Monitoring Target Engagement of Deubiquitylating Enzymes Using Activity Probes: Past, Present, and Future

Abstract

Deubiquitylating enzymes or DUBs are a class of enzymes that selectively remove the polypeptide posttranslational modification ubiquitin from a number of substrates. Approximately 100 DUBs exist in human cells and are involved in key regulatory cellular processes, which drive many disease states, making them attractive therapeutic targets. Several aspects of DUB biology have been studied through genetic knock-out or knock-down, genomic, or proteomic studies. However, investigation of enzyme activation and regulation requires additional tools to monitor cellular and physiological dynamics. A comparison between genetic ablation and dominant-negative target validation with pharmacological inhibition often leads to striking discrepancies. Activity probes have been used to profile classes of enzymes, including DUBs, and allow functional and dynamic properties to be assigned to individual proteins. The ability to directly monitor DUB activity within a native biological system is essential for understanding the physiological and pathological role of individual DUBs. We will discuss the evolution of DUB activity probes, from in vitro assay development to their use in monitoring DUB activity in cells and in animal tissues, as well as recent progress and prospects for assessing DUB inhibition in vivo.

Deubiquitylating enzymes and drug discovery: emerging opportunities

“…DUB substrates can be determined by biochemistry, yeast two-hybrid interactions, proteomic profiling and genetics 232 , but this is often challenging and time-consuming. Clearly, the ability to directly monitor DUB activity within a native biological system is essential to understanding the physiological and pathological role of individual DUBs as well as the effects of DUB inhibition 233 .…”

Recent Advances in the Discovery of Deubiquitinating Enzyme Inhibitors

“…These have now been optimised from low-throughput Western blot assays to much higher throughput versions. Potential inhibitors can be tested in whole-cell and tissue systems (followed by lysing and competing with the noncell-penetrant activity probes) which is more physiologically relevant than an isolated DUB in solution with an arbitrary reducing agent [46,47,[51][52][53].…”

Developing Targeted Therapies That Exploit Aberrant Histone Ubiquitination in Cancer

“…In addition, DUB inhibitors directed against the catalytic thiol group are often non-specific as DUB specificity is regulated by factors independent of the catalytic site (i.e., interactions with the ubiquitin and target protein, and allosteric regulation) [133]. However, new in vitro E3 ubiquitin ligase and DUB activity assays that better model the physiological conditions and reduce false-positive rates may expedite the development of histone E3 ubiquitin ligase and DUB inhibitors [131,134,135,136]. In addition, novel cell-based assays enabling high-throughput in vivo drug screening may facilitate the identification of inhibitors with acceptable pharmacological properties [136,137].…”

“…However, new in vitro E3 ubiquitin ligase and DUB activity assays that better model the physiological conditions and reduce false-positive rates may expedite the development of histone E3 ubiquitin ligase and DUB inhibitors [131,134,135,136]. In addition, novel cell-based assays enabling high-throughput in vivo drug screening may facilitate the identification of inhibitors with acceptable pharmacological properties [136,137]. Furthermore, better characterizing allosteric regulations of histone E3 ubiquitin ligases and DUBs may highlight new targeting opportunities.…”