In Vitro and In Vivo Proteasome Activity Profiles of Bortezomib and a Novel Proteasome Inhibitor NPI-0052.

Proteasome inhibition is an effective anti-cancer therapy. Proteasome function is mediated by three catalytic activities: chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L). Kinetics of inhibition of catalytic activities may define the pharmacologic utility of proteasome inhibitors. Here we utilized two structurally distinct proteasome inhibitors Bortezomib, a dipeptide boronic acid; and a non-peptide proteasome inhibitor NPI-0052 to determine their effect on proteasome activities in vitro and in animal model. Examination of the proteasome activity using human erythrocyte 20S proteasomes and fluorogenic substrates shows that NPI-0052 and Bortezomib inhibit all three proteasome activities, albeit at different concentrations: NPI-0052 inhibits CT-L and T-L activities at lower concentrations than Bortezomib (NPI-0052: EC₅₀ = 3.5 ± 0.3 nM versus Bortezomib: 7.9 ± 0.5 nM for CT-L activity; and NPI-0052: EC50 = 28 ± 2 nM versus Bortezomib: EC₅₀ = 590 ± 67 nM for T-L activity); in contrast, higher concentrations of NPI-0052 than Bortezomib are required to inhibit C-L activity (NPI-0052 EC₅₀ = 430 ± 34 nM versus Bortezomib: EC₅₀ = 53 ± 10 nM for C-L activity). We next compared the effects of NPI-0052 and Bortezomib on all three proteasome activities in vivo. Mice were treated with a single MTD dose of NPI-0052 (0.15 mg/kg i.v) or Bortezomib (1 mg/kg i.v); blood samples were collected at 90 mins, 24h, 48h, 72h, or 168h; and whole blood cells were then analyzed for proteasome activity. NPI-0052 completely inhibited CT-L activity by 90 mins, which was recoverable by 168h; whereas Bortezomib-inhibited CT-L activity is recoverable at 24h. T-L activity is significantly inhibited by NPI-0052 at 90 mins, 24h, 48h, and 72h; and is recoverable by 168h; in contrast, Bortezomib enhances T-L activity. Finally, NPI-0052 inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and this activity recovered at 168h, whereas Bortezomib significantly inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and is similarly recoverable at 168h. We next utilized a novel methodology to measure proteasome activity by immunoblotting using dansylAhx3L3VS as a probe (Berkers et al., Nature Methods, 2005), which also allow for determining subunit specificity of a proteasome inhibitor. Multiple myeloma (MM) cells were cultured in the presence or absence of various concentrations of either NPI-0052 (2 nM; 7 nM: IC₅₀; or 20 nM) or Bortezomib (2 nM; 5 nM: IC₅₀; or 20 nM). Competition experiments between either NPI-0052 or Bortezomib and dansylAhx3L3VS revealed that NPI-0052 (7 nM) markedly inhibits the CT-L activity represented by beta-5 subunit of the proteasome and decreased the dansylAhx3L3VS-labeling of the beta-1 (C-L activity) and -2 (T-L activity) subunits. Slightly higher concentrations of Bortezomib are necessary to markedly inhibit beta-5 and -1 subunits, whereas beta-2 subunits are not inhibited. Importantly, both agents trigger apoptosis in MM cells; however, NPI-0052 is remarkably less toxic to normal lymphocytes than Bortezomib. Our data show that NPI-0052, like Bortezomib, targets the proteasome, but triggers a proteasome activity profile distinct from Bortezomib. The mechanistic insights gained from these studies will allow for improved drug design based on targeting specific proteasome subunits.