Platinum(II) (Pt(II))-based drugs, including cisplatin (CDDP), carboplatin, and oxaliplatin (OXP), have been extensively used in cancer treatment for nearly 50 years since their introduction into clinical practice (Ghosh, 2019, Kelland, 2007). Unfortunately, their use is frequently limited by significant side effects, non-selective toxicity, and the emergence of drug resistance (Gatti et al., 2015, Han et al., 2015, Wlodarczyk et al., 2018). Recent studies have demonstrated the clinical potential of multi-target platinum (IV) complexes(Babu et al., 2023; Shi et al., 2024), which are emerging as ideal alternatives to overcome the shortcomings of clinical platinum (II) drugs, with the potential to achieve breakthroughs in the development of next-generation platinum-based therapies (Johnstone et al., 2016). By optimizing the ligands of platinum(IV) prodrugs in both axial and equatorial positions, multifunctionality could be achieved, which enhanced their selectivity for tumor cells while maintaining stability and resistance to nucleophilic substitution in circulation (Hall et al., 2007, Kastner et al., 2023). Typically, axial ligands imparted important properties to platinum (IV) prodrugs, such as prolonged drug action, increased lipophilicity, enhanced redox stability, and improved cellular uptake, all of which collectively contribute to minimizing side effects (Chen et al., 2018, Lappano et al., 2017, Novohradsky et al., 2017, Oldfield et al., 2007). Although recent studies had attempted to enhance the function of platinum (IV) complexes by selecting and optimizing axial compounds to provide additional targeted therapeutic effects beyond platinum (II)-mediated chemotherapy, with the aim of developing multi-target platinum (IV) drugs that combine chemotherapy and targeted therapy (Yu et al., 2024), no platinum (IV) prodrugs had advanced to the clinical stage yet (Sessa et al., 1998).

Among the numerous targets for cancer treatment, TGF-β plays a dual role in tumor biology. Initially, TGF-β functions as a tumor suppressor, inhibiting cellular proliferation (Johansen et al., 2024). However, as cancer progresses, TGF-β shifted its role to a pro-tumorigenic factor, facilitating tumor cell invasion, metastasis, and resistance to therapies (Derynck et al., 2021, Johansen et al., 2024). The TGF-β signaling pathway exerted its effects on tumor progression and treatment resistance through several mechanisms, including the modulation of tumor microenvironment, regulation of immune responses, enhancement of cancer stem cell properties, and promotion of epithelial-mesenchymal transition (EMT) (Deng et al., 2024, Wang et al., 2024, Zhou et al., 2024). TGF-β-mediated immune suppression significantly hampered the efficacy of immune checkpoint inhibitors and other immunotherapeutic strategies (Deng et al., 2024). Local delivery of TGF-β inhibitors within the TME had shown promise in promoting macrophage polarization toward the antitumor M1 phenotype, reversing CD8⁺ T cell exhaustion, and enhancing the response of cancer to anti-PD-1 therapy (Mariathasan et al., 2018, Zhong et al., 2017). Consequently, TGF-β has emerged as a promising target for cancer therapy, with various inhibitors currently undergoing clinical trials aimed at improving therapeutic outcomes (Brandes et al., 2016, Derynck et al., 2021, Kozono et al., 2013). Gramine, an indole alkaloid with antiviral, antifungal, antioxidant, anti-proliferative, and anti-inflammatory properties, has been demonstrated to have anti-tumor activity through the inhibition of TGF-β signaling (Xu et al., 2023). It effectively reduced the phosphorylation and nuclear translocation of Smad2 and Smad4 by blocking the activity of TGF-β receptors I and II, thereby inhibiting oral squamous cell carcinoma with reduced toxicity (Ramu et al., 2017). Therefore, gramine has the potential effect to inhibit TGF-β-mediated function (Xu et al., 2023). The NH bond of gramine can be modified without significantly impacting its biological activity, and its stable indole ring structure minimizes the generation of harmful byproducts in vivo, thereby demonstrating substantial potential for drug development and reverse drug resistance, particularly as its core bioactivity can be preserved while optimizing its pharmacokinetic properties and safety through chemical modifications (Lajarin-Cuesta et al., 2016, Perez-Ramirez et al., 2017). Given gramine's targeted inhibitory effect on TGF-β, it would be beneficial to develop gramine as an axial ligand in platinum (IV) complexes to design a multi-functional platinum (IV) prodrug with improved targetability, stability, reducibility, solubility, and other pharmacokinetic parameters, thereby creating a novel drug that could combine targeted therapy and chemotherapy.

Platinum-based drugs work by binding to DNA, causing DNA damage and triggering cell apoptosis. Simultaneously, they release damage-associated molecular patterns (DAMPs), which activate the immune system, stimulating dendritic cells and T-cell anti-tumor responses (Leonetti et al., 2019, Shu et al., 2016). As a result, platinum drugs not only directly kill tumor cells but also enhance immune activation, making them well-suited for combination with immunotherapy to achieve synergistic effects (Bartoletti et al., 2024, Zhou et al., 2021). An increasing number of preclinical and clinical studies demonstrate the value of combining platinum drugs with immune checkpoint inhibitors (ICIs) to significantly improve treatment outcomes. This strategy shows particular importance in immunotherapy resistant-tumor, where platinum-based multi-agent chemotherapy regimens combined with PD-1/PD-L1 inhibitors have yielded encouraging results, as evidenced by the KEYNOTE-522 trial for triple-negative breast cancer (TNBC) (Schmid et al., 2024, Schmid et al., 2022) and the KEYNOTE-966 trial for advanced biliary tract cancer (Kelley et al., 2023). Importantly, the combination of TGF-β inhibitors and immune checkpoint inhibitors (ICIs), such as anti-PD-1/PD-L1 therapies, shares similarities with the aforementioned studies and also shows tremendous potential in cancer treatment. For example, the TGF-β receptor inhibitor Galunisertib combined with anti-PD-1 therapy has shown enhanced T-cell-mediated tumor regression, suggesting a synergistic effect beneficial for resistant tumors (Li et al., 2021, Yamazaki et al., 2022). Additionally, bispecific agents like SHR-1701, which target both PD-L1 and TGF-β, exhibit antitumor activity in various advanced cancers while maintaining favorable safety profiles (Feng et al., 2022). These advancements reflect a growing interest in targeting TGF-β to mitigate immune suppression and improve the efficacy of ICIs, particularly in tumors with inherent resistance. Furthermore, NIS793, a new TGF-β-blocking antibody, has demonstrated preclinical efficacy when combined with FOLFIRINOX. This suggests that TGF-β inhibition in conjunction with platinum-based chemotherapy regimens may yield a synergistic therapeutic effect (Qiang et al., 2023). The previously mentioned studies suggest that platinum-based chemotherapy and TGF-β targeting have a strong synergistic effect on immunotherapy (Abdulla et al., 2023). If platinum (IV) complexes can be effectively combined with TGF-β targeting agents, it may amplify this synergistic effect, which would play a significant role in the development of new drugs. TGF-β-R1 is widely expressed in various cell types, raising concerns about potential off-target effects and the likelihood of unavoidable side effects in cancer patients due to unselective inhibition. However, the novel platinum (IV) prodrug demonstrates enhanced tumor accumulation and targetability, while minimizing adverse events.

In this study, we incorporate gramine (GM) into platinum(IV) precursors to develop a series of gramine-platinum (IV) prodrugs. The gramine modification enhances the drug's lipophilicity and targeting ability, leading to increased intracellular drug accumulation and a synergistic effect between TGF-β inhibition and the cytotoxic action of the platinum-based drug. In vivo, these prodrugs preferentially accumulate at the tumor site following intravenous injection, resulting in potent antitumor efficacy without significant toxicity. By releasing GM from these prodrugs, the TGF-β pathway is effectively inhibited, which overcomes CDDP resistance, enhances platinum-induced DNA damage, and reduces the accumulation of immunosuppressive cells. This transformation leads to the conversion of immunotherapy-resistant tumors into more immunogenic ones. Our studies highlight the in vivo effectiveness of this platinum-based therapy in treating both pancreatic ductal adenocarcinoma (PDAC) and TNBC, which is characterized by immunologically "cold" tumor microenvironments (TMEs) and marked by substantial myeloid cell infiltration and dysfunctional CD8⁺ T cells (Khosravi et al., 2024, Tharp et al., 2024). This combined strategy has the potential to significantly enhance antitumor immune responses and improve therapeutic outcomes for both chemotherapy and immunotherapy, offering a promising treatment approach for pancreatic and breast cancers.