RA-9 is a potent and selective proteasome-associated inhibitor of deubiquitinating enzymes (DUBs) with a favorable toxicity profile and anticancer activity, particularly effective in inducing apoptosis in ovarian cancer cell lines.

The characterization of RA-9 as a small-molecule inhibitor of proteasome-associated DUBs.?Treatment with RA-9 selectively induces onset of apoptosis in ovarian cancer cell lines and primary cultures derived from donors.?Loss of cell viability following RA-9 exposure is associated with an unfolded protein response as mechanism to compensate for unsustainable levels of proteotoxic stress.

In vivo treatment with RA-9 retards tumor growth, increases overall survival, and was well tolerated by the host.

Mice were inoculated i.p. with 100,000 ES-2 cells (in 100 μl DMEM) stably expressing GFP.?When tumor was detectable (approx.?6 days post inoculation), mice were randomly assigned into two groups receiving RA-9 or 0.9% saline.?Treatment with RA-9 was given i.p. on a one-day on, two-days off schedule.?The control group received the vehicle alone at the same schedule.?Working concentrations of RA-9 (10 mg/ml) were reached by dissolution in Cremophor EL and polyethylene glycol 400 (Sigma).?Prior to each injection RA-9 was further diluted in 0.9% saline (working concentration 1mg/ml).?To monitor for tumor growth, RA-9 treated and control mice were imaged with an IVIS SpectrumCT Pre-clinical in vivo imaging system (PerkinElmer) every other day.?Animals were sacrificed when abdomens became distended to twice normal size.