WEHI-092 is a potent and selective inhibitor of USP9X with IC50 of 254 nM, directly binds to USP9X catalytic domain with SPR KD of 1 uM, does not inhibit USP24 and other DUBs.
Incubation of the USP9X catalytic domain with 50 µM WEHI-092 inhibited cleavage of K48-linked di-ubiquitin and prevented the modification of USP9X with ubiquitin propargylamide (Ub-PA), a suicide substrate that covalently modifies the catalytic Cys1566 of USP9X.
WEHI-092 (50 uM) does not affect any of 49 human DUBs with the exception of USP9X.
WEHI-092 binds within the Fingers subdomain of USP9X.
WEHI-092 is cytotoxic in a small subset of cancer cell lines, destabilises the centrosomal protein CEP55 with IC50 of 1.3 uM.
WEHI-092 is non-lethal and non-toxic to the non-cancerous human cell lines studied (HUVECs and HDFs).
WEHI-092 (10 uM) caused 50% cell growth inhibition, including stalling cell growth completely in MOLT-4 and SK-MEL-2 cells.
WEHI-092 (10 uM) also led to reduced protein levels of CEP55, MKRN2, CEP131 and PCM1, in MDA-MB-231 cells.
WEHI-092 caused arrested mitosis in MDA-MB-231, MiaPaca2, and SK-MEL-2 cells.