Derivation and characterization of ubiquitin-specific protease 18 inhibitors

Blessing O. Ogunlade; Kevin N. Dalby; Samuel C. Okpechi; Eun Jeong Cho; Liliya Tyutyunyk-Massey; Zibo Chen; Xiuxia Liu; Joseph Ivanic; Brian Luke; Shyamal D. Desai; Yair Alfaro; Ashwini K. Devkota; Rae M. Sammons; Gilbert G. Privé; Xi Liu; Ethan Dmitrovsky

doi:10.1172/jci.insight.202853

Derivation and characterization of ubiquitin-specific protease 18 inhibitors

Blessing O. Ogunlade,¹ Kevin N. Dalby,² Samuel C. Okpechi,³ Eun Jeong Cho,² Liliya Tyutyunyk-Massey,³ Zibo Chen,³ Xiuxia Liu,³ Joseph Ivanic,⁴ Brian Luke,⁴ Shyamal D. Desai,⁵ Yair Alfaro,³ Ashwini K. Devkota,² Rae M. Sammons,² Gilbert G. Privé,⁶ Xi Liu,³ and Ethan Dmitrovsky³

Published May 19, 2026 - More info

View PDF

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. Interferon-Stimulated Response Element (ISRE) reporter assays following β-lapachone treatment activated this reporter. USP18 co-transfection antagonized this activity. β-lapachone treatments increased global ISGylation. RNA sequencing 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.

Derivation and characterization of ubiquitin-specific protease 18 inhibitors

Blessing O. Ogunlade,¹ Kevin N. Dalby,² Samuel C. Okpechi,³ Eun Jeong Cho,² Liliya Tyutyunyk-Massey,³ Zibo Chen,³ Xiuxia Liu,³ Joseph Ivanic,⁴ Brian Luke,⁴ Shyamal D. Desai,⁵ Yair Alfaro,³ Ashwini K. Devkota,² Rae M. Sammons,² Gilbert G. Privé,⁶ Xi Liu,³ and Ethan Dmitrovsky³

Article tools

Metrics

Go to

Derivation and characterization of ubiquitin-specific protease 18 inhibitors

Blessing O. Ogunlade,1 Kevin N. Dalby,2 Samuel C. Okpechi,3 Eun Jeong Cho,2 Liliya Tyutyunyk-Massey,3 Zibo Chen,3 Xiuxia Liu,3 Joseph Ivanic,4 Brian Luke,4 Shyamal D. Desai,5 Yair Alfaro,3 Ashwini K. Devkota,2 Rae M. Sammons,2 Gilbert G. Privé,6 Xi Liu,3 and Ethan Dmitrovsky3

Article tools

Metrics

Go to

Sign up for email alerts

Blessing O. Ogunlade,¹ Kevin N. Dalby,² Samuel C. Okpechi,³ Eun Jeong Cho,² Liliya Tyutyunyk-Massey,³ Zibo Chen,³ Xiuxia Liu,³ Joseph Ivanic,⁴ Brian Luke,⁴ Shyamal D. Desai,⁵ Yair Alfaro,³ Ashwini K. Devkota,² Rae M. Sammons,² Gilbert G. Privé,⁶ Xi Liu,³ and Ethan Dmitrovsky³