Among them Ala49 makes a direct hydrogen bond to bortezomib

Among them Ala49 makes a direct hydrogen bond to bortezomib explaining why this position is most frequently mutated in bortezomib-resistant cells. Furthermore, a Cys52Phe or Met45Val substitution results in a steric clash between the side chains of these two residues, leading to repulsion of bortezomib from the binding pocket. In contrast, these mutations should not affect the affinity of 1644060-37-6 K-7174 to subunit, because the binding site of K-7174 is spatially distinct from the bortezomib-binding pocket. Because K-7174 appears to inhibit proteasome activity with a distinct mode from bortezomib, it is anticipated that K-7174 is effective for bortezomib-resistant cells. Previous studies revealed that a mutation of the PSMB5 gene at nucleotide position 322, which corresponds to the substitution of Ala49 to Thr, induced conformational changes in the bortezomib-binding pocket of subunit and was responsible for acquired bortezomib resistance in T-cell acute lymphoblastic leukemia and myeloid leukemia cells. Recently, Ri reported the establishment of bortezomib-resistant MM cell lines by transduction with G322A-mutated PSMB5 cDNA. Taking the same approach, we established three mutant sublines from RPMI8226 cells lentivirally transduced with mutated PSMB5 along with a marker gene VENUS. As controls, three wild-type sublines were concomitantly established by mock transfection. We selected mutant-5F and WT-9G as representative sublines after determining the sensitivity to bortezomib. The expression levels of VENUS and PSMB5 were virtually identical in these sublines. As expected, sensitivity to bortezomib was significantly lower in the mutant subline than in the WT subline in MTT assays. In striking contrast, K-7174 induced cytotoxicity equally in WT and mutant sublines. In correlation with the results of MTT assays, K-7174 inhibited chymotrypsin-like activity similarly in both sublines, whereas bortezomib could only partially inhibit the activity in the mutant subline. These results were fully reproducible in other WT and mutant sublines. To confirm the effects on proteasome activities, we determined the accumulation of ubiquitinated proteins in these sublines. As shown in Fig. 5D, ubiquitinated proteins were accumulated to a lesser extent in mutant cells than WT cells when they were treated with bortezomib. In contrast, K-7174 similarly induced intracellular BMS-687453 citations protein ubiquitination in WT and mutant sublines. These results suggest that K-7174 can overcome bortezomib resistance. In the present study, we show that HPDs constitute a novel class of PIs with a unique mode of proteasome binding.