Melasolv™ was synthesized by AMOREPACIFIC, as previously described (Kang et al., 2003).

Human epidermal melanocytes derived from the neonatal foreskin of moderately pigmented donors (African-American males) were purchased from Cascade Biologics (Portland, OR, United States) and cultured in Medium 254 (Thermo Fisher Scientific, Waltham, MA, United States) supplemented with a human melanocyte growth supplement (Thermo Fisher Scientific) under humidified 5% CO₂ atmosphere. Cells from passages 2 to 5 were used in this study. For transcriptional profiling, melanocytes were treated with 10 μg/mL Melasolv™ for 1 day.

Senescence in melanocytes was induced as described in our previous study (Choi et al., 2018). In brief, melanocytes with the indicated concentration in Figure were exposed to sub-cytotoxic doses (20 mJ/cm²) of UVB twice, with a 24-h interval in between. Melasolv™ treatment was administered at the time of the first UVB exposure. After 2 weeks of culture, SA-β-galactosidase activity and melanin contents were analyzed.

Total RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA from the total RNA was fragmented, and the Illumina TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA, United States) was used for library preparation. RNA-seq was conducted by Macrogen (Seoul, Korea) using the NovaSeq 6,000 platform (Illumina) (GEO ID: GSE229700). Triommomatic version 0.38 (Bolger et al., 2014) was used for trimming and quality control, and HISAT2 version 2.1.0 (Kim et al., 2015) and Bowtie2 version 2.3.4.1 (Langmead and Salzberg, 2012) were used to map the reads to the human genome reference UCSC hg19 database. Raw read counts were quantified using STRING Tie version 1.3.4d (Pertea et al., 2015). Differentially expressed genes (DEGs) were then identified using the DEseq2 R package (Love et al., 2014). DEGs were defined using the following criteria: |log₂FoldChange (log₂FC)| ≥ 1 and adjusted p < 0.05.

Volcano plots for the expression levels of Melasolv™-treated melanocytes and melanocyte inducing transcription factor (MITF) targets were generated using the ggplot2 R package, and heatmaps were visualized using the pheatmap package in R. MITF targets of Melasolv™ were extracted using the DoRothEA R package (Garcia-Alonso et al., 2019). Targets with high confidence levels (A, B, and C) were selected.

Gene Ontology (GO) analysis of DEGs was performed using Metascape version 3.5 (Zhou et al., 2019). Representative terms were selected from the top GO terms or pathways (p < 0.01). The Gseapy Python package (Fang et al., 2023) was used for the Gene Set Enrichment Analysis (GSEA) using the libraries Reactome_2022 and MSigDB_hallmarks_2020. We ranked Melasolv™-induced gene expression profiles based on the log2FC value.

The CMap library, a collection of gene expression profiles of drug-induced human cancer cells, has been widely used in drug repurposing studies (https://clue.io/data/CMap2020#LINCS2020) (Lamb et al., 2006). In this study, the PharmacoGx R package (Smirnov et al., 2016) was used to determine connectivity scores. The CMap dataset provides gene expression profiles at different normalization levels ranging from 1 to 5. Level 5 data that represent transcriptomic signatures (i.e., differential gene expression in response to perturbation, e.g., chemical compounds, gene knockdown, gene knockout, and overexpression) were used. Only the signatures with a perturbation time exceeding 24 h were used as reference profiles. The input data for connectivity mapping analysis comprised upregulated and downregulated DEGs induced by Melasolv™ treatment. DEGs were compared with reference gene signatures using a pattern-matching algorithm based on the non-parametric rank-ordered Kolmogorov-Smirnov statistic. Compounds with significant positive connectivity scores may have an MOA similar to that of Melasolv™, and therefore, these compounds were used as reference compounds to explore possible novel biological functions of this drug.

Chilibot (Chen and Sharp, 2004) was used to estimate the relationship between the target genes of compounds with top connectivity scores and aging/senescence. Chilibot enables the retrieval of biological relationships between genes and biological processes using natural language processing integrated with biomedical knowledge. The results consisted of weights for interactive relationships, which reflect the number of abstracts obtained from PubMed; relationships with weights higher than 15 were considered. The selected interactive relationships between target genes and aging/senescence were visualized using Cytoscape (v.3.8.2, https://cytoscape.org/).

The extracted total RNA was reverse-transcribed to generate cDNAs using a Superscript Reverse Transcriptase II kit (Thermo Fisher Scientific). RT-qPCR was performed using the TaqMan Universal Master PCR mix and TaqMan Gene Expression Assays (Thermo Fisher Scientific) using an ABI7500 FAST real-time PCR system (Applied Biosystems, Thermo Fisher Scientific). The measurement of the relative expression of mRNAs was carried out using probes detailed in Supplementary Table S1. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 4333764F; Applied Biosystems) was also amplified and used to normalize variations in cDNA quantities.

The mammalian β-gal assay kit was purchased from Thermo Fisher Scientific. Two weeks after treatment with Melasolv™, proteins from melanocytes were extracted using a mammalian protein extraction reagent (M-PER; Thermo Fisher Scientific). After reaction with β-gal assay reagent, the absorbance at 405 nm was measured using Synergy H2 microplate reader (BioTek, Winooski, VT, United States). SA-β-gal staining was performed using a β-gal staining kit (K145501; Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. Images of five to ten random fields were obtained using an optical light microscope (Nikon Eclipse TS100, Tokyo, Japan) at a magnification of 200x.

Cells were counted and pellets w containing melanin were dissolved in 1 N NaOH and incubated for 30 min at 60 °C. The melanin contents were determined by measuring the absorbance at 450 nm using a Synergy H2 microplate reader (BioTek) and compared with that of a standard curve of synthetic melanin (Sgima).

Melanocytes were exposed to 20 mJ/cm² of UVB twice with a 24-h interval, and Melasolv™ treatment was administered at the time of first UVB exposure. Genomic DNAs were extracted from melanocytes using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, United States), according to the manufacturer’s instructions. To quantitatively measure the damage of DNA, the formation of 8-hydroxydeoxyguanosine, one of the oxidative DNA damage byproducts, was detected using OxiSelect™ Oxidative DNA Damage ELISA kit (Cell Biolabs Inc., San Diego, CA, United States) according to the manufacturer’s instructions.

All statistical data are presented as the mean ± SD from three independent experiments. A two-tailed Student’s t-test was used to analyze differences between the two groups. *p < 0.05, **p < 0.01.

We performed differential gene expression analysis to identify genes that were differentially expressed following Melasolv™ treatment in human melanocytes. A total of 255 DEGs were identified (|log2FC| > 1 and adjusted p-value <0.05), including 168 upregulated and 87 DEGs in Melasolv™-treated melanocytes compared to the control (Figures 1A,B and Supplementary Table S2). We validated the mRNA expression of selected DEGs using RT-qPCR (Supplementary Figure S1) and confirmed that the RNA-seq data is consistent. In our analysis, we first examined the changes in the expression of genes related to the depigmentation function of Melasolv™. Notably, the mRNA expression of dopachrome tautomerase (DCT), an enzyme that initiates melanogenesis by catalyzing tyrosine conversion, was significantly reduced (Figures 1D,E). Moreover, analysis of the collection of target genes of transcription factors obtained using DoRothEA tools (Garcia-Alonso et al., 2019) revealed that the target genes of MITF are significantly downregulated following treatment with Melasolv™ (Figure 1C and Supplementary Table S3).

To gain further insight into the biological functions of the identified DEGs, we performed enrichment analyses (Figures 1D,E). GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the genes upregulated following Melasolv™ treatment were related to cell differentiation, wound-healing responses, and extracellular matrix organization. Moreover, the downregulated genes in Melasolv™-treated cells were significantly enriched in processes such as cholesterol biosynthesis. These analyses indicate that Melasolv™ induces significant changes in genes associated with various biological processes. This result suggests that the potential biological effects of Melasolv™ may extend beyond its well-known anti-melanogenesis function.

The connectivity mapping approach is widely used to uncover unknown functions of drugs (Musa et al., 2018). The CMap database provides large-scale drug perturbation data, including transcriptomic profiles of reference signatures of drugs and their known MOAs and target genes. To infer potential MOAs associated with Melasolv™, we performed a connectivity mapping analysis. We used DEGs induced by Melasolv™ as input queries for the CMap database. Each reference signature was ranked according to its connectivity score, with the top-ranked signatures showing the strongest correlation with the Melasolv™ signature. The analysis revealed several compounds with high connectivity scores and potential MOAs, including PIM kinase inhibitors, TRPV agonists, retinoid receptor agonists, mTOR inhibitors, HDAC inhibitors, RAF inhibitors, and BCL inhibitors (Table 1). These compounds have previously been shown to have therapeutic effects in various disease conditions and may provide insights into the potential MOA of Melasolv™.

Among the top compounds identified by connectivity mapping analysis, CD-1530 (Thacher et al., 2000), sirolimus (Wang et al., 2017; Chung et al., 2019; Blagosklonny, 2022), and vorinostat (McIntyre et al., 2019) exhibit well-known anti-aging functions. Visualization of the query gene mapped onto reference signatures showed that many upregulated DEGs following Melasolv™ treatment were mapped near the top of the ranked reference signature list, whereas downregulated DEGs appeared at the bottom of the reference signature list (Figure 2A and Supplementary Table S4). We validated the mRNA expression of selected genes (ACAT2, MVD, and HMGCR) that significantly contributed to the high connectivity with anti-aging-associated compounds using RT-qPCR (Supplementary Figure S2). In addition, the results of GSEA showed that the senescence-associated secretory phenotype (SASP) and mTOR signaling pathways were significantly enriched among the downregulated genes (Figure 2B). These pathways play important roles in skin aging and senescence (Sharma and Padwad, 2019). Notably, in the enrichment analysis, we observed that genes upregulated by the treatment with Melasolv™ were significantly associated with processes of cellular differentiation (Guo et al., 2022) and wound-healing ability (Khalid et al., 2022). These processes are well-documented to diminish with aging. Conversely, cholesterol biosynthesis was enriched among downregulated genes, and previous research has indicated that cholesterol can induce melanogenesis and the release of cyclic adenosine monophosphate (cAMP) in epidermal melanocytes (Schallreuter et al., 2009). Furthermore, activation of the cAMP pathway is associated with the expression of p27 and p16 and the loss of E2F activity, which are markers of cellular senescence in human melanocytes (Haddad et al., 1999). These findings imply that Melasolv™ may could potentially inhibit the biological processes associated with aging. To further validate the link between the connectivity mapping results and aging and senescence, we used a text-mining approach using Chilibot (Chen and Sharp, 2004). From the PubMed database, we obtained the frequency of studies that related to each of the top-ten target genes involved in aging/senescence. These frequencies are depicted as dot sizes in the network shown in Figure 2C. Of these 30 genes, 22 genes were associated with aging/senescence. Taken together, the results of connectivity mapping, GSEA, and literature mining analyses suggest that Melasolv™ treatment may prevent the aging/senescence of melanocytes.

We subsequently examined the preventive effect of Melasolv™ on the senescence of melanocytes in vitro. We used a previously established melanocyte senescence model in which human epidermal melanocytes were exposed twice to 20 mJ/cm² UVB over a 24-h interval (Figure 3A) (Choi et al., 2018). At the time of first UVB exposure, cells were treated with Melasolv™. As previously shown, chronic UVB exposure induced a senescent phenotype, which included an increased number of flattened and enlarged cells and high production of pH-dependent SA-β-Gal after 2 weeks of cultivation. In contrast, treatment with Melasolv™ significantly blocked the senescent phenotypes (Figures 3B,C). This preventive effect was further assessed by analyzing the mRNA expression of the cell cycle regulator genes CDKN2A and CDKN1A, encoding cyclin-dependent kinase inhibitors, and two selected SASP-related genes, IL6 and CCL8. As shown in Figure 3D, treatment with Melasolv™ significantly reduced the upregulated expression of these genes. In addition, treatment with Melasolv™ effectively protected melanocytes against UVB-induced DNA damage. These results indicate that Melasolv™ can broadly block the damaging effects of UV, subsequently preventing the senescence process.

Melanin content is increased in senescent melanocytes (Choi et al., 2018; Park et al., 2023); therefore, we assessed changes in the melanin content in response to treatment with Melasolv™. Treatment with Melasolv™ mitigated the increased melanin content in senescent melanocytes (Figure 3E). Our results indicate that Melasolv™ not only exerts a significant depigmenting effect but also exhibits a potent efficacy in preventing senescence, suggesting its potential as an effective treatment for addressing age-related skin pigmentation concerns.