High-throughput Screening of Usp14 Inhibitors

For expression in mammalian cells we employed full-length Usp14 (Usp14-wt) and its splice variant lacking exon 4 (Usp14-SF) subcloned into pcDNA3.1 (Invitrogen) as previously described19 (link). The Usp14-C114A and Usp14-ΔUBL constructs were generated in the same vector by PCR-mediated mutagenesis. Human proteasomes were affinity-purified on a large scale from a stable HEK293 cell line harboring HTBH-tagged hRpn11 (a kind gift from L. Huang). 10 μl of Usp14 was dispensed into 384-well low volume plates in duplicate using a Wellmate dispenser. 33.3 nl of compound from the library was transferred into the wells using a Seiko pin transfer robotic system, followed by preincubation for ~30 min. To initiate the reaction, 10 μl of VS-proteasome plus Ub-AMC mixture was added to each well. The sources of compound libraries for screening were as follows: Maybridge, Asinex, ActiMol TimTec, ChemBridge, ChemDiv, Enamine, and MMV1. Primary hits were defined by ‘robust’ Z-score analysis (Supplementary Fig. 7). To obtain dose-response curves, curve fitting was performed by the four parameter logistic model or the three parameter fixed bottom model using SigmaPlot 9.0 according to guidelines from NIH Chemical Genomics Center. The gene trap allele, usp14^rrk114 (ref 19 (link)), is referred to here as usp14^−/−. For additional details see Supplementary Information.