treatment with PROTAC–Pt(IV) resulted in a significant reduction in the expression of BRD4 compared to carboplatin (Fig. 3C). This reduction was similar to results obtained with ARV771 alone or in combination with carboplatin, indicating the efficient release of intact ARV771 ligand upon the intracellular reduction of PROTAC–Pt(IV). In addition, both ARV771 and PROTAC–Pt(IV) degraded BRD4 in a concentration-dependent manner. Furthermore, PROTAC–Pt(IV) at a concentration of 200 nM completely eradicated BRD4 in A2780 cells (Fig. S14A†).

To assess the expression of BRD4 at a subcellular resolution, A2780 cells were subjected to immunofluorescence staining to detect this protein. The subcellular localization of BRD4, visualized via immunofluorescence, generally correlated well with DAPI nuclear staining, except in the nucleolus (Fig. 3D). This observation is consistent with the predominant cellular distribution of BRD4 in the nucleoplasm rather than the nucleolus.¹⁴ Notably, treatment with PROTAC–Pt(IV) resulted in a significant reduction in the immunofluorescence signal of BRD4 compared with the signal in the untreated and carboplatin-treated groups, demonstrating the effective degradation of BRD4 by PROTAC–Pt(IV) (Fig. 3D and S14B†). Similarly, effective degradation of BRD4 following treatment with PROTAC–Pt(IV) was also observed in A549 cells (Fig. S15†).

The metastasis of cancer cells is associated with a poor prognosis for cancer treatment. Platinum-based chemotherapy has been observed to enhance cancer cell migration, while the overexpression of BRD4 has been linked to the promotion of cancer cell migration and invasion.²³ Therefore, we evaluated the inhibitory effects of carboplatin, a mixture of ARV771 and carboplatin, JQ1–Pt(IV), and PROTAC–Pt(IV) on cell migration in a wound-healing assay (Fig. S16†). In the untreated, carboplatin-, and JQ1–Pt(IV)-treated groups, 38%, 46%, and 35% of the scratched area was repopulated by cells after 48 h of incubation, respectively. In contrast, following treatment with PROTAC–Pt(IV), the wound closure ratio decreased to 16%, indicating a significant reduction in cell migration.

Building on its role in promoting BRD4 degradation and effective DNA binding, PROTAC–Pt(IV) further demonstrated its compelling anticancer activity through a cascade of molecular events. Treatment with PROTAC–Pt(IV) significantly upregulated p53 (Fig. 4A), a critical mediator of apoptosis and cell cycle regulation,^24,25 triggering the cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP) (Fig. S17†), hallmark indicators of apoptosis,²⁶ and resulting in enhanced apoptotic cell death compared to carboplatin and JQ1–Pt(IV) treatments (Fig. 4B and S18†). Concurrently, PROTAC–Pt(IV) induced the upregulation of p21 (Fig. 4C), a cyclin-dependent kinase inhibitor,²⁷ which in turn downregulated cyclin A2 and cyclin B1 (Fig. S19†), critical regulators in cell cycle progression. These molecular alternations culminated in cell cycle arrest at both the G₂/M and G₀/G₁ phases (Fig. 4D and S20†). Collectively, these findings suggest that PROTAC–Pt(IV) inhibits cancer cell proliferation by inducing cell cycle arrest while simultaneously promoting apoptosis to effectively eliminate cancer cells.

Fig. 4 (A) Western blotting images and quantification of p53 in A2780 cells upon treatment with 1 μM of different complexes for 24 h. (B) Apoptosis of A2780 cells treated with 40 nM of different complexes for 48 h, analyzed using annexin V/PI double staining via flow cytometry. (C) Western blotting images and quantification of p21 in A2780 cells upon treatment with 1 μM of different complexes for 24 h. (D) Cell cycle distribution of A2780 cells treated with 40 nM of different complexes for 48 h. (E) Western blotting images of PD–L1 in A2780 and A2780cisR cells upon treatment with 1 μM of different complexes for 24 h. (F) Quantitative expression levels of PD–L1 in A2780cisR cells upon different treatments.

Programmed death ligand 1 (PD–L1) enables tumor cells to evade immune detection. Overexpression of PD–L1 on the surface of a cancer cell allows it to bind to the PD-1 receptor on T cells, resulting in the inhibition of T cell activity.²⁸ Treatment with platinum-based drugs has been shown to increase the expression of PD–L1, contributing to tumor cell resistance to platinum-based chemotherapy.²⁹ In contrast, the inhibition of BRD4 has been reported to downregulate PD–L1 expression in various cancer cells.³⁰ Accordingly, we examined the expression of PD–L1 in A2780cisR cells. Consistent with platinum-induced upregulation of PD–L1, we observed significantly increased expression of PD–L1, which was primarily localized on the plasma membrane, in the untreated A2780cisR cells. In contrast, treatment with ARV771 alone or in combination with carboplatin or PROTAC–Pt(IV) led to the downregulation of PD–L1 expression (Fig. 4E and F, and S21†). This downregulation may contribute to the immune-mediated elimination of cancer cells.

Despite exerting superior cytotoxicity, PROTAC molecules have been reported to exhibit suboptimal pharmacokinetic profiles, which have limited their further clinical advancement.² We next examined the circulation of carboplatin, ARV771, and PROTAC–Pt(IV) in mice. A single dose of each compound was administered via intravenous (i.v.) injection, after which venous blood was collected at different time points and analyzed to determine the drug concentration levels (Fig. 5A). Notably, PROTAC–Pt(IV) exhibited pharmacokinetic characteristics distinct from those of ARV771 (Table S2†). Specifically, PROTAC–Pt(IV) demonstrated a lower clearance (CL: 2.65 L h⁻¹) and longer half-life (t_1/2: 0.43 h) than ARV771 (CL: 15.54 L h⁻¹; t_1/2: 0.31 h), indicating prolonged systemic circulation and sustained effective drug levels. Moreover, its smaller volume of distribution (6.74 mL) indicates more limited tissue dispersion relative to ARV771 (38.54 mL). These properties collectively indicate that PROTAC–Pt(IV) achieved significantly greater systemic drug exposure, as evidenced by an area under the drug concentration-time curve (AUC) of 338.2 h μM, which surpassed that of ARV771 and carboplatin by 12.1 and 7.2 times, respectively (Fig. 5B). The distinct pharmacokinetic profile resulting from modification of the PROTAC molecule with a Pt(IV) moiety may have potentially enhanced the antitumor efficacy of PROTAC–Pt(IV) compared with the parent PROTAC molecule.

Fig. 5 In vivo antitumor effects of PROTAC–Pt(IV). (A) Compound concentrations in the blood of BALB/C mice at different time points after a single intravenous administration [2 mg-Pt kg⁻¹ for platinum complexes, 10.1 mg-ARV771 kg⁻¹ (same molar concentration as Pt)]. (B) Area under the drug concentration-time curve from the time of administration to infinity. (C) Tumor volume of 4T1 tumors in xenograft models (n = 6 per group) with different treatments [3 mg-Pt kg⁻¹ for platinum complexes, 15.2 mg-ARV771 kg⁻¹ (same molar concentration as Pt)]. (D) Photographs of excised tumors after different treatments for 20 days. (E) Tumor weights after treatment with vehicle, PROTAC–Pt(IV), carboplatin, and ARV771/carboplatin (1 : 1). (F) CD3 and CD4 staining for tumors from each group. Scale bars: 100 μm. (G) Body weight of mice (n = 6 per group) with different treatments. P values were calculated using a two-tailed, unpaired Student's t-test. *** p ≤ 0.001, ** p ≤ 0.01, * 0.01 < p ≤ 0.05.

The in vivo antitumor efficacy of PROTAC–Pt(IV) was further demonstrated using a xenograft tumor model established in BALB/c mice by the subcutaneous injection of 4T1 murine breast cancer cells. We initially assessed the toxicity of PROTAC–Pt(IV) in vivo to ensure its safety. Mice were administered various doses of PROTAC–Pt(IV) through a single i.v. injection. Remarkably, even at the highest dose of 3 mg-Pt kg⁻¹, the observed body weight in mice did not change significantly (Fig. S22†), demonstrating the safety of PROTAC–Pt(IV) for use in subsequent in vivo experiments. Subsequently, mice bearing 4T1 tumors were administered the same dose of carboplatin, a mixture of ARV771 and carboplatin (1 : 1), or PROTAC–Pt(IV) via i.v. injection. The tumor volumes and body weights of the mice were monitored every 2 days (Fig. S23 and S24†). Carboplatin alone or in combination with ARV771 exerted a moderate antitumor effect compared with the vehicle control. Strikingly, despite being less cytotoxic than ARV771 in vitro, PROTAC–Pt(IV) exhibited the most potent antitumor effect in vivo: the tumor size in mice treated with this drug reached only 45% of the tumor size in vehicle-treated mice at the end of the experiment (Fig. 5C and S25†). The tumors were subsequently collected, photographed, and weighted (Fig. 5D). Those from mice treated with PROTAC–Pt(IV) had the lowest average mass (Fig. 5E). The excised tumor tissues were subsequently subjected to staining analysis. PROTAC–Pt(IV) treatment led to decreased expression of the tumor proliferation factor Ki67 (Fig. S26†), indicating reduced tumor cell proliferation. Additionally, PROTAC–Pt(IV) was associated with the strongest apoptosis signal, as detected by terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining (Fig. S27†), suggesting increased tumor cell apoptosis. Notably, PROTAC–Pt(IV) led to increases in CD3 and CD4 expression (Fig. 5F and S28†), indicating T cell infiltration and T helper cell activation within tumors. Throughout the treatment period, no notable losses of body weight were observed (Fig. 5G), indicating that the treatment was tolerable. Additionally, hematoxylin–eosin (H&E) staining of main organ tissues did not reveal any systematic toxicity caused by PROTAC–Pt(IV) at the tested dosage (Fig. S29†), further indicating its safety in vivo.

Conclusions

In conclusion, we report the synthesis of PROTAC–Pt(IV) as a pioneering platinum(IV)-based PROTAC. This prodrug remains stable in buffers and is reduced in the presence of reducing agents to release a cytotoxic platinum(II) drug and intact PROTAC molecule. Cytotoxicity assays revealed a remarkable increase in potency of up to three orders of magnitude in cells treated with PROTAC–Pt(IV) compared with the traditional inhibitor-based Pt(IV) prodrug, JQ1–Pt(IV), and this increased potency is attributed to the ability of PROTAC–Pt(IV) to promote targeted protein degradation. Mechanistic investigations demonstrated links between the augmented cancer cell lethality of PROTAC–Pt(IV) and enhanced cellular accumulation and genomic DNA binding, the inhibition of migration and colony formation, and the induction of cell cycle arrest and apoptosis through the dysregulated expression of p53 and p21, as well as the cleavage of caspase-3 and PARP. Remarkably, PROTAC–Pt(IV) was shown to exert stronger tumor suppression than ARV771 in an in vivo tumor model, which was attributed to prolonged and elevated systemic drug exposure. Furthermore, the observed downregulation of PD–L1 expression in cancer cell membranes and enhanced immune activation in xenograft tumor model mice treated with PROTAC–Pt(IV) suggest a potential role for this drug in the immune-mediated eradication of cancer cells (Fig. 6). This study demonstrates that PROTAC–Pt(IV) represents a new class of dual-action Pt(IV) prodrugs that target protein degradation and induce DNA damage simultaneously to achieve enhanced tumor suppression. The use of a carbonate linkage ensures both stability and intact release upon reduction, suggesting that this strategy could be readily extended to other VHL-based PROTACs, particularly those targeting resistance pathways in platinum-based chemotherapy. Moreover, the incorporation of Pt(IV) complexes offers a feasible strategy for improving the pharmacokinetics of PROTAC molecules and achieving synergistic effects upon intracellular reduction.

Fig. 6 Proposed action mechanism of PROTAC–Pt(IV).

Author contributions

J. X. and S. C. contributed equally. J. X. and G. Z. designed the study. J. X. performed the experiments. S. C. and K.-Y. N. performed the in vivo experiments. J. X., W. C. F., and G. Z. analyzed the data. X. C. supports the manuscript writing. J. X. and G. Z. wrote the paper. All authors edited and approved the final manuscript.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We thank the Hong Kong Research Grants Council (Grant No. CityU 11303320, CityU 11302221, CityU 11313222, CityU 11304923, and C1018-23G), the National Natural Science Foundation of China (Grant No. 22077108 and 22277103), the Science Technology and Innovation Committee of Shenzhen Municipality (JCYJ20210324120004011), and the City University of Hong Kong (Grant No. 7020014) for funding support.

References

1. K. Li and C. M. Crews, PROTACs: past, present and future, Chem. Soc. Rev., 2022, 51, 5214–5236.
2. Y. Chen, I. Tandon, W. Heelan, Y. Wang, W. Tang and Q. Hu, Proteolysis-targeting chimera (PROTAC) delivery system: Advancing protein degraders towards clinical translation, Chem. Soc. Rev., 2022, 51, 5330–5350.
3. R. A. Ward, S. Fawell, N. Floc'h, V. Flemington, D. McKerrecher and P. D. Smith, Challenges and opportunities in cancer drug resistance, Chem. Rev., 2020, 121, 3297–3351.
4. P. Martín-Acosta and X. Xiao, PROTACs to address the challenges facing small molecule inhibitors, Eur. J. Med. Chem., 2021, 210, 112993.
5. M. Békés, D. R. Langley and C. M. Crews, PROTAC targeted protein degraders: the past is prologue, Nat. Rev. Drug Discovery, 2022, 21, 181–200.
6. T. C. Johnstone, K. Suntharalingam and S. J. Lippard, The next generation of platinum drugs: targeted Pt(II) agents, nanoparticle delivery, and Pt(IV) prodrugs, Chem. Rev., 2016, 116, 3436–3486.
7. S. Jin, N. Muhammad, Y. Sun, Y. Tan, H. Yuan, D. Song, Z. Guo and X. Wang, Multispecific Platinum(IV) Complex Deters Breast Cancer via Interposing Inflammation and Immunosuppression as an Inhibitor of COX-2 and PD-L1, Angew. Chem. Int. Ed., 2020, 59, 23313–23321.
8. Q. Cao, D. J. Zhou, Z. Y. Pan, G. G. Yang, H. Zhang, L. N. Ji and Z. W. Mao, CAIXplatins: highly potent platinum(IV) prodrugs selective against carbonic anhydrase IX for the treatment of hypoxic tumors, Angew. Chem. Int. Ed., 2020, 59, 18556–18562.
9. Z. Wang, Z. Xu and G. Zhu, A platinum(IV) anticancer prodrug targeting nucleotide excision repair to overcome cisplatin resistance, Angew. Chem. Int. Ed., 2016, 55, 15564–15568.
10. L. Markova, M. Maji, H. Kostrhunova, V. Novohradsky, J. Kasparkova, D. Gibson and V. Brabec, Multiaction Pt(IV) Prodrugs Releasing Cisplatin and Dasatinib Are Potent Anticancer and Anti-Invasive Agents Displaying Synergism between the Two Drugs, J. Med. Chem., 2024, 67, 9745–9758.
11. K. G. Z. Lee, M. V. Babak, A. Weiss, P. J. Dyson, P. Nowak-Sliwinska, D. Montagner and W. H. Ang, Development of an efficient dual-action GST-inhibiting anticancer platinum(IV) prodrug, ChemMedChem, 2018, 13, 1210–1217.
12. M. D. Hall, H. R. Mellor, R. Callaghan and T. W. Hambley, Basis for design and development of platinum(IV) anticancer complexes, J. Med. Chem., 2007, 50, 3403–3411.
13. L. Zhao, J. Zhao, K. Zhong, A. Tong and D. Jia, Targeted protein degradation: mechanisms, strategies and application, Signal Transduction Targeted Ther., 2022, 7, 113.
14. B. Donati, E. Lorenzini and A. Ciarrocchi, BRD4 and Cancer: going beyond transcriptional regulation, Mol. Cancer, 2018, 17, 164.
15. Z.-Q. Wang, Z.-C. Zhang, Y.-Y. Wu, Y.-N. Pi, S.-H. Lou, T.-B. Liu, G. Lou and C. Yang, Bromodomain and extraterminal (BET) proteins: biological functions, diseases, and targeted therapy, Signal Transduction Targeted Ther., 2023, 8, 420.
16. A. L. Drumond-Bock and M. Bieniasz, The role of distinct BRD4 isoforms and their contribution to high-grade serous ovarian carcinoma pathogenesis, Mol. Cancer, 2021, 20, 145.
17. K. Raina, J. Lu, Y. Qian, M. Altieri, D. Gordon, A. M. K. Rossi, J. Wang, X. Chen, H. Dong and K. Siu, PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer, Proc. Natl. Acad. Sci. U. S. A., 2016, 113, 7124–7129.
18. J. Krieger, F. J. Sorrell, A. A. Wegener, B. Leuthner, F. Machrouhi-Porcher, M. Hecht, E. M. Leibrock, J. E. Mueller, J. Eisert and I. V. Hartung, Systematic potency and property assessment of VHL ligands and implications on PROTAC design, ChemMedChem, 2023, 18, e202200615.
19. S. Chen, K.-Y. Ng, Q. Zhou, H. Yao, Z. Deng, M.-K. Tse and G. Zhu, The influence of different carbonate ligands on the hydrolytic stability and reduction of platinum(IV) prodrugs, Dalton Trans., 2022, 51, 885–897.
20. J. Rautio, H. Kumpulainen, T. Heimbach, R. Oliyai, D. Oh, T. Järvinen and J. Savolainen, Prodrugs: design and clinical applications, Nat. Rev. Drug Discovery, 2008, 7, 255–270.
21. S. Sant and P. A. Johnston, The production of 3D tumor spheroids for cancer drug discovery, Drug Discovery Today: Technol., 2017, 23, 27–36.
22. P. D. O'Dowd, G. P. Sullivan, D. A. Rodrigues, T. N. Chonghaile and D. M. Griffith, First-in-class metallo-PROTAC as an effective degrader of select Pt-binding proteins, Chem. Commun., 2023, 59, 12641–12644.
23. T. Liu, Z. Zhang, C. Wang, H. Huang and Y. Li, BRD4 promotes the migration and invasion of bladder cancer cells through the Sonic hedgehog signaling pathway and enhances cisplatin resistance, Biochem. Cell Biol., 2022, 100, 179–187.
24. D. Wang and S. J. Lippard, Cellular processing of platinum anticancer drugs, Nat. Rev. Drug Discovery, 2005, 4, 307–320.
25. A.-L. Latif, A. Newcombe, S. Li, K. Gilroy, N. A. Robertson, X. Lei, H. J. S. Stewart, J. Cole, M. T. Terradas and L. Rishi, BRD4-mediated repression of p53 is a target for combination therapy in AML, Nat. Commun., 2021, 12, 241.
26. G. M. Cohen, Caspases: the executioners of apoptosis, Biochem. J., 1997, 326, 1–16.
27. T. Abbas and A. Dutta, p21 in cancer: intricate networks and multiple activities, Nat. Rev. Cancer, 2009, 9, 400–414.
28. H. Yamaguchi, J.-M. Hsu, W.-H. Yang and M.-C. Hung, Mechanisms regulating PD-L1 expression in cancers and associated opportunities for novel small-molecule therapeutics, Nat. Rev. Clin. Oncol., 2022, 19, 287–305.
29. L. Fournel, Z. Wu, N. Stadler, D. Damotte, F. Lococo, G. Boulle, E. Ségal-Bendirdjian, A. Bobbio, P. Icard and J. Trédaniel, Cisplatin increases PD-L1 expression and optimizes immune check-point blockade in non-small cell lung cancer, Cancer Lett., 2019, 464, 5–14.
30. W. Mao, A. Ghasemzadeh, Z. T. Freeman, A. Obradovic, M. G. Chaimowitz, T. R. Nirschl, E. McKiernan, S. Yegnasubramanian and C. G. Drake, Immunogenicity of prostate cancer is augmented by BET bromodomain inhibition, J. Immunother. Cancer, 2019, 7, 1–14.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5qi00605h

‡ These authors contributed equally to this work.

---