PR-619

Deubiquitinases (DUBs) play a critical role in the regulation of various cellular processes, such as protein homeostasis and signaling, rendering them attractive drug targets. However, the generation of reagents for measuring DUB activity typically involves several steps and is not straightforward. Here, we report the development and characterization of a novel fluorescent polarization assay using an isopeptide bond substrate mimetic (IsoMim) that can be made recombinantly in high yields. The IsoMim assay was able to discern the differential activity of ubiquitin-specific protease family members (USP4, USP15, USP11, and USP2), the ubiquitin C-terminal hydrolase UCHL3, and the Machado-Joseph Domain deubiquitinase JOSD2. A competition assay format of the assay was developed that discerned differences between the close paralogues USP15, USP4, and USP11 in interacting with mono-ubiquitin, the isopeptide mimetic ubiquitin-GGG, and the C-terminal truncation variant ubiquitin (1-74). Moreover, dose-response curves and associated pIC50 values using the broad-spectrum inhibitor PR-619 confirmed differential inhibition in the low μM range for four tested DUBs. The successful discrimination of DUB activity and inhibition and the easily scalable generation of the substrate make the IsoMim assay method applicable for high-throughput screening (HTS). This was ascertained in a "pseudo HTS screen" for USP4 inhibitors in which PR-619 was successfully identified as a "pseudo hit." The developed assay provides a valuable tool for probing DUB activity and the identification and characterization of DUB inhibitors and has the potential to accelerate drug discovery efforts in this area.

Targeting oncogenic ubiquitin-specific proteases (USPs) has been regarded as a potential useful strategy for cancer treatment. In the current study, we aimed to screen candidate small molecule target oncogenic USPs in osteosarcoma. Initially, analysis of public datasets revealed that USP3 is significantly upregulated in osteosarcoma tissues. Functional studies further demonstrated that USP3 promotes osteosarcoma cell proliferation and tumor growth both in vitro and in vivo. Through structure-based virtual screening, we identified Vitamin B12 as a potential USP3 inhibitor. Biochemical assays confirmed that Vitamin B12 effectively suppresses USP3 activity at a level comparable to PR619, a broad-spectrum deubiquitinase inhibitor. Furthermore, Vitamin B12 mitigates the oncogenic effects of USP3 in osteosarcoma by inhibiting its deubiquitinase activity. In conclusion, our findings offer new insights into the role of USP3 in osteosarcoma and underscore the potential of Vitamin B12 as a promising therapeutic agent.