Establishment of assay systems for drug development against the oncogenic phosphatase WIP1

The serine/threonine phosphatase WIP1 (PPM1D) is a key negative regulator of the DNA damage response (DDR) and a recognized oncogene, frequently amplified or truncated in various cancers including breast, ovarian, neuroblastoma, and glioblastoma. Its inhibition restores DDR signaling, offering a promising therapeutic strategy. Despite initial efforts, including the development of the allosteric inhibitor GSK2830371, clinical translation has been limited. We report the establishment of a comprehensive assay platform to support drug development targeting WIP1. Biochemical assays were optimized using fluorescein diphosphate (FDP), malachite green with phosphopeptides (p53, H2AX, p38 MAPK), and TR-FRET formats, enabling robust activity profiling. Cellular mechanism-of-action assays were developed to monitor phosphorylation changes in WIP1 substrates, with pS15-p53 ELISA in U2OS cells selected for medium-throughput iterative compound profiling. Phenotypic assays demonstrated compound efficacy in 2D and 3D proliferation models, with leukemic cell lines showing pronounced sensitivity. Combination studies revealed strong synergy between WIP1 inhibitors and MDM2 antagonists (e.g., Nutlin3a), supporting a dual-targeting approach for p53 pathway reactivation. In vivo efficacy was confirmed in MV4-11 xenograft models, with dose-dependent tumor growth inhibition and favorable tolerability profiles. Our integrated assay suite enables iterative screening and lead optimization, culminating in the identification of a potent allosteric WIP1 inhibitor with nanomolar activity and promising preclinical efficacy. These findings support WIP1 as a viable target in oncology and provide a foundation for future clinical development.