Inhibitor Screen Of USP15/USP21 And The Improvement Of The Dub Activity Detection Assay

Modification of proteins by ubiquitin, the so-called ubiquitylation, is one of the most important post translational modifications of proteins. The ubiquitylated proteins can hydrolysis off their ubiquitin with the action of deubiquitinating enzyme. In the past 5 to 10 years, many researches show that the deubiquitinases being implicated in various physiological processes, and their dysfunctions are associated with many diseases, including oncology, neurodegeneration, haematology and infectious deseases. The hunt for deubiquitinase antagonizes is thus actively carried out by pharmaceutical companies and academics. The ubiquitin-specific protease USP15 is reported that can regulate the TGF-β signaling, promote the development and progression of tumor. USP15 also stabilized MDM2, which in turn negatively regulated T cell activation, as well as regulated p53 function, and inhibition its activity can induce the apoptosis of tumour cells, as well as the antitumor T cell responses. Thus targeting USP15 has being identified as a potential therapy for cancer. Furthermore, USP15 inhibition has also been reported as a therapeutic strategy for Parkinson’s disease (PD) cases caused by reduced Parkin levels. But, until now, no selective USP15 inhibitor has been discovered. Although PR-619 can effectively inhibit the activity of USP15, PR-619 is a broad-spectrum deubiquitinase inhibitor, its low specificity greatly limited its applications. USP21 has been reported can down regulate the NF-K B signaling pathway, as well as the RIG-I-mediated interferon signaling. And USP21 has also been reported can remove ubiquitin from histon H2A resulting in regulation of transcriptional initiation, can stabilize GATA3 which further increase the presence of FOXP3 in regulatory T cells (Tregs). In addition, our laboratory has found that USP21 plays an important role in inherent immunity and the maintenance of stem cells’stemness. However, like the USP15, the pan-inhibitor PR-619 can inhibit the USP21’s activity, but it is lake of specificity. Until now, there isn’t a selective inhibitor targeted USP21 has been reported. In this study, we use the Ub-Nanoluc deubiquitinase activity detection assay to screen the inhibitor of USP15 and USP21, and we proved the small molecules we found can inhibit the activity of USP15 and USP21 in vitro respectively.For the purpose of finding the inhibitor of deubiquitinase, there are many kinds of DUB assays to assessing deubiquitinase activity. Our laboratory use a new, steady, and high sensitive reporter Nanoluc to set up a deubiquitinase enzyme activity detection method, but because of the solid phase carrier used in this method is Ni-NTA beads, its operation is very complicated. In order to simplify the operation of this method, increase its throughput, in this study, we changed the carrier to Ni-coated 96-well white plate. The improved method has an easier operation, and is more suitable for high throughput screen.Above all, in this study, we have screened the inhibitors targeted USP15 and USP21 respectively, and demonstrated their inhibitions in vitro. And then, we improved the method we used to screen inhibitors to make it easier.