Derivation and characterization of ubiquitin-specific protease 18 inhibitors.

Abstract

Ubiquitin-Specific Protease 18 (USP18) is a deISGylation enzyme and antineoplastic target. To develop USP18 inhibitors, an enzymatically active human recombinant USP18 protein was engineered suitable for high-throughput screening of ~80,000 chemical compounds. Three of them substantially inhibited USP18 enzymatic activity, with β-lapachone having prominent antineoplastic activity. Independent β-lapachone treatments of murine and human lung cancer cell lines statistically significantly reduced proliferation and increased apoptosis. Gain of USP18 expression antagonized these effects. β-Lapachone treatments statistically significantly repressed lung cancer xenograft growth. β-Lapachone increased reactive oxygen species (ROS), but antineoplastic effects occurred at dosages with negligible ROS production. ROS scavenger treatments did not rescue β-lapachone effects at these concentrations, consistent with an ROS-independent mechanism. IFN-Stimulated Response Element (ISRE) reporter assays following β-lapachone treatment activated this reporter. USP18 cotransfection antagonized this activity. β-Lapachone treatments increased global ISGylation. RNA-seq of lung cancer cells engineered with or without enhanced USP18 expression showed specific pathways affected by β-lapachone treatment. Proteomic analysis of these treated cells revealed known and new ISGylated proteins. In silico modeling identified a unique USP18 pocket where these USP18 inhibitors bind. Engineered mutation of this pocket disrupted β-lapachone activity. Taken together, β-lapachone is an antineoplastic tool compound useful for USP18 inhibitor development.