Discovery of a novel PLK1 inhibitor with high inhibitory potency using a combined virtual screening strategy

Abstract

PLK1 is essential for cell cycle regulation and proliferation, and its elevated expression in prostate cancer is associated with high tumour grade. Therefore, PLK1 inhibition is considered a promising strategy for the treatment of prostate cancer. Here, we identified five compounds (Hits 1-5) targeting the kinase domain (KD) of PLK1 using a combined virtual screening approach. Hits 1-5 all had picomolar (pM) inhibitory potency against PLK1. Notably, Hit-4 showed the strongest inhibitory activity against PLK1 (IC₅₀ = 22.61 ± 1.12 pM) and displayed high selectivity for PLK1. Meanwhile, molecular dynamics (MD) simulations revealed that the complex formed by Hit-4 and PLK1 remained stable. Importantly, Hit-4 exhibited potent inhibitory effects on the proliferation of DU-145 prostate cancer cells (IC₅₀ = 0.09 ± 0.01 nM). In conclusion, Hit-4 is a potent and highly selective antitumor candidate with therapeutic potential for prostate cancer.

Introduction

Prostate cancer (PCa) is the second most commonly diagnosed malignancy and the fifth leading cause of cancer death in men worldwide¹. Current treatments for prostate cancer include chemotherapy, radiotherapy, surgery, and hormone therapy^2,3. Although a number of drugs have been tested and approved for use in the early stages of the disease, the heterogeneity of tumours and drug resistance remain a major challenge in the treatment process^2,4. Androgen deprivation therapy initially shows a period of efficacy, but it is common for the disease to gradually progress to castration-resistant prostate cancer (CRPC)^5,6. In addition, most of these treatments have limited efficacy in patients with advanced disease and are associated with significant side effects^2,7. Therefore, the discovery of potential drug candidates with low side effects and high efficacy for prostate cancer therapy is necessary.

Polo-like kinase 1 (PLK1), a serine/threonine kinase, is central to the regulation of the cell cycle and cell proliferation^8,9. PLK1 is localised to critical mitotic structures and functions as a key regulator of the mitotic process, involved in mitotic entry, centrosome maturation, sister chromatid separation, and the initiation of cytokinesis^10–12. Given its important role in mitosis, PLK1 is overexpressed in many types of tumours and the elevated expression levels of PLK1 seem to be associated with the aggressiveness and poor prognosis of a wide range of cancers^13–16. Research shows that the expression levels of PLK1 are significantly elevated in prostate cancer tissue compared to normal prostate cells and correlate with high tumour grade, suggesting that PLK1 is an important factor in the growth and progression of prostate cancer^17–19. Furthermore, PLK1 is one of the top five signalling pathways upregulated after castration, and downregulation of the PLK1 gene inhibits mitosis in prostate cancer cells^19–21. Therefore, a therapeutic approach targeting PLK1 may be a promising approach for the treatment of prostate cancer.

DU-145 cells are derived from prostate cancer that has metastasised to the brain and have intermediate metastatic potential²². DU-145 cells are androgen insensitive and do not express prostate specific antigen (PSA), which is particularly important for studying the role of PLK1 inhibitors in CRPC²³. Meanwhile, DU-145 cells showed high levels of PLK1 mRNA and protein expression, and several PLK1 inhibitors significantly inhibited the proliferation of DU-145 cells^24,25. Therefore, DU-145 cells were used in this study to evaluate the antiproliferative activity.

PLK1 contains a highly conserved catalytic kinase domain (KD) at the N-terminus and a Polo-box domain (PBD) at the C-terminus^26–28. The KD is responsible for the kinase activity of PLK1 and serves as a key factor in the modulation of cell cycle progression by PLK1, while the PBD modulates the subcellular localisation and function of PLK1 by binding to specific phosphorylated substrates^29–31. The KD and PBD domains of PLK1 are intrinsic targets for the development of PLK1 inhibitors^32,33. To date, several PLK1 inhibitors have entered the testing phase of clinical trials, such as volasertib³⁴, rigosertib³⁵, and GSK461364³⁶ (Figure 1). In particular, the KD inhibitor volasertib (BI6727) has been granted orphan drug designation by the US FDA^37,38. However, the lack of specificity that characterises many other KD inhibitors results in dose-limiting toxicities, severely restricting their development³³.

Figure 1.

Reported PLK1 inhibitors.

In our research, we discovered a novel, potent, and highly selective small molecule inhibitor targeting the KD domain of PLK1. First, the optimal pharmacophore models were constructed through the conformational relationship analysis of the KD domain structure of PLK1. Based on the key pharmacophore features, candidate molecules were screened from the compound database for conformational matches. Subsequently, the molecular docking screening of these compounds was performed based on the KD domain of PLK1, and five potential compounds (Hits 1–5) were identified. Hits 1–5 showed pM inhibitory potency against PLK1. Among them, Hit-4 was the most potent inhibitor of PLK1 and showed selective inhibition of PLK1. In addition, Hit-4 exhibited significant antiproliferative activity against DU-145 prostate cancer cells. In conclusion, our study has identified a small molecule inhibitor targeting PLK1 with high selectivity and inhibitory activity, which is worthy of further investigation.

Materials and methods

Cell culture and materials

The DU-145 prostate cancer cells used in this study were purchased from the American Type Culture Collection (ATCC), which is a reputable cell repository. ATCC follows strict guidelines and ethical protocols for the collection, authentication, and distribution of cell lines. We utilised these cells to perform in vitro antiproliferation assays to test the inhibitory effects of the tested compounds on cell proliferation. The cells were cultured in medium supplemented with 10% (v/v) foetal bovine serum, 50 units/ml of penicillin, and 50 µg/ml of streptomycin. The cell culture was maintained at 37 °C in a humid environment with 5% CO₂. Hit compounds were purchased from WuXi AppTec. Recombinant human PLK1 protein was purchased from Abcam (Cambridge, MA, USA).

Pharmacophore construction

The crystal structure of the PLK1 protein with its ligand (PDB ID: 3FC2) was retrieved from the Protein Data Bank (PDB) and was imported into the Molecular Operating Environment (MOE) for processing. First, the above structure was subjected to optimisation by the Quick Prep function in MOE, including the removal of unbound water molecules, the determination of partial charges, the attachment of polar hydrogen atoms, and energy minimisation processes. Then, the Ligand Interactions tool in MOE was utilised to analyse the interaction between PLK1 and the ligand to identify the key active binding sites. The Pharmacophore Query Editor tool in MOE was used to construct pharmacophore models based on the interaction analysis. In the Scheme menu, the EHT pharmacophore scheme was selected. Next, the key interaction point in the ligand was identified and the Feature tool was used to generate a series of pharmacophore features, including aromatic centres, hydrogen-bond acceptors, and hydrogen-bond donors.

Virtual screening

A chemical database of 35,000 compounds was constructed using combinatorial chemistry methods³⁹. The two-dimensional (2D) structures of these compounds were then converted into three-dimensional (3D) conformations using the Conformation Import tool in MOE. The conformations were generated using the MMFF94x force field, and the Energy Minimisation module was used to ensure conformational stability. Then, the pharmacophore models constructed above for PLK1 were applied to perform a virtual screening of the compound database using the Pharmacophore Search panel. The Pharmacophore Search panel can be accessed directly from the Pharmacophore Query Editor. The conformation database was imported, and the Search module was used to initiate the search process. Next, the hits identified through pharmacophore screening were subjected to molecular docking into the KD of PLK1 for further evaluation. The Dock module within MOE was utilised to position each compound within the active site of PLK1. The docking results were evaluated using the Triangle Matcher method in association with the London dG scoring function. Comparing the binding free energies for different compound conformations, a lower docking score indicated a higher binding affinity.

PLK1 kinase inhibition assay

The assay was conducted as previously described⁴⁰. The kinase reaction was performed in 384-well plates with a total assay volume of 5 μL per well, each well containing a reaction mixture consisting of 50 μM DTT, kinase and kinase substrate (25 ng PLK1 and 0.5 μg casein), 50 μM ATP and the tested compound at designated concentration (2.5, 5, 10, 20, 40, 80, 160, 320, 640, 1280, 2560 and 5120 pM). The tested compound solution (1 μL) was replaced with 5% DMSO for positive control wells, while negative control wells contained neither the tested compound nor enzyme. Each well was incubated for 60 min at room temperature. Subsequently, 5 μL of ADP-Glo reagent was added to each well and incubated for 40 min to terminate the reaction. ADP was then converted to ATP by adding 10 μL of kinase detection reagent and incubating for 30 min. Then, the luminescence of ATP was measured using the Victor Nivo Multimode Microplate Reader, and the following formula was used to calculate kinase activity: kinase activity% = [(luminescence of compound - luminescence of negative control)/(luminescence of positive control - luminescence of negative control)] × 100%. IC₅₀ values were determined by analysing dose-response curves using Graph Prism 8.0 software.

Selectivity assays

The selectivity profile of Hit-4 was determined by evaluating its inhibitory activity against a panel of 69 kinases containing other polo-like kinases. The experiment was conducted by ICE Bioscience Inc. (Beijing, China). The LanthaScreen^TM Eu Kinase Binding Assay was performed to evaluate the selectivity profile of Hit-4 on a panel of 69 kinases.

Molecular dynamics simulation

The crystal structures of PLK1 (PDB ID: 3FC2) was obtained from the PDB. MD simulations were performed using GROMACS (version 2022). The topology of PLK1 was generated under the AMBER99SB-ILDN force field. Hit-1 and Hit-4 were processed through the Acpype Server (www.bio2byte.be) to generate their respective topology files. Subsequently, the topology files for the ligands were merged with the protein topology file to create the complete complex system, respective. The system was solvated using the SPC/E water model within a cubic box of 1.0 nm. The sodium (Na⁺) and chloride (Cl^-) ions were added to maintain charge neutrality. Next, the steepest descent algorithm with 5000 steps was used to minimise the system energy. The system temperature was maintained at 300 K during a 100 ps NVT simulation. A 100 ps NPT simulation was then performed to keep the pressure at 1 bar. Finally, a 50 ns MD simulation was performed on the system. The 50 ns MD simulation has been used in several previous protein-ligand interaction stability studies by our research group and it was found that energy equilibrium was reached and the simulation results were highly reliable at 50 ns MD simulation duration^41,42. These data were processed using Prism 8.0 software.

In vitro antiproliferation assay

A density of 5 × 10⁴ cells per well was inoculated into 96-well plates and incubated overnight. Varying concentrations of Hits 1–5 were introduced into each well, and the plates were incubated at 37 °C for 72 h. Following incubation, the culture medium was aspirated and MTT solution (with a concentration of 5 mg/mL) was dispensed into each well for a further incubation period of 4 h. The supernatant was then removed via centrifugation, followed by the addition of DMSO to solubilise the precipitated crystals. Ultimately, absorbance was measured at 570 nm using a microplate reader. The non-linear regression was used to plot a dose-response curve and determine the IC₅₀ value. Data analysis was performed using Prism 8.0 software.

Results and discussion

Pharmacophore construction

The pharmacophore models were constructed based on a detailed analysis of the binding site interaction between PLK1 and its ligand BI6727 (PDB ID: 3FC2). First, the Ligand Interaction module within MOE was employed to assess the structure-activity relationship between PLK1 and BI6727, and identified key interaction elements for pharmacophore models construction (Figure 2a). The ligand made hydrophobic contacts with the residues Phe58, Ala80, Val114, Leu130, Leu132, and Phe183 of PLK1, prompting the inclusion of aromatic features in the pharmacophore model. In addition, the amide CO group of the ligand formed a hydrogen bond with Arg57 of PLK1, providing a hydrogen-bond acceptor feature, while NH group of the ligand created a hydrogen bond with the key residue Leu59 of PLK1, establishing a hydrogen-bond donor feature. Consequently, four critical pharmacophore features were constructed: two aromatic features (F1 and F2: Aro), one hydrogen-bond acceptor feature (F3: Acc), and one hydrogen-bond donor feature (F4: Don). These pharmacophore models with essential chemical features are pivotal for the identification of potential PLK1 inhibitors.

Figure 2.

(a) The constructed pharmacophore models. (b) The workflow of combined virtual screening.

Virtual screening

In this study, a combined virtual screening approach including pharmacophore screening, molecular docking, MD simulation, and biological assays was employed to discover novel PLK1 inhibitors. The virtual screening workflow is illustrated in Figure 2b. First, the 2D database consisting of 35,000 compounds was converted into 3D structure. The above four pharmacophore features (F1 and F2: Aro, F3: Acc, F4: Don) were then applied to filter the database, and a total of 92 compounds were identified that matched the Aro, Acc, and Don features. Subsequently, these 92 compounds were docked into the KD domain of PLK1, with docking scores indicating binding affinity—lower scores corresponding to stronger affinities. The top five compounds with the lowest docking scores were selected as optimal hits. As shown in Figure 3, five top hits (Hits 1–5) with the lowest scores were selected from 92 compounds. Notably, the docking scores of Hits 1–5 were lower than the known PLK1 inhibitor BI-6727 (docking score of −9.31 kcal/mol), suggesting superior binding affinities. Among these, Hit-4 demonstrated the lowest docking score, indicative of the strongest binding to PLK1. The structures of Hits 1–5 are presented in Figure 4. Next, the molecular interactions of these five compounds with PLK1 were analysed in detail to validate the screening findings.

Figure 3.

The binding free energy (kcal/mol) of five compounds (Hits 1–5). Data are expressed as mean ± SD, n = 3. ***P < 0.001 indicates a significant difference from the control group BI-6727.

Figure 4.

(a-e) The 2D interactions of Hits 1–5 with PLK1, respectively. Green spheres represent hydrophobic amino acids in PLK1, purple spheres represent polar amino acids in PLK1, green and blue dashed arrows represent hydrogen bonds.

Interaction analysis

The molecular interactions of the five hits (Hits 1–5) with PLK1 were analysed and the possible binding modes are shown in Figures 4 and 5. The hydrogen bonding and hydrophobic interactions between Hits 1–5 and PLK1 are shown in Table 1. The ligands formed hydrophobic interactions with the key residues Phe58, Ala80, Val114, Leu130, Leu132, and Phe183, consistent with the Aro (F1 and F2) pharmacophore features. In particular, the 1-methyl-1-(trifluoromethyl)cyclopentane group at the terminal end of Hit-1 and the spiro-nonane group at the terminal end of Hit-4 are relatively hydrophobic. Meanwhile, the amide group in the ligand formed hydrogen bond interactions with the residues Leu59 and Arg57 of PLK1, corresponding to the Acc (F3) and Don (F4) pharmacophore features, respectively. Moreover, the surface maps showed that the PLK1 active pocket was well matched in shape to Hits 1–5. Notably, the 1-methyl-1-(trifluoromethyl)cyclopentane group at the end of Hit-1 and the spiro-nonane group at the end of Hit-4 showed excellent shape complementarity with the active pocket of PLK1. In conclusion, Hits 1–5 showed significant interactions with key residues within the active sites of PLK1 and perfectly matched to the binding pocket of PLK1, especially Hit-1 and Hit-4. Based on the favourable binding pattern analysis, the inhibitory potential of Hits 1–5 was evaluated.

Figure 5.

The binding modes of Hits 1–5 within the PLK1 binding pocket are shown as follows: (a, b) Hit-1 is shown in green; (c, d) Hit-2 is shown in yellow; (e, f) Hit-3 is shown in purple; (g, h) Hit-4 is shown in orange; (i, j) Hit-5 is shown in blue. Active site residues are shown as pink sticks. Hydrogen bonds are represented in black dashed lines.

Table 1.

PLK1 residues forming key binding interactions with Hits 1–5.

Name	Hydrogen bonding interactions			Hydrophobic interactions
	Amino acid involved	Hydrogen bonds	Bond distance (Å)
Hit-1	Arg57	O•••H-N	2.83	Phe58, Ala80, Val114, Leu130, Leu132, Phe183
	Leu59	H-N•••O	2.97
Hit-2	Arg57	O•••H-N	2.83	Phe58, Ala80, Val114, Leu130, Leu132, Phe183
	Leu59	H-N•••O	3.00
Hit-3	Arg57	O•••H-N	2.85	Phe58, Ala80, Val114, Leu130, Leu132, Phe183
	Leu59	H-N•••O	2.97
Hit-4	Arg57	O•••H-N	2.84	Phe58, Ala80, Val114, Leu130, Leu132, Phe183
	Leu59	H-N•••O	3.00
Hit-5	Arg57	O•••H-N	2.83	Phe58, Ala80, Val114, Leu130, Leu132, Phe183
	Leu59	H-N•••O	2.97

In vitro PLK1 inhibitory activity

Enzyme inhibition assay was performed to evaluate inhibitory potency of Hits 1–5 on PLK1. As shown in Table 2, Hits 1–5 exhibited significant inhibitory activity against PLK1 with IC₅₀ values ranging from approximately 22.61 to 68.32 pM, which were superior to those of the positive control BI-6727. Notably, Hit-4 had the strongest inhibitory activity against PLK1 (IC₅₀ = 22.61 ± 1.12 pM) with an approximately 38-fold lower IC₅₀ value compared to BI-6727. In addition, we performed a comparison of the IC₅₀ values between the reported PLK1 KD inhibitors (0.83-4 nM) and Hits 1–5 (22.61-68.32 pM). The results indicate that Hits 1–5 exhibited significantly stronger inhibitory activity than the reported PLK1 KD inhibitors (Table S1).

Table 2.

The inhibitory effects of Hits 1–5 on PLK1 and DU-145 cells.

Name	PLK1 (IC50, pM)a	DU-145 (IC50, nM)b
Hit-1	39.46 ± 2.79	0.18 ± 0.02
Hit-2	47.35 ± 3.21	0.23 ± 0.04
Hit-3	68.32 ± 5.28	0.35 ± 0.05
Hit-4	22.61 ± 1.12	0.09 ± 0.01
Hit-5	56.82 ± 3.34	0.47 ± 0.06
BI-6727	871.04 ± 16.15	4.03 ± 0.15

^a IC₅₀ (pM) is the concentration of compound needed to achieve 50% inhibition of enzyme activity treated with Hits 1–5.

^b IC₅₀ (nM) is the concentration of compound needed to reduce cell growth by 50% after 72 h treatment with Hits 1–5.

The data are presented as the mean ± SD, n = 3.

In addition, selectivity assays were conducted on a panel of 69 kinases to further evaluate the selective inhibitory effect of Hit-4. As shown in Table 3, Hit-4 exhibited an inhibition rate of less than 10% against these kinases at a concentration of 10 μM. These results suggest that Hit-4 selectively targets PLK1 for inhibition.

Table 3.

Kinase selectivity profile of Hit-4 at a concentration of 10 µM on a panel of 69 kinases.

Target	% Inhibition at 10 μM	Target	% Inhibition at 10 μM	Target	% Inhibition at 10 μM
ABL1	0.70 ± 0.01	FES	4.79 ± 0.12	LTK	3.51 ± 0.09
ABL2	0.08 ± 0.02	FGFR1	0.87 ± 0.03	LYN	0.62 ± 0.04
AXL	0.40 ± 0.05	FGFR2	0.75 ± 0.04	MERTK	0.19 ± 0.01
BLK	0.69 ± 0.03	FGFR3	5.51 ± 0.16	MET	8.12 ± 0.17
BMX	2.03 ± 0.06	FGFR4	2.84 ± 0.07	MST1R	7.45 ± 0.11
BTK	3.10 ± 0.08	FGR	0.45 ± 0.03	MUSK	0.94 ± 0.05
CSF1R	1.22 ± 0.05	FRK	6.49 ± 0.22	NTRK1	0.75 ± 0.06
EGFR	0.49 ± 0.04	FYN	0.08 ± 0.01	NTRK2	1.81 ± 0.09
DDR1	0.19 ± 0.01	PIM1	0.32 ± 0.03	NTRK3	5.03 ± 0.17
DDR2	3.48 ± 0.09	RAF1	6.19 ± 0.24	PDGFRA	0.61 ± 0.04
ALK	0.91 ± 0.03	ROS1	0.66 ± 0.05	PDGFRB	5.71 ± 0.12
EPHA1	0.96 ± 0.05	ZAK	0.33 ± 0.02	PTK2	3.08 ± 0.06
EPHA2	0.61 ± 0.04	TYRO3	0.90 ± 0.04	CDK5	0.68 ± 0.04
EPHA3	0.79 ± 0.07	YES1	0.20 ± 0.01	CDK6	4.18 ± 0.08
EPHA4	0.45 ± 0.03	ZAP70	6.09 ± 0.17	CDK7	7.58 ± 0.13
EPHA5	2.86 ± 0.08	HCK	1.44 ± 0.08	CDK8	5.28 ± 0.09
EPHA6	4.17 ± 0.07	IGF1R	0.49 ± 0.03	KIT	0.77 ± 0.04
EPHA7	0.30 ± 0.01	INSR	0.15 ± 0.01	KDR	0.14 ± 0.01
EPHA8	0.73 ± 0.04	INSRR	0.34 ± 0.04	ERBB2	6.41 ± 0.15
EPHB1	0.93 ± 0.03	ITK	0.70 ± 0.05	PLK2	0.73 ± 0.03
EPHB2	0.68 ± 0.06	JAK1	0.90 ± 0.03	PLK3	0.24 ± 0.01
EPHB3	0.53 ± 0.02	JAK2	7.69 ± 0.27	PLK4	1.87 ± 0.07
EPHB4	2.77 ± 0.08	JAK3	6.51 ± 0.18	PLK5	0.48 ± 0.02

The data are presented as the mean ± SD, n = 3.

MD simulation

Since Hit-1 and Hit-4 were perfectly matched to the active pocket of PLK1 and both showed strong inhibitory activity against PLK1, MD simulations were performed using GROMACS to assess their binding stability to PLK1. The smaller variation of the root mean square deviation (RMSD) value indicates the better binding stability of the complex. Figure 6a and 6b showed that the RMSD of both PLK-Hit-1 and PLK-Hit-4 complexes reached a stable value with slight fluctuations around 0.2 nm. This indicates that the binding of Hit-1 and Hit-4 to PLK1 is stable. The root mean square fluctuation (RMSF) provides insight into the flexibility of amino acids involved in ligand binding. In Figure 6c and 6d, the RMSF values of the key residues Arg57, Phe58, Leu59, Ala80, Val114, Leu130, Leu132, and Phe183 in the binding site of PLK1 were less than 0.13 nm, reflecting the stable binding of the key amino acids of PLK1 to Hit-1 and Hit-4. In addition, Figure 6e and 6f) showed that the radius of gyration (Rg) value of PLK1 was stable at around 2.05 nm, indicating that the protein maintained structural integrity during the simulation. In Figure 6g and 6h, no significant changes in the secondary structure of the PLK1 were observed, indicating that the protein remained structurally stable after complexation with Hit-1 and Hit-4. In summary, Hit-1 and Hit-4 are able to bind stably to the active site of PLK1 and maintain the structural stability of the complex throughout the simulation.

Figure 6.

MD simulation of PLK1 in complex with Hit-1 and Hit-4. (a) RMSD of Hit-1 in PLK1-Hit-1 complex. (b) RMSD of Hit-4 in PLK1-Hit-4 complex. (c, d) RMSF of PLK1 residues in the complex of PLK1-Hit-1 and PLK1-Hit-4, respectively. (e, f) Rg of PLK1 in the complex of PLK1-Hit-1 and PLK1-Hit-4, respectively. (g, h) The secondary structures of PLK1 in the complex of PLK1-Hit-1 and PLK1-Hit-4, respectively.

Inhibition of cell growth

MTT assays were conducted to assess the inhibitory effects of Hits 1–5 on the proliferation of DU-145 prostate cancer cells. As shown in Table 2, Hits 1–5 and the positive control BI-6727 exhibited different degrees of antiproliferative activity against DU-145 cells, and the inhibitory activity of Hits 1–5 was significantly higher than that of BI-6727. Among them, Hit-4 was the most potent compound in inhibiting the growth of DU-145 cells (IC₅₀ = 0.09 ± 0.01 nM). These data suggest that Hit-4 has potential therapeutic efficacy, making it a promising candidate for prostate cancer treatment.

Conclusions

PLK1 is overexpressed in prostate cancer and is one of the top five signalling pathways upregulated in castration-resistant prostate cancer, highlighting the importance of developing potent PLK1 inhibitors as a therapeutic strategy for prostate cancer. In this study, we successfully identified five novel and potent PLK1 inhibitors (Hits 1–5) using a combined virtual screening protocol. Based on the constructed pharmacophore features of PLK1, we screened the candidate molecules from a compound database. These compounds were then docked into the KD domain of PLK1 and five potential compounds (Hits 1–5) were identified. The structures of Hits 1–5 showed compatibility with the active site pocket of PLK1, especially Hit-4. Enzyme inhibition assay showed that Hits 1–5 exhibited pM inhibitory potency against PLK1, and Hit-4 had the strongest inhibitory activity. Notably, Hit-4 selectively targeted PLK1 for inhibition without significant inhibitory effects on other kinases. The concordance between inhibitory activity and docking scores demonstrated the effectiveness of the combined virtual screening approach in predicting the activity of lead compounds. Importantly, Hit-4 displayed significant antiproliferative activity against DU-145 prostate cancer cells.

Given the excellent in vitro antiproliferative activity of Hit-4 against DU-145 cells, we believe that Hit-4 is a promising new anti-prostate cancer candidate worthy of further in vivo evaluation in mice to assess its therapeutic potential and safety profile. In addition, the development of Hit-4 may face some challenges, including the potential emergence of drug resistance and the need for combination therapies to improve therapeutic efficacy. Combinations of some PLK1 inhibitors with chemotherapy and targeted therapies have shown promising results in numerous cancer treatment studies both in vitro and in vivo⁴³. This suggests that the combination of Hit-4 with other therapeutic agents may offer superior efficacy compared to monotherapy, making it an attractive strategy to enhance treatment outcomes. Therefore, advancing Hit-4 into in vivo studies in mice is essential to evaluate its safety profile and address potential challenges in drug development. In conclusion, we have identified Hit-4 as a highly selective PLK1 inhibitor with pM inhibitory activity, demonstrating significant potential for the treatment of prostate cancer and warranting further investigation.

Funding Statement

This research was supported by the Taizhou People’s Hospital (Taizhou, Jiangsu, China; Grant No. ZL202220).

Author contributions

ZX, LG, and YW performed experiments and acquired data. ZX, LG, and MMN collected experimental samples. ZX, MMN, and YR performed statistical analysis. CX and LY analysed data and wrote the paper. All authors had edited and approved the final manuscript.

Ethical statement

The cell culture and experimental procedures were conducted in accordance with standard laboratory practices and guidelines. We did not perform any procedures that would require additional specific ethical approvals beyond the standard practices for cell culture and experimentation in a laboratory setting, as this study used commercially available cell lines and the experiments did not involve any procedures that could potentially harm humans or animals.

Disclosure statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Data availability statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.