Synthesis and structure-activity relationships of USP48 deubiquitinylase inhibitors

Ubiquitin-specific proteases represent a family of enzymes that catalyze the cleavage of ubiquitin from specific substrate proteins to regulate their activity. USP48 is a rarely studied USP, which has recently been linked to inflammatory signaling via regulation of the transcription factor nuclear factor kappa B. Nonetheless, a crystal structure of USP48 has not yet been resolved and potent inhibitors are not known. We screened a set of 14 commercially available USP inhibitors for their activity against USP48 and identified the USP2 inhibitor "ML364" as a candidate for further optimization. Using a ligand-based approach, we derived and synthesized a series of ML364 analogs. The IC₅₀ concentrations of the new compounds to inhibit USP48 were determined in a deubiquitinylase activity assay by measuring the fluorescence intensity using tetra-ubiquitin rhodamine110 as substrate. A compound containing a carboxylic acid functionalization (17e) inhibited USP48 activity toward tetra-ubiquitin rhodamine110 with an IC₅₀ of 12.6 µM. Further structure-based refinements are required to improve the inhibition activity and specificity.