Rpn11 is not the only potential drug target in the 19S RP. The first reported inhibitor of the 19S RP was ubistatin, which blocks the binding of ubiquitin chains to their receptors [
151]. Ubistatins A and B compete for the polyUb receptor Rpn10 and the shuttle protein Rad23 for their interactions with polyUb chains (
Table 3 and
Figure 3) [
151]. As a result, ubistatins inhibit protein degradation in vitro [
151]. More recently, a structural study revealed an interaction between ubistatin and ubiquitin. Ubistatin directly interacts with a hydrophobic patch and the surrounding basic/polar residues on the ubiquitin surface. Ubistatin shows a strong preference for K48 linkages over K11 and K63 linkages due to differences in the conformation and distribution of the hydrophobic patches on the surfaces [
152]. The major challenge to further develop ubistatins as therapeutics is their negative charge, which precludes efficient membrane penetration, thereby limiting their potency in cells.
Another polyUb receptor in the 19S RP, Rpn13, is an attractive target for the development of alternative proteasome inhibitors [
39]. Rpn13 and the deubiquitinating enzyme that it activates, Uch37, have been found to be important for cell cycle progression in cell culture [
153], and increased expression of the gene encoding Rpn13 (
ADRM1) has been identified in several forms of cancer [
154,
155,
156,
157]. Multiple efforts have been put forth to look for Rpn13 inhibitors. RA190, a bis-benzylidine piperidone, was the first identified inhibitor of Rpn13. It was believed to covalently interact with Cys88 of Rpn13 and block the binding of polyUb substrates (
Table 3) [
158]. RA190 induces the accumulation of polyUb proteins, causes ER stress, and stabilizes proteasome substrates, including p53 [
158]. It showed anti-tumor effects in mouse models for MM and ovarian cancer [
158]. A subsequent study suggests that RA190 has two synergistic mechanisms of action: targeting Rpn13 when it is not bound to the proteasome (at Cys88), and inactivating Uch37 at the proteasome [
159]. Recently, an improved compound with higher potency, RA183, was developed and tested in mice [
160]. Similar to RA190, RA183 is an irreversible inhibitor targeting Cys88 of Rpn13 and inhibits tumor growth in a xenograft mouse model of ovarian cancer (
Table 3) [
160]. In addition to RA190/RA183, a peptoid inhibitor, KDT-11, was identified as a Rpn13 inhibitor that binds with a modest K
d of 1.7 µM (
Table 3) [
161]. KDT-11 causes ubiquitin accumulation and acts synergistically with bortezomib in treating MM cells. KDT-11 is a reversible inhibitor that does not compete with RA190, indicating a different binding mechanism [
161]. It should be noted that “enones” like RA190 have been described as “pan-assay interference compounds,” or PAINS, which raises concern about their specificity [
162]. However, demonstrated examples of ways to circumvent the specificity problem with thiol-reactive compounds are described below (
Section 3.2.2). Irrespective of the type of inhibitor, further support for inhibiting Rpn13 comes from the recent development of a proteolysis-targeting chimeric molecule (PROTAC), WL40, that fuses RA190 with thalidomide, targeting Rpn13 for degradation [
163]. This molecule successfully inhibits MM cell growth in a mouse xenograft model. In fact, the molecule shows improved MM cell death compared to Rpn13 inhibitors. This presents a conundrum, though, because the PROTAC works by targeting Rpn13 for proteasomal degradation, but the effect of the drug is to hamper proteasomal degradation. Nonetheless, this proof-of-principle study shows that there could be some benefit to degrading a proteasomal or proteasome-associated target rather than functionally hindering it. There is a lot of excitement surrounding PROTACS like WL40 and their uses in selectively degrading proteins by linking a ubiquitin ligase to a particular target; the advantages and disadvantages of this strategy are reviewed elsewhere [
164,
165,
166].
Table 3.
Inhibitors of non-20S UPS components.
Name | Target | IC50/Kd | Mechanism |
---|
Capzimin | Rpn11 | 0.3 µM (In vitro Rpn11 assay); 0.6 µM (UbG76V-GFP degradation assay in cells) | Chelating the zinc ion in Rpn11, inactivating its DUB activity [40] |
Ubistatin B | polyUb | 10 µM (CFTR ubiquitination assay in cells) | Binding to the polyUb chain and blocking the substrate/Ub receptor interaction [151,152] |
RA190 | Rpn13 | Not Applicable | Covalently binding to cysteine residue 88 (Cys88) of Rpn13 when it is not bound to the proteasome and Uch37 at the proteasome [158,159,160] |
RA183 |
KDT-11 | Rpn13 | 1.7 µM (Kd) | Peptoid ligand binding to Rpn13 [161] |
RIP-1 | Rpt4 | 3.0 µM (proteasome-mediated stripping of the Gal4-VP16 protein from DNA in vitro) | Inhibiting the protein unfolding activity of the 19S RP [167,168] |