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. 2025 Jun 4;105(6):e70136. doi: 10.1111/cbdd.70136

Identification of a Selective Cell‐Active Inhibitor of Protein Arginine Methyltransferase 5 (PRMT5) for the Treatment of Prostate Cancer by Structure‐Based Virtual Screening

TongXiang Diao 1,2, Chen Feng 1,3, Shuai Liu 1,2, Jia‐Li Song 4, Kong‐kai Zhu 4,✉, Cheng‐Shi Jiang 4,✉, Qiang Fu 1,2,✉
PMCID: PMC12138182  PMID: 40468526

ABSTRACT

Protein arginine methyltransferase 5 (PRMT5) is an epigenetic‐related enzyme that has been shown to be a promising target for the treatment of human cancers. In prostate cancer, gene knockout has been shown to inhibit cancer cells by regulating the androgen receptor (AR), but this method has no effect on advanced prostate cancer without AR expression, and existing anticancer drugs are effective only in the current stage and promote the progression of cancer to advanced prostate cancer. We hope to design and synthesize a new compound that can inhibit prostate cancer at different stages. A series of candidate PRMT5 inhibitor molecules were designed on the basis of virtual molecular docking screening, and the binding mode was predicted via molecular docking simulation. Prostate cancer cell proliferation was detected by CCK‐8, EdU, and flow assays, which verified the changes in the cancer cell cycle. Migration and invasion assays verified the effects of the compounds on the metastatic function of prostate cancer cells. Finally, Western blotting was used to detect the mechanism of action of the compounds in the treatment of prostate cancer. In prostate cancer, gene knockout has been shown to inhibit cancer cells by regulating the AR, but it has no effect on advanced prostate cancer without AR expression, and existing anticancer drugs are effective only in the current stage and promote the progression of cancer to advanced prostate cancer. SJL2‐1 may be a promising compound for novel therapies for early androgen‐sensitive prostate cancer and advanced castration‐resistant prostate cancer (CRPC).

Keywords: arginine methylation, molecular docking, PRMT5 inhibitor, prostate cancer, virtual screening

Combining previous research, compound SJL2‐1 was identified through virtual screening based on molecular docking. By reducing the expression levels of PRMT5 and AR, it exhibits anti‐proliferative effects, inhibits cell migration and invasion abilities, blocks the cell cycle, and induces apoptosis in AR‐sensitive prostate cancer and CRPC.

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1. Introduction

Prostate cancer is one of the most common nonskin cancers among men worldwide (Sung et al. 2021). Although many molecules and signaling pathways are closely associated with prostate cancer development and progression, androgen receptor (AR) signaling remains the most important factor driving prostate cancer development and progression (Lamb et al. 2014; Thoma 2017). In addition to prostatectomy or radiation therapy, androgen deprivation therapy (ADT) is the first‐line standard of care for patients with locally advanced and metastatic high‐risk prostate cancer; however, although ADT is initially effective, most patients develop resistance and eventually castration‐resistant prostate cancer (CRPC) (Howard et al. 2019; Yap et al. 2016). Treatment options for CRPC include abiraterone, enzalutamide, and either ADT in combination with docetaxel or ADT in combination with abiraterone, which has a greater survival benefit for patients with this type of prostate cancer (James et al. 2016). AR proteins continue to be expressed in most CRPC patients, and AR signaling still plays an important role in tumors. Therefore, effectively slowing different progressive prostate cancers by targeting AR signaling has great therapeutic benefit for patients.

Protein arginine methyltransferase 5 (PRMT5) belongs to the protein arginine methyltransferase (PRMT) family. PRMT5 catalyzes the symmetric arginine dimethylation of its substrate protein, including histone and nonhistone proteins (Stopa et al. 2015). PRMT5 has recently been found to be upregulated in many human malignancies, such as lung cancer (Wei et al. 2012), colorectal cancer (Zheng et al. 2013), lymphomas (Chung et al. 2013; Wang et al. 2008; Pal et al. 2007), and breast cancer (Powers et al. 2011). In addition, potent antiproliferative effects were reported when PRMT5 was knocked down in mantle cell lymphoma (MCL) cell lines (Chung et al. 2013; Wang et al. 2008; Pal et al. 2007). PRMT5 is believed to play many important roles in cell cycle progression, cell proliferation, cell growth, and death (Karkhanis et al. 2011). Although the mechanism of tumorigenesis by PRMT5 is still unknown, it has been regarded as the most promising anticancer target in the epigenetic field. Until now, dozens of PRMT5 inhibitors have been reported and mainly could be classified into three mechanistic categories: S‐adenosylmethionine (SAM)‐competitive inhibitors (e.g., LLY‐283, PF‐06939999, and JNJ64619178) and substrate‐competitive inhibitors (e.g., EPZ015666 and GSK332695) (Figure S1). However, these nucleoside‐based inhibitors, which mimic the SAM cofactor, exhibit limited therapeutic potential due to poor membrane permeability. While non‐nucleoside inhibitors such as EPZ015666 and the clinical‐stage compound GSK3326595 demonstrate enhanced pharmacokinetic profiles compared to their nucleoside counterparts, GSK3326595 has shown significant safety concerns in Phase I clinical trials, with hematological toxicity observed in 89% of patients and emerging evidence of dose‐limiting toxicities such as hepatic dysfunction. Consequently, advancing novel PRMT5 inhibitors with improved safety while maintaining therapeutic efficacy remains a critical unmet need in epigenetic drug discovery (Li et al. 2025).

Given that the progression of prostate cancer is variable and difficult to detect, failure to change drugs in a timely manner in response to disease progression often results in less than expected therapeutic effects on cancer (Mottet et al. 2021). However, increasing evidence indicates that long‐term use of conventional anti‐prostate cancer drugs such as bicalutamide and abiraterone may promote the conversion of hormone‐sensitive prostate cancer to CRPC with a poor prognosis (McCrea et al. 2016). Therefore, we desire a compound or small‐molecule drug that can inhibit both androgen‐sensitive prostate cancer and castration‐resistant prostate cancer. The role of epigenetics in prostate cancer has received increasing attention (Goel et al. 2022; Kumaraswamy et al. 2021), and PRMT5, an epigenetically related enzyme, has been shown to be associated with AR expression and activity (Deng et al. 2017; Beketova et al. 2020). In this study, we designed and screened a new small molecule inhibitor of PRMT5 to explore its effects on prostate cancer cell activity and function.

2. Materials and Methods

2.1. Molecular Docking‐Based Virtual Screening

To perform virtual screening, a molecular docking model based on the crystal structure of the PRMT5:MEP50 complex with its inhibitor EPZ015666 (PDB code: 4X61) (Chan‐Penebre et al. 2015) was constructed. Our in‐house compound database was then screened using this model. First, the 3D structures of the compounds in this database were generated via the LigPrep panel (version 2.3, Schrödinger LLC, New York, NY). A docking grid (centered on EPZ015666) was subsequently produced. Finally, the prepared compound database was docked to the grid in XP mode. Molecular docking simulations were performed with the Glide program (Friesner et al. 2004). The docking score was calculated via the XP G‐score.

2.2. In Vitro Enzymatic Assay

The in vitro enzymology of PRMT5 was tested via radioactive methylation assays (Zhu et al. 2018). The following materials were purchased: PRMT5/MEP50 (Cat. No. 51045, BPS Bioscience), [3H]‐SAM (Lot. No. 2146246, PerkinElmer); SAM (Cat. No. A7007, Sigma); SAH (Cat. No. A9384‐25MG, Sigma); 384‐well plate (Cat. No. 6007299, Perkin Elmer); and 5× epigenetic buffer (Cat. No. AL008C, Perkin Elmer). IC50 values were derived by testing different concentrations of compounds (starting from 100 μM, 3‐fold dilution) and are shown as the means ± SDs of three parallel replicates. SAH was used as a control. The tested compounds were dissolved and diluted to 20 mM with DMSO for storage. The known PRMT5 inhibitor, EPZ015666, was used as a positive control in the enzyme inhibitory bioassay, and the IC50 value of EPZ015666 was determined to be 0.047 μM.

2.3. Chemistry

The NMR data of SJL2‐1 were measured on a Bruker AVANCE DRX600 spectrometer. ESI–MS analyses were performed on an Agilent 1260–6460 Triple Quadrupole LC–MS instrument (Agilent, Waldbronn, Germany), and the elemental analysis was recorded on a Vario EL III (Elementar, Langenselbold, Germany). 1H NMR (600 MHz, CDCl3) δ 7.58 (dd, J = 8.0, 2.7 Hz, 1H), 7.34 (dd, J = 12.5, 8.2 Hz, 1H), 7.24–7.19 (m, 1H), 7.17–7.11 (m, 3H), 7.09 (t, J = 6.6 Hz, 1H), 7.06–7.01 (m, 1H), 7.00 (s, 1H), 4.25–4.03 (m, 3H), 3.84 (dq, J = 49.8, 6.9 Hz, 1H), 3.17–3.04 (m, 1H), 2.98–2.83 (m, 1H), 2.79 (ddd, J = 12.5, 6.1, 1.1 Hz, 1H), 2.71 (ddd, J = 12.5, 4.9, 3.7 Hz, 1H), 2.65 (dt, J = 10.5, 4.9 Hz, 1H), 2.45 (ddd, J = 58.9, 12.5, 10.1 Hz, 1H), 2.35 (s, 3H), 1.33 (dd, J = 15.0, 6.7 Hz, 3H). ESI–MS m/z: 335 [M + H]+. Anal. Calcd. for C22H26N2O (334.4630): C, 79.00; H, 7.84; N, 8.38. Found: C, 79.15; H, 7.79; N, 8.31.

2.4. Western Blot Analysis

Treated prostate cancer cells were washed three times with cold PBS, total protein was extracted via lysis buffer and phosphatase inhibitor, and protein concentrations were detected via a bicinchoninic acid (BCA) assay. For Western blot analysis, 20 μg of total cell lysate was loaded onto 10% or 15% SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Subsequently, the PVDF membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. The membranes were subsequently incubated with horseradish peroxidase‐conjugated secondary antibodies for 1 h at room temperature. After the samples were washed with TBST, they were observed with an LAS3000 image analyzer (Fujifilm, Tokyo, Japan), and the band density was quantified via ImageJ 2x software. The primary antibodies used for immunoblotting were as follows: rabbit anti‐PRMT5 antibody (1:1000) (Abcam # ab‐109,451), rabbit anti‐androgen receptor antibody (1:1000) (Abcam # ab‐108,341), mouse monoclonal antibody against GAPDH (1: 1000) (Abcam # ab‐8245), rabbit anti‐SDMA Somatic Antibody (1:1000) (CST, 13222 s), Bcl‐2 (1:1000) (Cell Signaling Technology), Bax (1:1000) (Abcam), caspase 3 (1:1000) (Cell Signaling Technology), and pro‐caspase 3 (1:1000) (Cell Signaling Technology).

2.5. Cell Viability Assay

The compounds were dissolved with dimethyl sulfoxide (DMSO) to 20 mmol/L, and the solutions were diluted with F‐12 K and 1640 culture plus 10% fetal bovine serum, respectively, to obtain the final concentrations. PC3, 22RV1, and LNCAP cells were inoculated into 96‐well plates at densities of 3 × 104 mL−1, 7 × 104 mL−1, and 5 × 104 mL−1, respectively. The cells were incubated at 37°C and 5% CO2 for 24 h. The cells were treated with different concentrations of the PRMT5 inhibitor before incubation for 24, 48, 72, and 96 h. Subsequently, 100 μL of a mixture of CCK‐8 reaction reagent (Solarbio, CA1210, Beijing, China) and serum‐free medium was added to each well at a ratio of 1:10, and the cells were incubated for 4 h. Finally, a microplate reader (Bio‐Rad, Hercules, USA) was used to measure the absorbance at 450 nm for each condition.

2.6. 5‐Ethynyl‐2′‐Deoxyuridine (EdU) Incorporation Assay

To further assess the effects of the compounds on the proliferation of prostate cancer cells, PC3, 22RV1, and LNCAP cells were seeded in 96‐well plates and cultured for 1 day at 37°C with 5% CO2 in complete medium. The next day, the cells were treated with half the IC50 concentration of the compound. Twenty‐four hours after treatment, cell proliferation was measured via an EdU Cell Proliferation Assay Kit (KaiJi, Nanjing, China), which measures the binding rate of EdU according to the manufacturer's instructions. Nuclei were stained with Hoechst 33342 (KaiJi, Nanjing, China), diluted with PBS at a ratio of 1:1000, and incubated for 10 min at room temperature in the dark. The proportion of cells incorporated into the EdU solution was detected with an inverted fluorescence microscope (Multiskan FC, Thermo Scientific).

2.7. Cell Migration and Invasion Assays

To verify the effects of the compounds on the migration and invasive ability of prostate cancer cells, a Transwell assay was used. To treat PC‐3, 22RV1, and LNCaP cells, the compounds were added to medium supplemented with 10% fetal bovine serum to form cultures with concentrations of half the IC50, while the control cells were treated with medium supplemented with the same amount of DMSO. We collected the cells 48 h after treatment and resuspended them in serum‐free medium. The invasion assay was performed by prefilling the upper chamber of a 24‐well Transwell device with 80 μL of a 1:8 mixture of matrix gel and serum‐free solution and stabilizing it at 37°C and 5% CO2 for 1 h. For the migration assay, the upper chamber did not contain matrix gel. In the serum‐free medium, 180 μL of cells were seeded into the upper chamber at a concentration of 1.5 × 105, and 600 μL of medium containing 20% FBS was added to the lower chamber. After incubation in a CO2 incubator at 37°C for 48 h, the cells in the upper chamber were wiped off with a cotton swab, fixed with 4% paraformaldehyde for 30 min, and stained with 1% crystal violet for 30 min. Migrating and invading cells were counted under an ortho‐fluorescence microscope in five randomly selected microscopic fields (100×) on each membrane.

2.8. Cell Cycle Analysis

PC3, 22RV1, and LNCAP cells were treated with different concentrations of compounds (0, 15, 30, and 45 μM) for approximately 48 h. Then, the cells were digested and collected, fixed overnight by cooling with 70% cold ethanol at 4°C, washed three times with cold PBS before the prostate cancer cells were incubated with RNase A at 37°C for 30 min, and then incubated with propidium iodide (50 μg/mL) (Sigma–Aldrich, St. Louis, MO, USA) for 30 min in the dark. The stained cells were detected by a flow cytometry system (BD FACSCalibur) to determine the phase of the cycle they were in.

2.9. Statistical Analysis

SPSS 25 software and GraphPad Prism 6.0 software were used for statistical analysis; all the experiments were repeated at least three times, and all the data are expressed as the means ± standard deviations. Student's t test and ANOVA were used to calculate p values. p values were two‐sided and considered statistically significant when p < 0.05.

3. Results

3.1. Molecular Docking‐Based Virtual Screening Led to the Discovery of the Hit Compound SJL2‐1

We identified several PRMT5 inhibitors through a virtual screening platform [13–16]. In the present study, an in‐house database (containing 1000 compounds) was screened for PRMT5 inhibitors. The virtual screening workflow is shown in Figure 1A. A total of 50 candidates were ultimately selected for further PRMT5 enzymatic activity tests. The results of the enzymatic PRMT5 inhibitory activity test (Figure 1B) revealed that SJL2‐1 had the most potent inhibitory activity, with an IC50 of 1.56 μM (Figure 1C).

FIGURE 1.

FIGURE 1

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Virtual screening workflow (A), chemical structure (B), and IC50 curves (C) of SJL2‐1.

The binding mode of SJL2‐1 with PRMT5 is shown in Figure 2 to present the detailed interactions between SJL2‐1 and PRMT5. These results indicate that SJ2‐1 is located in the substrate binding pocket of PRMT5 and forms potent hydrophobic interactions with residues Q309, L312, L319, Y324, F327, K333, V503, S578, W579, and F580. In addition, SJL2‐1 established H‐bond interactions with residues E444 and W579. The XP G‐score of SJ2‐1 was −11.215, which indicated a strong binding affinity between SJL2‐1 and PRMT5.

FIGURE 2.

FIGURE 2

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Predicted binding mode of SJL2‐1 with PRMT5. (A) The hydrophobic interactions (shown as starbursts) and H‐bond interactions (depicted by dotted green lines) are displayed in a schematic representation. (B) Close‐up view of SJL2‐1 binding to PRMT5. A cartoon representation of PRMT5 is shown, whereas SJL2‐1 and the interaction residues are depicted as stick representations.

3.2. Compound SJL2‐1 Inhibited the Proliferation, Migration, and Invasion of PCa Cells

To investigate the functional role of SJL2‐1 in PCa cells, we performed CCK‐8 cell proliferation assays by treating 22RV1, PC‐3, and LNCaP cell lines with different concentrations of SJL2‐1, as shown in Figure 3A. The IC50 values at 48 h were 40.07, 37.29, and 4.29 μM, respectively. Consistently, immunofluorescence staining assays for the EdU incorporation assay revealed that inhibition of cell proliferation and a reduction in active DNA replication were observed with SJL2‐1 treatment (Figure 3B,C). Functional validation through transwell migration and matrigel invasion assays demonstrated that the metastatic phenotype mediated by SJL2‐1 was attenuated, with a significant reduction in the number of migratory cells in PC‐3, 22RV1, and LNCaP cells (Figure 3D,E), and invasion assays further confirmed the inhibitory effect of SJL2‐1 on cell invasion ability (Figure 3F,G), suggesting the impact of SJL2‐1 on the overall motility of cancer cells. Notably, SJL2‐1 exhibited no detectable effects on viability, migratory potential, or invasive capacity in normal prostate epithelial cells (RWPE‐1) (Figure S2), reinforcing its tumor‐specific cytotoxic profile. Collectively, these findings demonstrate that SJL2‐1, functioning as a potent PRMT5 inhibitor, effectively impairs critical oncogenic traits in prostate cancer cells, including proliferation, migration, and invasion.

FIGURE 3.

FIGURE 3

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Compound SJL2‐1 suppresses proliferation and metastatic competence in prostate cancer cells. (A) CCK‐8 assay demonstrated SJL2‐1‐induced proliferation inhibition in PC3, 22RV1, and LNCaP cells in a concentration‐ and time‐dependent manner. (B) EdU incorporation assay validating anti‐proliferative effects of SJL2‐1. (C) Quantitative analysis showing reduction in EdU‐positive cells. (D) Transwell migration assay assessing impact of SJL2‐1 on prostate cancer cell motility. (E) Statistical quantification of migrated cells. (F) Matrigel invasion assay evaluating SJL2‐1‐mediated suppression of tumor cell invasive potential. (G) Bar graphs confirming significant attenuation of invaded cell counts. Two groups were compared via t tests; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3.3. SJL2‐1 Promotes Apoptosis and Blocks the Cell Cycle in the G0/G1 Phase

These results indicated that SJL2‐1 inhibited the proliferation and motility functions of 22RV1, PC‐3, and LNCAP cells. To further investigate whether apoptosis occurs in SJL2‐1‐treated cells, we examined the changes in Bax, Bcl2, caspase‐3, and pro‐caspase‐3 levels in the three cell lines via protein blotting. Following 48‐h treatment with escalating concentrations of SJL2‐1, we observed a concentration‐dependent upregulation of the pro‐apoptotic effector Bax and elevated levels of both pro‐caspase‐3 and its activated form (caspase‐3)—key executioners of apoptosis—alongside a concomitant reduction in anti‐apoptotic Bcl‐2 expression (Figure 4A,B).

FIGURE 4.

FIGURE 4

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Effects of different concentrations of SJL2‐1 on the apoptosis of three types of prostate cancer cells. (A) Western blot of Bax, Bcl‐2, Caspase‐3, and Pro‐Caspase‐3 protein levels. (B) Quantification of protein expression normalized to β‐Actin. Multiple groups were compared by one‐way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

In addition, to verify whether SJL2‐1 induces apoptosis while arresting the cancer cell cycle, we used flow cytometry to analyze the effect of SJL2‐1 on the cell cycle. Analysis of the cell cycle distribution revealed that treatment of cells with different concentrations (15, 30, and 45 μM) of SJL2‐1 for 48 h impeded the progression of 22RV1, PC3, and LNCAP cells to the G1 phase in a dose‐dependent manner, with a significant accumulation of cells in the G0/G1 phase and a concomitant decrease in the number of cells in the G2 phase (Figure 5A–C). Compared with the control treatment (39.1%, 53.08%, 53.59%), SJL2‐1 treatment resulted in 61.59%, 69.97%, and 71.59% arrest of 22RV1, PC‐3, and LNCAP cells at 45 μM, respectively. These data suggest that SJL2‐1 inhibits the proliferation of PC3, 22RV1, and LNCAP cells by inducing G0/G1 phase cell cycle arrest and promoting apoptosis.

FIGURE 5.

FIGURE 5

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Flow cytometry analysis revealed that SJL2‐1 inhibited the cell cycle. Separate treatments with different concentrations of SJL2‐1 were applied for 48 h. 22RV1 (A), PC3 (B), and LNCAP (C) stalled in the G0/G1 phase.

3.4. SJL2‐1 Has an Inhibitory Effect on Cytosolic Symmetric Dimethylation and Androgen Receptor Expression

To verify the efficacy of SJL2‐1 as a potent PRMT5 inhibitor, we examined the effects of SJL2‐1 on the expression levels of PRMT5 and symmetrical dimethylarginine dimethylation in 22RV1, PC‐3, and LNCAP cells by immunoblotting with a PRMT5 antibody and a symmetrical dimethylarginine (SDMA) antibody (Figure 6A,B). These results suggest that SJL2‐1 treatment resulted in a concentration‐dependent decrease in the intensity of the PRMT5 protein bands and multiple bands corresponding to symmetrical dimethylarginine, including the PRMT5 substrate SMD3 protein. As AR is a major factor that plays an important role in the growth of androgen‐dependent prostate cancer, a protein blotting assay revealed that SJL2‐1 significantly reduced the expression levels of ARV‐7 in 22RV1 cells and the androgen receptor in LNCAP cells, demonstrating that SJL2‐1 can target the binding of PRMT5 in cells and inhibit the methylation and expression of the androgen receptor.

FIGURE 6.

FIGURE 6

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SJL2‐1 affects PRMT5 and AR expression and arginine methylation. (A) SJL2‐1 significantly inhibited PRMT5 expression and downstream SDMA and SmD3me2S protein expression and decreased the expression level of the androgen receptor in AR‐positive PCa cells. (B) Quantitative analysis of the WB results. Three independent assays with three technical replicates were performed. Multiple groups were compared by one‐way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

4. Discussion

The treatment of prostate cancer is one of the challenging problems to be solved in urology (Bray et al. 2018). Currently, AR remains the main focus of prostate cancer treatment because of the important role of AR signaling in prostate cancer (Olson et al. 2017), and for hormone‐dependent prostate cancer, the first‐line treatment for PCa is mainly androgen deprivation therapy and surgery (Litwin and Tan 2017). However, patients with advanced prostate cancer usually develop CRPC due to various mechanisms, such as AR gene amplification and splice variants (Watson et al. 2015; Devlies et al. 2021; Attard and Antonarakis 2016), and the untimely detection of disease transformation creates difficulties in the selection of therapeutic agents. In addition, the long‐term use of single conventional anticancer agents can lead to a worse CRPC outcome for prostate cancer patients (Tilki et al. 2016). Therefore, finding a method based on the regulation of AR expression and activity that is effective in both hormone‐sensitive patients and CRPC patients is necessary.

During the catalytic process of PRMT5, one or two methyl groups from S‐adenosyl‐L‐methionine (SAM) are transferred to the target arginine residue, with ω‐NG monomethyl arginine (ω‐MMA) and symmetric ω‐NG, NG‐dimethylarginine (ω‐sDMA) produced (Pollack et al. 1999). We constructed a new series of PRMT5 inhibitors through a molecular docking‐based virtual screen and simulated their mode of action via molecular docking. To further validate the effects of these compounds, we used three cell lines, PC3, 22RV1, and LNCAP, to simulate the progression of different prostate cancers. SJL2‐1 affected cell migration, invasion, and cell cycle inhibition at the G0/G1 phase and promoted apoptosis. SJL2‐1 decreased PRMT5 and cytosolic symmetric arginine dimethylation levels, including those of SMD3, and reduced AR expression in a dose‐dependent manner. SJL2‐1 exhibited no significant cytotoxic effects toward normal prostate epithelial cells (RWPE‐1). These results indicate that SJL2‐1 possesses selective tumor‐inhibitory activity. In conclusion, our data support the tumor suppressive role of SJL2‐1 in regulating PCa progression.

PRMT5, an emerging protein arginine methyltransferase, is an important regulator of several key processes, including cell proliferation (Lu et al. 2018), cell cycle progression (Otani et al. 2021), and apoptosis (Jing et al. 2018), and is overexpressed in multiple cancers (Wu et al. 2021). Indeed, Deng et al. reported that PRMT5 regulates AR transcription by knocking down the PRMT5 gene in prostate cancer in an epigenetic control manner, which in turn inhibits the proliferation of LNCAP and C4‐2 cells expressing the AR receptor (Deng et al. 2017). In addition, PRMT5 promotes AR transcription in 22RV1 cells expressing ARV‐7 in a pICln‐dependent manner (Beketova et al. 2020), but PRMT5 gene knockdown does not affect PC3, a type of prostate cancer similar to DU145. Interestingly, in contrast to the above findings, our experimental results verified that the newly synthesized small‐molecule compound SJL2‐1 also had inhibitory effects on AR‐negative PC3 cells (representing CRPC types), suggesting that the newly synthesized compound SJL2‐1 had inhibitory effects on different stages and the progression of prostate cancer. This may be related to the effect of this compound on the methylation of posttranslational signaling molecules, such as EGFR and p53, via the inhibition of PRMT5 (Yan et al. 2021; Scoumanne et al. 2009).

Several questions are also raised by our study that require further investigation. First, the inhibitory effect of SJL2‐1 on prostate cancer was only demonstrated at the cellular level in this study, and its antitumor efficacy was not verified at the animal level. In addition, the specific pathways through which SJL2‐1 exerts its effects need to be explored to confirm its role in regulating tumorigenesis and to investigate the specific mechanism by which SJL2‐1 distinguishes itself from previous results in which it inhibited PC3, a CRPC cell line. Finally, new PRMT5 inhibitor‐like compounds suitable for prostate cancer treatment with superior IC50 values and antitumor capacity need to be systematically developed.

5. Conclusion

In conclusion, by combining reported PRMT5 inhibitors, new PRMT5 inhibitors were successfully synthesized in this study, and SJL2‐1 was further shown to have the most potent PRMT5 inhibitory activity. The compound showed anti‐proliferative activity against both AR‐expressing LNCAP and 22RV1 cells and PC3 cells representing CRPC. In addition, SJL2‐1 inhibited the migration and invasion ability of the cells. Flow cytometry and WB analysis revealed that SJL2‐1 had significant cell cycle‐arresting and apoptosis‐inducing effects. In addition, SJL2‐1 decreased the symmetric arginine dimethylation level and AR expression level of the cells. Finally, the binding mode of SJL2‐1 to PRMT5 was explored via molecular docking simulation, and this compound provides a new scaffold for the treatment of prostate cancer at different stages and the further development of PRMT5 inhibitors.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1. Reported representative PRMT5 inhibitors.

Figure S2. SJL2‐1 exhibits no significant impact on normal prostate epithelial cells RWPE‐1. (A) CCK‐8 assay demonstrating SJL2‐1‐induced non‐cytotoxic profile with unaltered RWPE‐1 proliferation. (B) EdU incorporation assay confirming negligible effects on DNA synthesis. (C) Quantitative analysis of EdU‐positive cells reveals comparable proportions across cohorts.

Figure S3. 1H NMR spectrum of SJL2‐1.

Figure S4. 13C NMR spectrum of SJL2‐1.

CBDD-105-e70136-s001.docx (557.2KB, docx)

Funding: This research work was financially supported by the Natural Science Foundation of Shandong Province (Nos. ZR2021QH366, ZR2023QH325, and ZR2020MB103), the Key Technology Research and Development Program of Shandong Province (No. 2019GSF108043), and the Natural Science Foundation of China (No. 81803438).

TongXiang Diao and Chen Feng share co‐first authorship.

Contributor Information

Kong‐kai Zhu, Email: hkhhh.k@163.com.

Cheng‐Shi Jiang, Email: bio_jiangcs@ujn.edu.cn.

Qiang Fu, Email: qiangfu68@163.com.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Reported representative PRMT5 inhibitors.

Figure S2. SJL2‐1 exhibits no significant impact on normal prostate epithelial cells RWPE‐1. (A) CCK‐8 assay demonstrating SJL2‐1‐induced non‐cytotoxic profile with unaltered RWPE‐1 proliferation. (B) EdU incorporation assay confirming negligible effects on DNA synthesis. (C) Quantitative analysis of EdU‐positive cells reveals comparable proportions across cohorts.

Figure S3. 1H NMR spectrum of SJL2‐1.

Figure S4. 13C NMR spectrum of SJL2‐1.

CBDD-105-e70136-s001.docx (557.2KB, docx)

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.

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