Discovery and biological evaluation of WEE1 inhibitors through virtual screening and medicinal chemistry approaches for cancer treatment

Wee1-like protein kinase (Wee1) is a serine/threonine kinase essential for cell cycle regulation and the DNA damage response (DDR), acting as a tumor suppressor in normal cells. Its overexpression in cancers such as melanoma, hepatocellular carcinoma, and glioblastoma underscores its potential as a therapeutic target. Wee1 inhibition has emerged as a strategy to sensitize tumor cells to genotoxic therapies, though no inhibitors have yet received broad clinical approval. In the first round of screening, we employed ESSENCE-Dock virtual screening to identify potential Wee1 inhibitors from the Eurofins-Villapharma library. Of the 12 compounds experimentally validated, six exhibited IC50 values below 100 µM, with compound 1 demonstrating promising activity (IC50 = 240 nM). Based on this initial hit, we synthesized and tested additional compounds, culminating in compound 20, which showed an almost order of magnitude improvement in potency (IC50 = 33 nM). Structure–activity relationship (SAR) analysis highlighted key activity determinants, including the effects of methyl substitution on the phenylpiperazine and double-chlorine halogenation on the terminal phenyl group. Compound 20 compared favorably to known Wee1 inhibitors such as MK-1775, while maintaining a distinct chemical scaffold. To further validate these findings, molecular dynamics simulations were employed to elucidate the binding interactions and conformational dynamics of the high-affinity compounds. These simulations revealed stabilizing interactions that aligned with the observed SAR trends, solidifying compound 20 as a strong lead for further development. In addition, in vitro ADME profiling demonstrated favorable physicochemical properties and drug-likeness, including high solubility and metabolic stability, supporting its potential as a drug candidate. Kinome-wide profiling at 10 µM revealed modest selectivity, highlighting a key consideration for future optimization.