Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11

Abstract

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond, and those that do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop novel drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs less activity towards metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.

Discovery of an Inhibitor of the Proteasome Subunit Rpn11

“…Incubation of this substrate, Ub 4 peptide OG , with proteasome resulted in depolarization of Oregon Green fluorescence due to release of the peptide OG from Ub 4 . 25 Initial evaluation of the MBP library revealed three compounds with >50% inhibition, with the majority of the compounds exhibiting 0-30% inhibition (Figure 2). One fragment ( 8TQ , Figure 1) demonstrated essentially complete inhibition at a concentration of 200 μM.…”

“…The findings of the synthetic campaign described in this report prompted an extensive biological study that is described elsewhere. 25 This work opens the door to development of a novel class of proteasome inhibitors for cancer chemotherapy.…”

A Bioinorganic Approach to Fragment-Based Drug Discovery Targeting Metalloenzymes

“…In collaboration with Raymond Deshaies and co-workers,[22,27] we screened the CFL library against Rpn11. Indeed, a biochemical screen of ~100 compounds revealed a single, highly active hit 8-thioquinoline ( 8TQ , Figure 5).…”

“…[22] Upon optimizing the spacer length between the MBP and the heterocycle an inhibitor given the name ‘capzimin’ was identified. [27] Capzimin has an IC 50 value of 340 nM against Rpn11 and shows good selectivity over related Zn-dependent proteases. More importantly, capzimin induced accumulation of polyubiquitinated substrates (consistent with proteasome inhibition) and showed antiproliferative activity against several cancer cell lines, was confirmed to induce apoptosis in some of these cell lines, and showed activity against cell lines that were resistant against proteasome inhibitors that target the 20S CP (consistent with a novel mechanism of proteasome inhibition, i.e., Rpn11 inhibition in the 19S RP).…”

“…More importantly, capzimin induced accumulation of polyubiquitinated substrates (consistent with proteasome inhibition) and showed antiproliferative activity against several cancer cell lines, was confirmed to induce apoptosis in some of these cell lines, and showed activity against cell lines that were resistant against proteasome inhibitors that target the 20S CP (consistent with a novel mechanism of proteasome inhibition, i.e., Rpn11 inhibition in the 19S RP). [27] This is just one of many case studies of how FBDD using an MBP library has been used to successfully identify a novel metalloenzyme inhibitor. Capzimin is particularly notable as large scale HTS failed to produce viable lead compounds, but our FBDD approach resulted in a first-in-class inhibitor of the high value target Rpn11.…”