Wee1-targeting inhibitors: From virtual screening to lead discovery.

Wee1-like protein kinase (Wee1) is a validated target for cancer therapy, though no inhibitors have yet received regulatory approval. To identify new Wee1 inhibitors, we conducted a virtual screening campaign using the Eurofins-Villapharma library. Based on the initial hit Compound 1 (IC50 = 240 nM in the biochemical assay), we synthesized and tested additional compounds. Analog 20 showed a significant improvement in potency (IC50 = 33 nM). It was also the most potent in cellular assays, inhibiting the proliferation of lung carcinoma cell lines A427 (IC50 = 0.761 μM) and NCI-H23 (IC50 = 0.449 μM), OVCAR-3 (IC50 = 0.627 μM), and MDA-MB-231 (IC50 = 0.842 μM). Compound 18 (cell-free IC50 = 55 nM) showed weaker cellular activity, with IC50 values of 1.67 μM (A427), 1.38 μM (NCI-H23), 1.58 μM (OVCAR-3), and 2.93 μM (MDA-MB-231). In a kinome screen, Compound 18 was the most selective of the tested compounds, with an S(1) score of 0.025. Structure-activity relationship analysis highlighted key activity determinants, including the effects of methyl substitution on the phenylpiperazine and of double‑chlorine halogenation on the terminal phenyl group. Molecular dynamics simulations were used 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. In vitro ADME compound profiling demonstrated favorable physicochemical properties and drug-likeness, including high solubility and metabolic stability, supporting their potential as lead candidates.