Identification and Biological Evaluation of a Novel CLK4 Inhibitor Targeting Alternative Splicing in Pancreatic Cancer Using Structure-Based Virtual Screening

Abstract

Pancreatic cancer is an aggressive malignancy with a poor prognosis and limited treatment options. Cdc-like kinase 4 (CLK4), a kinase that regulates alternative splicing by phosphorylating spliceosome components, is implicated in aberrant splicing events driving pancreatic cancer progression. In this study, we established a computational model that integrates pharmacological interactions of CLK4 inhibitors with an improved hit rate. Through this model, we identified a novel CLK4 inhibitor, compound 150441, with a 50% inhibitory concentration (IC₅₀) value of 21.4 nm. Structure-activity relationship analysis was performed to investigate key interactions and functional groups. Kinase profiling revealed that compound 150441 is selective for CLK4. Subsequent in vitro assays demonstrated that this inhibitor effectively suppressed cell growth and viability of pancreatic cancer cells. In addition, it inhibited the phosphorylation of key splicing factors, including serine- and arginine-rich splicing factor (SRSF) 4 and SRSF6. Cell cycle analysis further indicated that the compound induced G2/M arrest, leading to apoptosis. RNA-seq analysis revealed that the compound induced significant changes in alternative splicing and key biological pathways, including RNA processing, DNA replication, DNA damage, and mitosis. These findings suggest that compound 150441 has promising potential for further development as a novel pancreatic cancer treatment.

Keywords: CLK4; alternative splicing; kinase inhibitor; pancreatic cancer; structure‐based virtual screening.

Figure 1

Workflow of the study. Initially, a computational model for identifying CLK4 inhibitors was first developed. Then, the model was applied to identify potential inhibitors. Selected compounds were validated using enzymatic assays. The effects of identified inhibitors on cancer cells were further evaluated.

Figure 2

Analysis of pharmacological interactions for CLK4. The analysis includes two types of interactions: A) hydrogen‐bonding and B) hydrophobic interactions. Interactions occurring with a frequency of 50% or higher are considered pharmacological interactions. C) Docking pose of ATP. Hydrogen bonds are shown as green dashes. Binding site residues are depicted as lines and labeled as shown. D) Comparison of scoring functions using docking score alone and pharmacological interaction score.

Figure 3

Docking pose and interactions of compound 150 441 in the CLK4 binding site. A) Compound 150441 (salmon) shows favorable occupation of the CLK4‐binding site (gray). Hydrogen bonds are represented as green dash lines. Binding site residues are listed as shown. B) The 2D interaction figure shows hinge residues (blue) interact with isoquinoline, interior residues (pink) interact with indole, and exterior residues (lime) interact with dimethylethylamine. Hydrogen bonds are represented as green dash lines, and hydrophobic interactions are represented as gray dot lines.

Figure 4

SAR analysis of 150441 and its analogs. A) Interaction profile of the compounds. The compounds were categorized into three classes based on their inhibitory activity. The most potent class is shown as green, and the weakest class is shown as orange. Residues in the CLK4 binding pocket are categorized into exterior, hinge, and interior sites. HB, A, and M stand for hydrogen‐bonding interaction, alkyl hydrophobic interaction, and mixed hydrophobic interaction, respectively. B) Docking poses of the compounds. Hydrogen bonds are shown as green dashes. Exterior, hinge, and interior residues are shown in lime, blue, and pink, respectively.

Figure 5

The anticancer effect of CLK4 inhibitors. A–D) Pancreatic cancer cells were treated with compounds at 0.3, 1, 3, and 10 µm for 72 hours. Cell viabilities were determined by 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, and antiproliferation effects were measured by sulforhodamine B (SRB) assay. E) Colony formation analysis. Cells were incubated with compound 150441 at 0.03, 0.1, 0.3, 1, and 3 µm for 12 days. F) Cells were treated with 150441 at 1, 3, and 10 µm or SM08502 (SM) at 0.1 µm for 2 hours, and the phosphorylation of SR protein was detected by western blotting. SM08502 as a reference compound for SR protein activity inhibition. These results were repeated in at least three independent experiments. ^* p < 0.05, ^** p < 0.01 compared to the control (ctrl., untreated) group.

Figure 6

Cancer cell apoptosis and G2/M arrest while 150441 treatment. Flow cytometry analysis was used to assess the distribution of pancreatic cancer cell lines Mia Paca‐2 and Panc‐1 in various cell cycle phases (sub‐G1, G0/G1, S, and G2/M). A,B) After treatment with indicated concentrations of compound 150441 (0.3, 1, 3, and 10 µm) for 72 hours and C) the proportion of cells in the sub‐G1 phase. D) The expression of apoptotic proteins, Mcl‐1, Bcl‐2, PARP, caspase 9, and caspase 3, detected by western blotting. E) Cells were treated with 3 µm compound 150441 for 8, 16, 24, 48, and 72 hours, and the proportion of cells in the G2/M phase. F) The expression levels of G2/M regulatory proteins were assessed following treatment with 10 µm of 150441 for 16 hours. These results were repeated in at least three independent experiments. ^* p < 0.05 compared to the control (ctrl., untreated) group.

Figure 7

Selectivity profile of compound 150441. A) Inhibitory activity of 150441 on a panel of 60 kinases. B) A kinome tree composed of seven families shows the kinase inhibitory activity of the compound. The compound was tested at a concentration of 30 nm. Among all tested kinases, only the inhibition percentage for CLK4 exceeded 50%, indicated by a red dot.

Figure 8

Compound 150441‐induced alternative splicing events in Mia PaCa‐2 cells. Cells were treated with 3 µm compound 150441 for 6 hours and then subjected to RNA‐seq analysis. A) Distribution of splicing event. The number of splicing events caused by compound 150441. B) Gene enrichment analysis. Pathway enrichment of genes affected by 150441‐induced splicing events, analyzed using DAVID gene functional classification. The top ten most enriched biological processes are ranked by fold enrichment. C) Top ten genes in mitosis ranked by |IncLevelDifference|. The genes most affected by 150441 in the mitosis pathway based on splicing inclusion level differences. D) List of genes with |IncLevelDifference| > 0.3 in mitosis.