Biology subjects

Kooij, R.

Publications and source records attributed to Kooij, R..

3 recordsLinked to original sources

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↗

bioRxiv2019

A Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos

Many reagents have been emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement allowing investigations of the function of individual enzymes in a cellular context. We here report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and whole animals. The probe labels active Ubiquitin Carboxy-terminal Hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1-2% of total brain protein. UCHL1 variants have been linked with the neurodegenerative disorders Parkinsons and Alzheimers disease. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1 specific research tools exist. We show that the reagents reported here are specific for UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 irreversibly in an activity-dependent manner. Its use is demonstrated by labelling of UCHL1 both in vitro and in cells. We furthermore show that this probe can report UCHL1 activity during the development of zebrafish embryos.

biochemistry↗