DHPS‐Mediated Hypusination Regulates METTL3 Self‐m6A‐Methylation Modification to Promote Melanoma Proliferation and the Development of Novel Inhibitors

Abstract

Discovering new treatments for melanoma will benefit human health. The mechanism by which deoxyhypusine synthase (DHPS) promotes melanoma development remains elucidated. Multi‐omics studies have revealed that DHPS regulates m6A modification and maintains mRNA stability in melanoma cells. Mechanistically, DHPS activates the hypusination of eukaryotic translation initiation factor 5A (eIF5A) to assist METTL3 localizing on its mRNA for m6A modification, then promoting METTL3 expression. Structure‐based design, synthesis, and activity screening yielded the hit compound GL‐1 as a DHPS inhibitor. Notably, GL‐1 directly inhibits DHPS binding to eIF5A, whereas GC‐7 cannot. Based on the clarification of the mode of action of GL‐1 on DHPS, it is found that GL‐1 can promote the accumulation of intracellular Cu²⁺ to induce apoptosis, and antibody microarray analysis shows that GL‐1 inhibits the expression of several cytokines. GL‐1 shows promising antitumor activity with good bioavailability in a xenograft tumor model. These findings clarify the molecular mechanisms by which DHPS regulates melanoma proliferation and demonstrate the potential of GL‐1 for clinical melanoma therapy.

Introduction

Melanoma is a highly malignant form of cancer with increasing incidence and mortality rates.^[1–6] Early surgical resection is the only effective treatment for malignant melanoma, and unfortunately, the prognosis is poor.^[7–9] Therefore, it is crucial to discover the development mechanism of melanoma and develop effective therapeutic drugs to improve patient outcomes.

Deoxyhypusine synthase (DHPS), as an intracellularly conserved enzyme, catalyzes the hypusination of eukaryotic translation initiation factor 5A (eIF5A).^[10–12] Our team was the first to report the inhibitory effect of DHPS inhibitors on melanoma.^[13] In recent years, it has been found that DHPS is highly expressed in some cancers (e.g., colorectal cancer, leukemia) and promotes the protein translation process by activating eIF5A (eIF5A-Hyp), which maintains the hyperproliferation of cancer cells.^[14–16] eIF5A promotes peptide chain elongation and release by occupying the E-site of the ribosome, thus regulating the translation process.^[17] However, not all proteins require eIF5A regulation, and the expression level of DHPS varies in different cancer cells.^[13–16] Therefore, discovering the regulatory mechanism of DHPS/eIF5A in melanoma is an essential basis for clinical chemotherapy.

Since DHPS regulates the protein translation process of mRNAs by promoting the hypusination of eIF5A, it remains to be elucidated whether this process is associated with epitope modification. N6-methyladenosine (m6A) is a dynamic and reversible methylation modification at the N6 site of adenosine that is important for maintaining the dynamic equilibrium of mRNA.^[18–20] There have been limited studies on the role of m6A-related regulatory proteins in melanoma, including “writers,” “erasers,” and “readers.”^[21–25] Methyltransferase-like 3 (METTL3) of the “Writers” plays a vital role in promoting melanoma tumorigenesis;^[26–28] obesity-associated protein (FTO) in “Erasers”^[29] acts as an, as an m6A demethylase, plays a crucial role in promoting melanoma tumorigenesis and anti-PD-1 resistance.^[29,30] Additionally, YTH domain family 2 (YTHDF2) of the “Readers” promotes the development of ocular melanoma by reducing mRNA degradation through reduced m6A recognition.^[31] Thus, studying the regulatory relationship the study of the regulatory relationship between DHPS/eIF5A-Hyp and m6A modification will provide a theoretical basis for the clinical application of DHPS in the treatment of melanoma and serve as a mechanistic reference for developing novel DHPS inhibitors.

Here, we aimed to uncover the pro-oncogenic mechanism of DHPS in melanoma by mediating the hypusination of eIF5A to promote the m6A modification of METTL3 itself through a multi-omics approach to elucidate the value of DHPS as a therapeutic target for melanoma. Based on the oncogenic mechanism of DHPS, a series of novel DHPS allosteric inhibitors were obtained through structure-based compound design and synthesis. The screening of the hit compound (GL-1) at the enzyme and cellular levels, in vivo studies were conducted to determine the ability of GL-1 to target DHPS and its efficacy against melanoma.

Result

Discovery and Validation of DHPS as a Target in Skin Melanoma

Analysis of The Cancer Genome Atlas (TCGA) database revealed that DHPS is highly expressed in cancer tissues, particularly in skin cancer (Figure 1A).^[32] Furthermore, a human melanoma tissue chip (111 melanoma samples and 18 adjacent normal tissues) was used for analysis. It was found that DHPS is more highly expressed in melanoma than in normal skin tissue (Figure 1B). DHPS-mediated hypusination modifications were also differentially expressed in melanoma compared to normal skin tissue (Figure 1C). When the DHPS gene was knocked down, the proliferation of melanoma cells was inhibited. In contrast, the proliferative ability of normal skin cells was unaffected (Figure 1D).

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DHPS Affects the Stability of mRNA by Regulating m6A Modifications in Melanoma

To explore the mechanism by which DHPS regulates melanoma development, high-throughput sequencing of melanoma cells after knockdown of the DHPS gene was performed here using mRNA-seq. After the knockdown of the DHPS gene, the mRNA expression of 4750 genes was up-regulated, and 466 genes were down-regulated in A375 cells (Figure 2A,B). GESA analysis revealed that the differential genes mainly focused on mRNA stability pathways (e.g., silencing, degradation, and metabolism) (Figure 2C). However, the colony formation assay in Figure 1D suggests that DHPS is an oncogene contrary to the mRNA overexpression shown by the high-throughput sequencing results. Thus, the phenomenon of mRNA overexpression in melanoma cells with proliferation inhibition suggests that the regulatory mechanism of DHPS may be related to the stability mechanism of mRNA.

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The m6A modification is a crucial factor that affects mRNA stability. To understand whether DHPS is associated with the m6A modification mechanism of mRNAs, a high-throughput analysis of m6A methylation levels was performed on knockdown DHPS and normal A375 cells. The results of MeRIP-seq showed that in A375 cells with the DHPS gene knockdown, the m6A peak abundance of 2551 genes decreased, while that of 246 genes increased (Figure 2D). Subsequent investigation on the m6A peak distributions revealed that total m6A distribution patterns were the same in the control groups and DHPS knockdown groups (Figure 2E). The consensus motif “RRACH,” which is highly concentrated at m6A sites, was present in both the control and DHPS knockdown cells (Figure 2F).^[33] Enrichment analysis showed that the regulation of melanoma cell signaling pathways by DHPS may be related to RNA stability (Figure 2G,H). Additionally, western blot analysis revealed that the expression of three vital proteins that regulate m6A modification, namely METTL3, YTHDF2, and YTHDC1, was reduced in melanoma cells after DHPS knockdown (Figure 2I,J).

These findings suggest that DHPS may drive melanoma by modifying the gene through m6A methylation.

DHPS Regulates the Self-m6A-Methylation of METTL3 Through the Catalysis of the Hypusination of eIF5A to Maintain mRNA Stability

To identify critical downstream targets of DHPS-mediated m6A modifications in A375 cells, we analyzed the frequency and distribution of m6A modifications in differentially expressed mRNA by MeRIP-seq and RNA-seq tandem analysis. This involved the selection of mRNAs with m6A peaks and mRNA abundance changes of >1.0-fold at p < 0.05. Further analysis revealed that the methylation of METTL3 itself was downregulated, with no significant difference in mRNA content. The analysis of the m6A methylation site of METTL3 also revealed that the abundance of its 3′UTR and CDS region was reduced significantly (Figure 3A,B). Meanwhile, the mRNA expression levels of m6A modification of YTHDF2 or YTHDC1 were not significantly different (Figure 3A). Consequently, we postulated that the regulatory mechanism of DHPS on METTL3 might be distinct from that of DHPS on YTHDC1 or YTHDF2. To ascertain whether the regulatory effect of DHPS on YTHDC1 or YTHDF2 involves the participation of METTL3, the expression levels of DHPS, YTHDC1, and YTHDF2 proteins were analyzed following overexpression of METTL3 in melanoma cells. Further overexpression of the METTL3 protein resulted in no significant changes in DHPS, YTHDC1, and YTHDF2 proteins, as shown in Figure 3C,D. These results indicated that DHPS may regulate the mRNA self-m6A-methylation modification of METTL3. It was determined that the translational regulation of YTHDF2 and YTHDC1 by DHPS did not involve m6A-methylation modification.

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Considering METTL3's role as a methyltransferase, we hypothesized that METTL3's m6A modification might be “written” by itself and that DHPS or eIF5A-Hyp could mediate this “writing” process. To investigate this, we conducted Co-IP and RIP assays to determine whether DHPS or eIF5A-Hyp regulates the m6A modification of METTL3. The Co-IP assays revealed that DHPS could not directly bind to METTL3 in melanoma cells, while eIF5A-Hyp could bind to METTL3. Interestingly, this binding was hindered when the hypusination of eIF5A was inhibited (Figure 3E–G). Additionally, analysis of the m6A peak region of METTL3 uncovered the presence of two similar “RRACH” sequences in the CDS region near the 5′UTR (Figure 3H). Based on this discovery, we designed specific RNA primers for RIP experiments (for details, Scheme S1, Supporting Information), and the results showed that METTL3 could bind to its mRNA in melanoma and eIF5A-Hyp could also bind to the METTL3 mRNA (Figure 3I). However, this binding was disrupted when the hypusination of eIF5A was inhibited. Furthermore, neither eIF5A-Hyp nor METTL3 could bind to the METTL3 mRNA after the mutation of A to C in the “RRACH” (Figure 3I). Moreover, the knockdown of DHPS and the mutation of the m6A site of METTL3 resulted in reduced mRNA stability of Ki67 and METTL3 (Figure 3J).

Taken together, these results suggest that DHPS mediates the hypusination of eIF5A by assisting METTL3 in recognizing its m6A site for methylation modification and maintaining the stability of mRNAs in melanoma cells (Figure 3H).

Discovery of GL-1 as a Novel DHPS Inhibitor

Based on the new mechanism of DHPS regulation in melanoma, the target potential of DHPS has been identified, which makes the development of DHPS-targeted inhibitors of great significance. A series of DHPS allosteric inhibitors with novel structures were designed by fragment-based structure optimization (Scheme S2, Supporting Information). A total of 14 target compounds were obtained by directed synthesis (Figure 4A). Structural confirmation of the target compounds is detailed in the Supporting Information (Scheme S3, Figures S1–S48, and Tables S1–S16). DHPS enzyme assay and CCK-8 assay screening (Table S17, Supporting Information) were used to obtain the hit compound 7f (GL-1). GL-1 inhibited DHPS enzyme activity more than GC-7 (IC_50 GL-1  = 0.21 ± 0.07 µm, IC_50 GC-7  = 1.58 ± 0.02 µm) and has a high affinity for DHPS enzymes (KD _GL-1  = 2.42 × 10⁻⁵, KD _GC-7  = 3.11 × 10⁻⁵) (Figure 4B,C; Figure S49, Supporting Information). Notably, the ability of GL-1 to inhibit cell viability at the same concentration was more prominent compared to GC-7, especially for A375 cells (Figure 4D). Moreover, GL-1 inhibited hypusination of eIF5A in a concentration-dependent manner (Figure 4E).

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The ability of GL-1 to stably bind to DHPS in cells was confirmed by CETSA and DARTS assays (Figure 4F,G). Docking studies revealed that GL-1 could interact with amino acid residues in the active pocket of DHPS in three poses (K329, V129, L281, and D238). A single-point mutation of the DHPS protein was performed, and the results showed that the interaction of GL-1 with all four amino acid residues inhibited the cell viability of either A375 or SK-MEL-28 cells. The D238 site was first identified as the critical amino acid site for DHPS activity, although its role was slightly weaker.

Simultaneously, since DHPS can directly bind to eIF5A to exert hypusination function, the Co-IP assay was used to detect the binding ability of DHPS to eIF5A after GL-1 treatment. The results were shown in Figure 4J, GL-1 could inhibit the binding of DHPS to eIF5A. However, GC-7 could not inhibit the binding of DHPS to eIF5A (Figure S50, Supporting Information).

The above results indicate that GL-1 is a potential DHPS inhibitor that could inhibit eIF5A's hypusination by directly interacting with DHPS (Figure 4K).

GL-1 Exhibits Promising Antimelanoma Efficacy In Vitro

To verify the antiproliferative ability of GL-1, we conducted a colony formation assay to explore the effect of GL-1 on the proliferative ability of melanoma cells. The results, shown in Figure 5A,B, indicate that GL-1 has a concentration-dependent inhibitory effect on the proliferation of A375, SK-MEL-28, and B16-F10 cells. Furthermore, GL-1 was more effective than GC-7 at the same concentration (2.0 µm).

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Flow cytometry analysis was utilized to detect apoptosis induced by GL-1 in melanoma cells (Figure 5C,D). However, GL-1 had a significantly stronger regulatory effect on B16-F10 than on A375 and SK-MEL-28. In A375 cells, treatment with GL-1 at a concentration of 2.0 µm for 24 h resulted in 35.1% cell survival, while GC-7 showed 92.4% survival at the same concentration. Western blot analysis revealed that GL-1 inhibited the expression of TYMS protein in a concentration-dependent manner in A375 or SK-MEL-28 cells. Similar results were observed in B16-F10 cells (Figure 5E–H). Additionally, GL-1 inhibited the expression of caspase9 protein and induced the activation of caspase3.

In summary, GL-1 effectively inhibited the proliferation of melanoma cells by inhibiting the expression of the proliferative protein TYMS and affecting the content of caspase9 and the activation of caspase3.

GL-1 Destroyed Cu²⁺ Homeostasis and Inhibited Melanoma Cells from Secreting a Variety of Cytokines

However, GL-1 did not regulate the protein expression of caspase9 and activated caspase3 in A375, SK-MEL-28, and B16-F10 cells in a concentration-dependent manner, suggesting that other apoptosis-related regulatory mechanisms may exist. In recent years, metal ions have been shown to play an important role in cell proliferation and apoptosis. We are concerned that copper ion homeostasis may directly influence apoptotic signaling in melanoma. To study the impact of GL-1 on copper ion homeostasis in melanoma cells, ELISA was used to quantify intracellular Cu²⁺ levels. The results showed that GL-1 could disrupt intracellular Cu²⁺ homeostasis in A375 and SK-MEL-28 cells in a concentration-dependent manner, resulting in large amounts of Cu²⁺ retained in the cells (Figure 6A). However, this effect was not evident for B16-F10 cells.

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The study investigated the effect of GL-1 on the protein expression of METTL3, YTHDF2, and YTHDC1, which are regulated by DHPS. The results indicated that GL-1 inhibited the protein expression of these three proteins in a concentration-dependent manner in A375 and SK-MEL-28 cells (Figure 6B,C), consistent with the findings of DHPS knockdown. Cu²⁺ plays a role in several cancer-related processes, such as generating and transporting cancer-related cytokines. Here, we used proteomics to investigate the relevant mechanisms of DHPS and cytokine regulation. Solid-state antibody chip results, including 105 antibodies, showed that GC-7 and GL-1 affected the expression of melanoma cytokines, among which ANG, IGFBP3, CSF2, CD71, CXCL1, and MMP9 were the most significant(Figure 6D; Figure S51, Supporting Information). IGFBP3, CSF2, CD71, and MMP9 are all factors associated with cell proliferation and migration. This suggests that when DHPS is inhibited, it directly affects the metastasis of melanoma cells. Further scratch assay also confirmed the ability of GL-1 to inhibit cell migration in a concentration-dependent manner (Figure S52, Supporting Information).

Since ANG, as an angiogenesis factor, directly affects melanoma angiogenesis, we used angiogenesis antibody microarrays containing 55 antibodies to explore the anti-angiogenic effects of GL-1 and GC-7. The results showed that GL-1 and GC-7 effectively inhibited the expression of ANG, PAI-1, MMP9, TSP-1, PDGF, VEGF, and FGF2 factors. The result suggested that inhibition of DHPS activity interferes with the expression of cytokines, particularly angiogenesis factors, and may disrupt the angiogenesis pathway.

GL-1 Effectively Inhibits Melanoma Development In Vivo and Shows Favorable Bioavailability

A pharmacokinetic study was conducted to determine the drug-gable characteristics of GL-1. Blood concentrations were analyzed at various time points following intravenous injection of 5 mg kg⁻¹ and intraperitoneal injection of 40 mg kg⁻¹. The results showed that GL-1 is stable and has high bioavailability (F = 55.07%) in vivo (Figure 7A). Since there was no significant difference in the in vivo half-life between intravenous and intraperitoneal administration (T_1/2, i.v. = 9.46 h, T_1/2, i.p. = 9.77 h), we chose the milder intraperitoneal administration for the in vivo pharmacodynamic study based on animal welfare considerations.

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A xenograft mouse model was constructed using A375 cells to verify the role of GL-1 as a DHPS inhibitor against melanoma in vivo. The inhibitory effect of GL-1 on melanoma tissues was investigated at the same dose (40 mg kg⁻¹) using GC-7 as a positive reference. The results are shown in Figure 7B–E; after 18 days of administration at 3-day intervals, GL-1 effectively inhibited the proliferation of tumor tissues with a TGI of 59% while having less effect on the body weight of the model mice (Figure 7F). Moreover, there was no significant difference in the effects on liver and kidney function (Figure 7G). The low toxicity of GL-1 was also confirmed by HE stains (Figure S54, Supporting Information). In addition, IHC analysis of tumor tissues showed that GL-1 inhibited the expression of Ki67, promoted the expression of cleaved-caspase3, and inhibited hypusiantion in tumor tissues (Figure 7H). The IHC and western blot results showed that both GL-1 and GC-7, as DHPS inhibitors, inhibited METTL3, YTHDF2, and YTHDC1 in tumor tissues (Figure 7H,I).

Discussion

Melanoma's high degree of malignancy results in a low survival rate due to the lack of effective treatments. The discovery of new therapeutic mechanisms and targeted drugs is crucial to bring hope for patients' survival. DHPS has received extensive attention in recent years.^[34,35] DHPS catalyzes the hypusination of eIF5A, an essential regulator of protein translation.^[36–40] However, there have been few reports on how DHPS is regulated in melanoma.

In this study, mRNA-seq analysis showed that the number of genes up-regulated by mRNA expression after DHPS knockdown was higher than the number of genes down-regulated, suggesting that DHPS is closely related to the mRNA stability in melanoma cells. In contrast, MeRIP-seq results showed that the number of genes down-regulated by m6A after DHPS knockdown was lower than that of control in melanoma cells. This contradictory mechanism suggests that the oncogenic mechanism of DHPS is intricately linked to m6A modification.

The m6A modification is widely present in cancer cells, but the various levels of m6A in different cancers also predict the diversity of regulatory mechanisms.^[41–43] Hypomethylation in conjunctival melanoma has been shown to promote melanoma development due to reduced recognition of m6A by YTHDF2, which inhibits mRNA degradation and promotes the translation process.^[31] However, our studies on cutaneous melanoma have demonstrated that the knockdown of DHPS reduces both YTHDF2 and METTL3 proteins. This reduction directly affects the intracellular modification of m6A, which may be the main reason for the changes in mRNA stability observed in cutaneous melanoma cells. Subsequent studies revealed that METTL3 could not regulate the expression of YTHDC1/YTHDF2 proteins in melanoma, which also predicts that the methylation modifications regulated by METTL3 are not required for all genes, but that METTL3-mediated m6A-methylation modification is dependent on DHPS. Mechanistic studies revealed that the binding of METTL3 to eIF5A-Hyp induced METTL3 to modify its self-m6A-methylation, and DHPS was a crucial factor in promoting eIF5A-Hyp expression. Therefore, our results suggest that DHPS is a promising anti-melanoma target that regulates intracellular m6A-methylation modification and influences protein translation (Figure 8).

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The development of new drugs represents a significant objective of mechanistic studies. Traditional DHPS inhibitors are spermidine analogs, of which GC-7 is the most widely used.^[44] In recent years, both Takeda Pharmaceutical Laboratories and our team have been working on developing novel DHPS allosteric inhibitors.^[45,46] In this study, a series of novel DHPS inhibitors were optimized and synthesized based on the lead structure, and the hit compound GL-1 was obtained by enzyme activity assay and cell viability assay. GL-1 inhibits the catalytic effect of DHPS on eIF5A by interacting with four amino acid residues in the active pocket of DHPS to form a stable binding. Of these four amino acid residues, K329 is the most important, as it is the essential site for the attachment of hypusine. Meanwhile, point mutation experiments showed that V129 and L286, like K329, could abolish the inhibitory effect of GL-1 on cell viability, and compared with these three sites, the mutation of the D238 site was slightly less effective. These results support that GL-1 is a DHPS inhibitor and provide a reference for the design of future DHPS inhibitors.

Surprisingly, both GL-1 and GC-7 could promote Cu²⁺ accumulation in melanoma cells, and this finding inspires the subsequent study of the regulatory relationship between DHPS and copper metabolism. Meanwhile, the results of the cellular multifactor assays showed that both GL-1 and GC-7 inhibited the expression of ANG, PAI-1, TSP-1, PDGF, VEGF, FGF2, IGFBP-3, CSF2, CD71, CXCL1, and MMP9; This also suggests that the inhibition of DHPS activity could effectively inhibit the angiogenesis process of melanoma, which provides a new reference for the indication of DHPS inhibitors. The excellent bioavailability of GL-1, low toxicity, and impressive TGI make GL-1 a promising candidate for inhibitor development.

Our study has some limitations. Mechanistically, the regulation of METTL3 by eIF5A-Hyp was significantly different from that of YTHDF2, suggesting that eIF5A-Hyp may have a unique translational regulation mechanism independent of epitope modification. Meanwhile, the effect of DHPS inhibitors on Cu²⁺ homeostasis in melanoma cells was found in the study of the anticancer mechanism of GL-1, and DHPS may be involved in the metabolic regulation of melanoma cells. These unanticipated findings provide new directions for subsequent studies of the DHPS pro-carcinogenic mechanism.

In conclusion, this study has innovatively identified DHPS-mediated m6A methylation modification in melanoma and obtained a novel DHPS allosteric inhibitor, GL-1, which had high efficiency, low toxicity, and good bioavailability in vivo and in vitro. These results provide a new reference for the pathogenesis of melanoma and the clinical treatment of melanoma.

Experimental Section

Bioinformatics Analysis

Cancer Genome Atlas (TCGA) was used to analyze tumor/normal differential expression.^[32]

Tissue Microarray

The expression levels of DHPS and Hypusine in melanoma samples were determined using IHC analysis. Tissue microarrays consisting of 111 melanoma samples and 18 adjacent normal tissues were used. Further information on this can be found in the Section S3.1 (Supporting Information).

Cell Lines and Transfection

A375, SK-MEL-28, B16-F10, and HACAT. The following cell lines were used in this study: All cell lines underwent STR identification and were examined for mycoplasma contamination. The DHPS shRNA knockdown plasmid was constructed using the target sequence “CCACATACTTGGGCGAGTTTA.” The plasmid was then transformed into receptor cells, and single clones were selected and shaken overnight. The DHPS knockdown plasmid was extracted using the Plasmid Extraction Kit (Tiangen, China) and identified through enzymatic sequencing. Following the lentiviral packaging kit instructions, the target or control plasmid and virus were co-transfected into 293T cells. After 48 h, the virus solution was concentrated, and quality testing and titer determination were performed. To transfect cells, they were spread into 24-well plates and the appropriate amount of virus was added based on the previously measured titer value. Infection occurred within 24 h after A375 and SK-MEL-28 cell attachment, and fluorescence was observed 72 h later. Infection efficiency was determined using qPCR and Western blot. Details regarding the cell line supplier and culture conditions can be found in Section S5.2 (Supporting Information).

mRNA-Seq and MeRIP-Seq

The mRNA was separated from A375 cells, and the samples were sequenced and analyzed by mRNA. Purified RNA was quantified, and the quality of the mRNA was assessed. The m6A Immunoprecipitation (MeRIP) procedure was used according to the instructions issued by the manufacturer. Detailed information can be found in the Section S3.3 (Supporting Information).

RNA Immunoprecipitation (RIP) and RNA Stability Assays

The Magna RIP RNA-binding protein immunoprecipitation kit (# 17–700, Millipore, MA) was used for RIP detection, following the manufacturer's instructions. The experimental procedures are detailed in Section S3.4 (Supporting Information). The primer sequence for METTL3 mRNA is as follows: forward primer 5′-TGGAAGGGTGTTTTGGAGGA-3′; reverse primer 5′-GGTCAACTCCCTGTCCTGAA-3′.

A375 and SK-MEL-28 cells were transfected with shDHPS and MT-siMETTL3, respectively, while untreated controls were set up. The above cells were treated with a final concentration of 10 µg mL⁻¹ of actinomycin D (ActD, CAS#:50-76-0, Bioss), and intracellular total RNA was extracted and subjected to qRT-PCR at 0, 1, 2, 4, and 6 h time points according to the RNA extraction kit instructions (CAS#64-17-5, Takara). The half-life of Ki67 and METTL3 were analyzed using the one-phase decay model assay in GraphPad Prism 6 (GraphPad InC, USA).

Colony Formation Assay

A375, SK-MEL-28, and B16-F10 cells were inoculated in the six-well plate at a density of 400 cells per well. 24 h later, compounds GL-1(0, 0.5, 1.5, 2.0 µm) and GC-7 (2.0 µm) with different concentrations were added for 10 days. The cell colonies were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet for 15 min. After washing with PBS 3 times, the number of cell colonies was detected.

Western Blot and Co-Immunoprecipitation (Co-IP) Assays

Protein was extracted from A375, SK-MEL-28, or B16-F10 cells after treatment with different concentrations of compounds GL-1 or GC-7 for 24 h. For Western blot, we used 10% or 15% polyacrylamide electrophoresis gel to separate and transfer to polyvinyl fluoride (PVDF) membrane for routine experiments. Co-IP assays (#635721, Takara) were operated by the vendor's guidelines. The primary antibodies used are detailed in Table S18 (Supporting Information).

General Methods

The chemicals and solvents needed for chemical synthesis were used directly. The methods for synthesis and data confirming the structure can be found in “Sections S1–S3” (Supporting Information). The instruments used for thin-layer chromatography, HRMS data collection, and melting point measurement were used according to the literature with minor modifications.^[13]

Deoxyhypusine Synthase (DHPS) Assays

The NAD/NADH-Glo assay (Promega, USA) was used in this study, as per the manufacturer's instructions. Detailed steps were conducted according to the literature with minor modifications.^[13] The concentrations of compounds were 1, 5, 10, 50, 100, 200, 400, 800, 1600, 2000 nm.

Surface Plasmon Resonance (SPR)

The experiment was conducted at 25 °C using a CM5 sensor chip on the BIAcore T200, and data collection and analysis were completed using the BIAcore T200 evaluation software (GE Healthcare) according to the manufacturer's instructions. It was exported to Origin 7 software (v.7.0552, Origin Lab) to generate the final data. Detailed steps are provided in the Section S3.5 (Supporting Information).

Cell Counting Kit-8 (CCK-8) Assays

Cell counting kit 8 (CCK-8, GlpBio, USA) was used to evaluate the growth inhibition effects of target compounds and GC-7 on A375, SK-mel-28, HACAT, and B16-F10 cells, and to establish a blank and control group, the concentrations of the compounds were 0.001, 0.01, 0.02, 0.035, 0.0625, 0.125, 0.25, 0.50, 1.00, 2.00, 4.00, 8.00 µm. The absorbance of the cells was measured at 450 nm by enzyme-labeled apparatus. The number of living cells was proportional to absorbance.

Cellular Thermal Shift Assay (CETSA) and Drug Affinity Responsive Target Stability (DARTS) Assay

For the CETSA assay, the cell lysate was divided into two equal parts. One part was used as a control, while the other was incubated with GL-1 (0.2 µm) at room temperature for 1 h. In the DARTS assay, 3 µL of GL-1 was added to a mixture of 297 µL of cell lysate supernatant and TNC solution. After incubation for 1 h, the mixture was divided into 6 centrifuge tubes with 50 µL each. Different proportions of pronase (#537088, Millipore, USA) solutions were added according to BCA results. The source of reagents and the experimental procedure for CETSA and DARTS were conducted according to the literature with minor modifications.^[47]

Docking

Molecular docking of DHPS with the inhibitor GL-1 was studied using MOE (Molecular Operating Environment, version 2016.08, Chemical Computing Group Inc., Canada). The docking site was set to “ligand” (GL-1), and the parameters were left at their default state. After optimization and Protonate 3D, the docking structure was minimized for energy in the Amber10: EHT force field.

Analysis of Apoptosis

The Annexin V-FITC/PI Apoptosis detection kit was purchased from Vzyme BioTech Co. (#N401-01, Vzyme). A375, SK-MEL-28, and B16-F10 cells were stained with Annexin V-FITC and PI for 15 min. The stained cells' spontaneous apoptosis was detected and analyzed using LSRFortessa flow cytometry (BD Biosciences, USA). The experiment was repeated three times.

Cell Copper (Cu) Colorimetric Assay Kit

The Cell Copper (Cu) Colorimetric Assay Kit (Complexing Method) (#E-BC-K318-M, Elabscience) was utilized to quantify the amount of Cu²⁺ present in the cells. A total of 2 × 106 cells were added to 200 uL of lysate. After sample processing, 100 uL of cell homogenate was taken for detection following the reagent's operation table.

Human High-Throughput Antibody Microarray Array

Rinse the cells with PBS and be sure to remove all PBS completely before adding the lysis buffer. A375 cells were dissolved with lysis buffer at a concentration of 1 × 107 cells mL⁻¹. After resuspension, gently shake the lysate at 2–8 °C for 30 min. Microcentrifuge 14 000 × g for 5 min and transfer the supernatant to a clean test tube. It was recommended to use a total protein assay to determine the protein concentration of samples. Perform experiments following the instructions provided by the reagent manufacturer (Catalog #: ARY007, Catalog #: ARY0022B, R&D Systems Hong Kong Limited). For detailed procedures, refer to Section S3.6 (Supporting Information).

Tumor Xenograft Model

BALB/c nude mice (4–5 weeks of age) were provided by HFK BIOSCIENCE CO., LTD (Beijing, China) in a pathogen-free environment. 1 × 107 A375 cells were subcutaneously implanted into nude mice with 100 µL PBS. Detailed experimental procedures are in the Section S3.7 (Supporting Information). The calculation formula for tumor volume is as follows:length×width2/2, (Liaoning Province, China).Tumor growth inhibition (TGI) = [1- relative tumor volume (GL-group)/relative tumor volume (control group)]*100%.

Pharmacokinetic Studies

The pharmacokinetics of GL-1 were studied through both intravenous and intraperitoneal injection. The procedure and dosage details are provided in Section S3.8 (Supporting Information).

Immunochemistry (IHC)

Melanoma tissue samples were fixed with formaldehyde, embedded in paraffin wax, sectioned, and immunostained with antibodies against target proteins. The primary antibodies were diluted at 1:2000, and their types were specified in Table S19 (Supporting Information). For IHC staining, streptavidin-peroxidase complexes were used, and images were obtained with a light microscope.

Statistical Analysis

Statistical analysis was conducted using GraphPad Prism 6 (GraphPad Inc, USA) following the method described in the literature with minor modifications.^[13]

Ethical Statement

The human tissue microarrays were purchased from Shanghai Outdo Biotech Company (Shanghai, China) and were reviewed by the Ethics Committee of the Shanghai Outdo Biotech Company Biobank (Ethical Number: SHYJS-CP-1910015). The animal experiment scheme was reviewed and approved by the Committee on the Use of Live Animals in Teaching and Research of Shengjing Hospital of China Medical University (Shenyang, Liaoning, China, Ethical Number: 2023PS1081K).

Acknowledgements

J.G., J.M., and X.Z. contributed equally to the work. This work was financial support for this work from the National Natural Science Foundation of China (No. 82104449), and the Science and Technology Program-Joint Program (Fund) of Liaoning Province (No. 2023JH21/101700160). This work was also funded by the China Postdoctoral Science Foundation (2021M703607). The authors thank the China Medical University High-Level Talent Program for its financial support.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

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