Compounds inhibit proteasome function in vitro and vivo.
(A) SKOV3 cells were treated with compounds for 4 h and analyzed by Western blot with antibody to ubiquitin to assess the accumulation of high molecular polyubiquitinated proteins associated with proteasomal inhibition. (B) Fold increase in bioluminiscence of ES2 cells stably expressing 4UbFL at 4 h post-treatment of compounds, as measured by luminometer. (C & D) Leg muscle was electroporated after i.m. injection with 4UbFL plasmid. 3 days later IVIS200 imaging was performed upon injection of D-luciferin to assess the bioluminescence of female CD1 mice leg muscle before (0 h) and 4, 24, 48, and 72h after drug treatment. Increased bioluminescence was observed after treatment with RA413S; the first dose utilized PEG formulation, and the second dose 72 h later used a b-HPCD formulation. (C, figure showing fold increase) or RA414 or bortezomib (D, figure showing bioluminescence units for individual mice).