Pol, V. | bioRxiv Science

bioRxiv2025

Huib32: A Potent and Selective USP32 Inhibitor Modulating Endosomal Processes and Advancing Cell-Permeable USP32 Probes

Deubiquitinating enzymes (DUBs) are pivotal regulators of ubiquitination, a vital post-translational modification essential for cellular processes. Dysregulated DUB activity disrupts cellular homeostasis, driving diseases like cancer and neurodegeneration. Ubiquitin-specific protease 32 (USP32) has emerged as a promising therapeutic target due to its role in endosomal and autophagosomal dynamics and its association with breast, ovarian, and lung cancers. Here, we describe Huib32 (Human deUbiquitinase Inhibitor 32) as a USP32 inhibitor. Cyanimide-containing Huib32 potently and selectively inhibits USP32 by covalently binding to the active site Cys743 in vitro and in cells, enhancing substrate ubiquitination, altering endosomal morphology, and mimicking USP32 depletion. Additionally, we present two activity-based probes (ABPs), Huib32*1 and Huib32*2, which enable precise detection of USP32 activity and confirm probe selectivity via mass spectrometry. Together, Huib32 and its probes represent a unique approach for targeting USP32, offering new research tools and potential therapeutic avenues for cancer and disorders involving endocytic trafficking.

biochemistry↗

bioRxiv2025

High-Throughput Synthesis and Screening of a Cyanimide Library Identifies Selective Inhibitors of ISG15-specific Protease USP18

High-throughput screening (HTS) of (large) compound collections is a critical early step in many drug discovery programs, enabling the rapid identification of lead molecules with desirable biological activity. The success of HTS depends heavily on the quality of the compound libraries used, and as such, the development of targeted libraries has emerged as a promising approach to enhance the effectiveness of HTS efforts. However, the acquisition and synthesis of such libraries remain costly and labor-intensive, often yielding compound quantities far exceeding the small amounts required for screening. To address these challenges, we present a high-throughput synthesis-to-screening method for the efficient inplate generation and immediate HTS of a deubiquitinase (DUB)-focused compound library. Central to our approach is the use of an Echo acoustic liquid handler, which enables precise nanoliter-scale transfers of DMSO-based solutions, facilitating efficient and miniaturized synthesis directly in 1,536-well plates. Using this platform, we constructed a library of 7,536 compounds featuring a DUB-privileged cyanimide warhead and screened it against a panel of twelve DUBs and ubiquitin-like proteases. This identified two structurally related molecules with selective inhibitory activity against the interferon-stimulated gene 15 (ISG15) protease USP18, which we further developed into a first-in-class USP18 inhibitor with 35 nM potency. This compound, BB07CA902, demonstrated exceptional specificity for USP18 across a panel of 41 DUBs and effectively increased ISGylation levels in cells by inhibiting USP18 activity. With our technology, we enabled the efficient preparation of large DUB-targeted cyanimide-based libraries, which will accelerate future DUB inhibitor development.

biochemistry↗