Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound

Abstract

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.

Discovery and Mechanistic Elucidation of NQO1‐Bioactivatable Small Molecules That Overcome Resistance to Degraders

“…Importantly, the initial comparative chemical screening was the key to uncovering this vulnerability, which would not have been identifiable through regular CRISPR synthetic lethality screening. Several small molecules with a degree of chemical analogy to RBS‐10 have recently been reported and linked by some means to NQO1, in some cases as potential inhibitors [56,58–59] . Our findings with RBS‐10 support the study of the mechanistic role that NQO1 may play in facilitating the toxicity of those small molecules through redox‐activation.…”

“…Several small molecules with a degree of chemical analogy to RBS-10 have recently been reported and linked by some means to NQO1, in some cases as potential inhibitors. [56,[58][59] Our findings with RBS-10 support the study of the mechanistic role that NQO1 may play in facilitating the toxicity of those small molecules through redox-activation.…”