Abstract
We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome’s 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma.
Highlights
The eukaryotic 26S proteasome is a 2.5MDa complex of 47 proteins arranged in a symmetrical barrel of two central 20S catalytic subunits (CS) capped at either end by 19S regulatory particles (RP) that act together to mediate targeted degradation of proteins via a coordinated multistep process
To further elucidate the pharmacophore and minimal structural features necessary for RPN13 binding and antineoplastic activity, we synthesized a new series of compounds based upon bisbenzylidine ring moieties with various modifications in and around the ring (S1 Table)
Biotinylated RA190 (RA190B, Fig 1A) is utilized as a probe to detect covalent binding to its cellular target(s) [10] in detergent lysates of cancer cell lines
Summary
The eukaryotic 26S proteasome is a 2.5MDa complex of 47 proteins arranged in a symmetrical barrel of two central 20S catalytic subunits (CS) capped at either end by 19S regulatory particles (RP) that act together to mediate targeted degradation of proteins via a coordinated multistep process. The proteasome performs key homeostatic cellular functions, targeted inhibition of the 20S proteasome chymotryptic catalytic subunit PSMB5 is used for the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL), and three such inhibitors have been licensed (bortezomib, ixazomib and carfilzomib). To date, they have failed to demonstrate efficacy against solid tumors, possibly reflecting limited drug assess, and MM and MCL patients frequently develop resistant disease and treatmentrelated peripheral neuropathy [2, 3]. There is considerable interest in addressing the limitations associated with 20S proteasome inhibitors by targeting upstream proteasome activities in the 19S RP including substrate recognition, deubiquitination enzymes (DUBs) and/or ATPdependent unfolding [4]
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