Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily

Abstract

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.

Maintaining protein stability of ∆Np63 via USP 28 is required by squamous cancer cells

“…Recently, small‐molecule inhibitors of ubiquitin‐specific proteases, including USP28, have become available (Lamberto et al , ; Wrigley et al , ). Therefore, we tested the available inhibitor AZ1 to target USP28 activity and, subsequently, to deregulate ∆Np63 protein stability in SCC.…”

“…Therefore, inhibiting the catalytic activity of USP28 is likely to be a suitable mechanism to target ∆Np63 in SCC tumours. That the DUB family presents a viable therapeutic option was tested by using a pan‐DUB inhibitor, PR‐619, as well as the USP25/USP28 dual‐specific inhibitor AZ1 (Wrigley et al , ), in cell culture as well as in vivo . SCC cells exposed to the pan‐inhibitor PR‐619 showed a strong decrease in ∆Np63 levels.…”

The USP28-ΔNp63 axis is a vulnerability of squamous tumours

“…Since available USP28 inhibitors (Wrigley et al, 2017) are not yet suited for in vivo application, we used genetic tools to interrogate the role of USP28 in induction and maintenance of SCC.…”

“…SCC cells exposed to PR-619 showed a strong decrease of ΔNp63 levels. USP25/28 dual specific as well as specific inhibitors for other DUBs have been identified and first-generation inhibitors have been reported (Wrigley et al, 2017). Given the crucial dependency of SCC tumour from different tissues on ΔNp63, including those of head-and-neck, cervix, oesophagus, vulva, skin and lung, targeting the dependence of ΔNp63 on USP28, revealed in this study, is a promising strategy to target this tumour entity independent of tissue origin.…”

Inhibition of USP28 overcomes Cisplatin-Resistance of Squamous Tumors by Suppression of the Fanconi Anemia Pathway

“…To address the potential involvement of USP28 in chemoresistance, we assessed the impact of USP28 depletion on DDR protein abundance. USP28 was thus either silenced in A431 cells by an inducible shRNA sequence, or the cells were treated with AZ1, a dual specific USP25/28 inhibitor (Wrigley et al, 2017). Changes in protein abundance were measured by whole proteome mass spectrometry ( Figure S3C and S3D).…”