lts 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 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. Although many kinds of small molecular PIs with various chemical structures have been developed, this is the first demonstration of the proteasome-inhibitory activity of HPDs. In addition, most of the previous PIs mainly acted on one or two catalytic subunits and their mechanisms of action are not fully understood. In contrast, we have demonstrated that HPDs act on all three catalytic subunits of the proteasome by direct binding to the active pockets of the ?1, ?2 and ?5 subunits with a similar binding mode and AZD1152-HQPA kinetics. These results indicate the unique features of homopiperazine-derived PIs in chemical structures and effects on the proteasome. Moreover, we have identified the critical chemical structure of homopiperazine-derived PIs; therefore, these observations may contribute to the development of novel PIs with higher activity and specificity. The high concentrations to trigger cytotoxicity might be the obstacle for clinical application of K-7174. Crystal structure analyses revealed that K-7174 interacts with ? subunits largely via hydrophobic interaction, whereas bortezomib binds to the ?5 subunit via a hydrogen-bond network, explaining why higher concentrations are required for HPDs compared with bortezomib. Therefore, the development of novel HPDs with higher activity and specificity is essential for clinical translation. Our finding on the chemical structure of homopiperazine-derived PIs may be of great help in this regard. Despite the great success of bortezomib in the treatment of refractory malignancies such as MM and mantle cell lymphoma, we still intend to develop orally bioavailable PIs with 1187187-10-5 cost distinct mechanisms of action from bortezomi
