Discovering Small-Molecule WEE1 Binders through Iterative Virtual Screening

Abstract Wee1-like protein kinase (WEE1) is a key regulator of the G2/M cell cycle checkpoint, preventing premature mitosis by phosphorylating cyclin-dependent kinase 1 (CDK1) and allowing time for DNA repair before division. Small-molecule Wee1 inhibitors are being investigated as a way to induce mitotic failure in cancer cells. Although several WEE1 inhibitors have entered clinical trials (phases I/II), none has received regulatory approval, underscoring the need for more selective and effective compounds. Here, complementary virtual screening strategies were used to identify novel small-molecule binders of WEE1. Structure-based screening of the ATP-binding pocket, combining docking, pharmacophore modeling, pose filtering, and evaluation of two protein structures, selected compounds for experimental testing. Microscale thermophoresis (MST) detected binding for 65 of 115 tested compounds (57%); ten had dissociation constants below 10 µM. These relatively small compounds (267–399 g/mol) are structurally distinct from known WEE1 inhibitors and provide starting points for future optimization.