Structure of CEP-18 770 and analyses of CEP boronic acid proteasome inhibitors in human multiple myeloma (MM) cells. (A) Structure of CEP-18770; (B) Cellular proteasome inhibitory profile in cell lysates obtained from RPMI-8226 MM incubated with increasing concentrations of bortezomib, CEP-18770, and a related analog, compound 19 (Dorsey et al, manuscript submitted). Inhibition was measured using fluorogenic substrates for the different catalytic activities of the proteasome. Results were plotted as percentage inhibition compared with nontreated lysates. Experiments were performed in triplicate. Error bars represent SD. (C) Ubiquitination assay: RPMI-8226 cells were treated for 24 hours with compounds 17, 24, 19 described previously (Dorsey et al, 2007) as well as CEP-18770 and bortezomib at the indicated concentrations; the accumulation of ubiquitinated proteins induced by proteasome inhibitors was assayed by Western blotting (WB) with antiubiquitin antibody. α-tubulin protein expression was included for protein loading normalization. (D) RPMI-8226, U266, and K562 cells were treated for different times with 20 nM CEP-18770 and 10 nM bortezomib (BOR); accumulation of ubiquitinated proteins was assayed by WB as described.