Molecular targeting of the deubiquitinase USP14 to circumvent cisplatin resistance in ovarian carcinoma and identification of novel inhibitors

<b>Aim:</b> This study aims to investigate the biological role of the proteasome-associated deubiquitinase ubiquitin-specific protease 14 (USP14) in ovarian carcinoma drug resistance and to identify novel USP14 inhibitors (USP14i) for further preclinical development. <b>Methods:</b> USP14 expression was evaluated in clinical samples from 134 ovarian carcinoma patients and in a broad panel of human ovarian carcinoma cell lines. Functional studies, including gain- and loss-of-function assays, migration and invasion, and apoptosis induction assays, were conducted using cisplatin-sensitive IGROV-1 cells and their cisplatin-resistant derivative IGROV-1/Pt1. A library of 1,056 small molecules was screened using an optimized hydrolysis assay. Docking and molecular dynamics simulations were employed to predict binding modes of candidate inhibitors within the USP14 domain. <b>Results:</b> In clinical specimens, USP14 mRNA expression was associated with tumor grade. Exogenous overexpression of USP14 enhanced the survival of cisplatin-resistant IGROV-1/Pt1 cells, but not parental IGROV-1 cells, upon cisplatin exposure. USP14 knockdown by small interfering RNAs in resistant cells reduced aggressive features and restored cisplatin sensitivity, whereas no sensitization was observed in IGROV-1 cells. Medium-throughput screening identified five candidate molecules, among which ARN12502 showed the strongest inhibitory activity against USP14. ARN12502 exhibited an IC₅₀ of 18.4 µM, and molecular dynamics simulations confirmed stable binding in two distinct modes. In proteasome sensor-expressing cells, ARN12502 displayed proteasome-inhibitory activity. <b>Conclusion:</b> USP14 contributes to the aggressiveness of ovarian carcinoma, particularly to the cisplatin-resistant phenotype, and represents a relevant promising druggable target. ARN12502 serves as a starting point for chemical optimization toward the development of more potent USP14i.