Figure 1. NNU219 and bortezomib differentially affect proteasome activities in vitro and in vivo. (A) In vitro effects of NNU219 or bortezomib on catalytic activities of the human constitutive proteasome (β1c, β2c, β5c) and the immunoproteasome (β1i, β2i, β5i). Data from at least three independent measurements were normalized to DMSO-treated controls and were presented as residual activities ± SD. (B) Inhibition of non-proteasomal proteases. NNU219 and bortezomib were tested at 10 μM against a panel of purified serines (Cathepsin G, Factor XIIa, Elastase), cysteine (Cathepsin B), aspartyl (Renin) and metallo (ACE) proteases. Percent inhibition was calculated based on the activities of compounds on protease subtracted with a substrate control without an enzyme. Data were presented as the mean inhibition ± SD relative to DMSO-treated controls (*, p < 0.5). (C) Selectivity of NNU219 in the active sites of human MM cell lines. MM cells were treated with various concentrations of NNU219 for 1 h and cytosolic extracts were analyzed for CT-L, C-L and T-L proteasome activities. Results were represented as percent activities of proteasome in drug-treated vs. vehicle treated cells ±SD. (D) Recovery of cellular proteasome activity following NNU219 or bortezomib treatment. Proteasome CT-L activity was determined in lysates prepared from RPMI 8226 (left panel) and ARH77 cells (right panel) at the indicated times following exposure to IC50 of NNU219 or bortezomib for 1 h. Mean values from three measurements are presented as the percent activity relative to control-treated cells ± SD (*, p < 0.5; ***, p < 0.001). (E) Proteasome active site selectivity of NNU219 in vivo. Mice (n=5) were treated with either NNU219 (0.2 mg/kg or 0.4 mg/kg i.v.) or bortezomib (1 mg/kg i.v.) for 1 h and whole blood was analyzed for CT-L, C-L and T-L proteasome activities. (F) Inhibition and recovery of proteasome activity in vivo. Mice (n=5) were treated with NNU219 (0.4 mg/kg i.v.), NNU546 (2 mg/kg i.g.) and bortezomib (1 mg/kg i.v.) at the indicated time-points and blood samples were analyzed for CT-L proteasome activities. The data are represented as the percent inhibition compared with vehicle treated animals from two independent experiments. (G) ARH77 tumor-bearing mice were administered a single dose of NNU219 (0.4 mg/kg i.v.), NNU546 (2 mg/kg i.g.) or bortezomib (1 mg/kg i.v.); mice were euthanized at 1 h and 24 h time points after treatment. Heart, brain and tumor were harvested. Protein extracts were prepared and the proteasome catalytic activity was evaluated with CT-L subunit-specific fluorescent peptide substrates. Values were presented as the mean percent activity relative to vehicle ± SD (3 mice per time-point). p values presented for bortezomib vs NNU219 or NNU546 (*, p < 0.5; **, p < 0.01; ***, p < 0.001). SD, standard deviation; i.v. intravenous administration; i.g. intragastric administration; CT-L, chymotrypsin-like; C-L, caspase-like; T-L, trypsin-like; MM, multiple myeloma.