Quinoxaline-based UCHL5 inhibitor as a next-generation proteasome-targeting anticancer agent beyond 20S inhibition

Quinoxaline-based small molecule DK-7 inhibits proteasomal deubiquitinase UCHL5, inducing ER stress and apoptosis, and effectively overcomes bortezomib resistance in multiple myeloma, highlighting its therapeutic potential. • DK-7 induces ER stress/UPR activation by accumulating Ub-proteins in cancer cells. • DK-7 inhibits 19S proteasomal deubiquitinase UCHL5 without affecting 20S activity. • DK-7 inhibits UCHL5 activity by direct binding. • DK-7 induces cellular apoptosis and exerts anti-cancer effects by inhibiting UCHL5. • DK-7 overcomes bortezomib-resistance in multiple myeloma. Cancer cells rely on the endoplasmic reticulum (ER) stress response and the unfolded protein response (UPR) to maintain protein homeostasis. However, prolonged activation of these pathways can lead to apoptosis. The ubiquitin–proteasome system (UPS) is a key regulator of protein balance and is often upregulated in tumors. Current proteasome inhibitors, which target the 20S catalytic core, have shown limited efficacy in solid tumors, and resistance often emerges, highlighting the need for alternative strategies. To identify new quinoxaline-derived small molecules that exhibit anticancer properties through selective inhibition of the proteasomal deubiquitinase UCHL5, a component of the 19S regulatory particle. This study also aimed to assess their potential to overcome resistance to the proteasome inhibitor bortezomib. A chemical library of quinoxaline derivatives was screened for compounds that induce ER stress. Enzyme assays, biophysical binding, molecular docking, and mass spectrometry were used to characterize the target of the lead compound, DK-7. The anticancer activity of DK-7 was assessed in various cancer cell lines, including bortezomib-resistant multiple myeloma (MM), and in xenograft mouse models. The quinoxaline derivative DK-7 was identified as a potent and selective inhibitor of UCHL5. Unlike conventional proteasome inhibitors, DK-7 did not inhibit 20S subunits but promoted accumulation of ubiquitinated proteins, induced ER stress, and activated apoptotic signaling. DK-7 significantly reduced cell viability in solid tumor and bortezomib-resistant MM cells. Furthermore, oral administration of DK-7 markedly suppressed tumor growth in HCT116 and bortezomib-resistant MM xenografts models. DK-7, a quinoxaline-based UCHL5 inhibitor, selectively triggers ER stress and apoptosis, effectively overcomes bortezomib resistance, and exhibits antitumor activity in solid tumors. These findings highlight UCHL5 as a promising therapeutic target and suggest quinoxaline-based DUB inhibitors as a next-generation proteasome-targeted strategy with broad clinical potential.