There has been a considerable interest in developing inhibitors targeting the ubiquitin-proteasome system. Here we describe our efforts in identifying novel small-molecule inhibitors that perturb the ubiquitin-proteasome system using GFP-p27 as a reporter protein. Through the screening of a small-molecule compound library, we have discovered that NSC624206 inhibits p27 ubiquitination in vitro and stabilizes p27 expression in cells. Furthermore, we have shown that NSC624206 inhibits the ubiquitin activating enzyme E1. Mechanistically, we have demonstrated that NSC624206 and PYR-41 specifically inhibit the ubiquitin-thioester formation step of the E1 activation reaction without having an effect on ubiquitin adenylation.
Results from the small-molecule screen lead to the identification of 15 compounds that prevented p27 degradation in cells. It is important to note that the mechanism of inhibition is unclear. For example, the compounds used in our screen could directly impair the function of the proteasome, perturb specific events in the ubiquitination pathway, or promote the activity of deubiquitination enzymes. The observed upregulation of the substrate may also be explained on the level of transcriptional upregulation. For example, the compound could inhibit histone deacetylase, as in the case of suberoylanilide hydroxamic acid (SAHA), which leads to transcriptional activation and subsequent upregulation of protein production.23
Thus, although the primary screen does not suggest a mechanism of action of the compounds, it also does not rely on any mechanistic assumption. Instead, these examples demonstrate that assays using reporter substrates are in fact functional assays that recapitulate the intricacy of the system in a single readout. Still, why the cells accumulated p27 in the presence of certain compounds was of interest. Therefore, a secondary in vitro screen that isolated the ubiquitination pathway was applied to the 15 compounds identified from the primary screen. Four of the 15 top hits showed inhibitory potential in the secondary p27 in vitro ubiquitination screen. Careful dissection of the p27 ubiquitination cascade revealed that one of the four identified compounds, NSC624206, had in vitro inhibitory activity against the ubiquitin activating enzyme E1. Degradation of p27Kip1 is known to be regulated by several cellular signaling pathways and ubiquitin E3 ligases. In this study, we focused on the characterization of NSC624206. The mechanism(s) of inhibition of p27Kip1 ubiquitination by the other three compounds identified needs to be further characterized in future studies. It is possible that these compounds may have inhibitory effects on other components of p27 ubiquitination such as E2, the E3 ligase specific to p27, or a component of the E3 ligase.
Whereas the catalysis of ubiquitin adenylation was unaffected, as measured by the [P]-PPi:[γ-32P]-ATP exchange assay, the production of AMP was greatly decreased, indicating that NSC624206 prevented the formation of the thioester bond between ubiquitin and the active site of the E1. Hence, we can rationalize that it is the nucleophilic nature of the active site cysteine that promotes an electrophilic attack on one of the sulfur atoms of the compound, which induces the displacement of the compound via a thiol-disulfide exchange reaction and ultimately leads to loss of E1 enzymatic activity (). This model is consistent with the observation that 2, which excludes the disulfide, failed to inhibit E1. Although this is a plausible explanation of the mechanism of E1 inhibition by NSC624206, we cannot rule out that NSC624206 binds to a nonactive site of E1 and prevents conformation changes associated with catalysis. To confirm , several studies could be carried out including the co-crystallization or mass spectrometry analysis of E1-NSC624206 conjugates. Future studies are necessary to distinguish the site of NSC624206 binding.
Figure 8 Proposed reaction mechanism of 1 with E1. Reaction of disulfide compound 1 with the active site cysteine of E1 causes the release of p-chlorobenzyl thiol (R1) and decylamino ethane thiol-E1 (R2), which results in inhibition of the enzyme. It is also possible (more ...)
In general, the thiol of cysteine residues can participate in oxidation reduction reactions. Also, cysteine can coordinate metal ions, as seen in zinc finger proteins, or it can react with electrophiles due to its nucleophilic character. Modification of cysteine by all of these processes strongly alters the properties of the related protein, which may be exploited in designing pharmacological agents. For example, several compounds that target tubulin, a protein whose cysteine residue upon oxidation prevents tubulin from polymerizing,24
have been reported to covalently modify Cys-239 via a nucleophilic aromatic substitution reaction while exerting a cytotoxic effect in many cancers.25
In addition, the asymmetric 1-methylpropyl-2-imidazolyl disulfide (IV-4), also known as PX-12, inhibits the small redox protein thiore-doxin-1 (Trx-1) through irreversible thioalkylation of a critical cysteine located outside of the catalytic site of the enzyme.26
Trx-1 is overexpressed in tumors; it promotes growth, apoptosis, and angiogenesis and is associated with decreased patient survival.27
It is encouraging to report that PX-12 has progressed into phase II clinical trials for the treatment of advanced pancreatic carcinoma. Further findings about PX-12 suggest that this compound also affects tubulin polymerization and causes inhibition of papain and ficin, cysteine proteases, indicating a wider range of PX-12 activity.28
Numerous cellular proteins rely on their active cysteines to function properly, so it is not surprising that PX-12 or the similarly configured disulfide compound presented here could engage in nonspecific activities. This should not undervalue the usefulness of such compounds, as indicated by the early clinical trial success of PX-12, thus validating this approach for drug discovery. Given these precedents, there is a possibility that NSC624206 could be further developed as a lead compound for E1 therapeutics in vivo.
A few ubiquitin E1 inhibitors have been reported in the literature. Panepophenanthrin, a natural compound derived from the mushroom strain Panus rudis
, inhibits E1 in vitro with an IC50
Himeic acid A, derived from the marine fungus Aspergillus
, has also been shown to inhibit E1 in vitro with IC50
PYR-41 inhibits ubiquitin E1 both in vitro and in cells.12
of PYR-41 is around 5 μM and thus more potent than the compounds described here. However, the exact mechanism of panepophenanthrin, himeric acid A, and PYR-41 inhibition is not known. Our findings suggest that PYR-41 blocked the formation of the thioester linkage between the active site cysteine of E1 and ubiquitin while not disturbing the release of PPi during adenylation. During the characterization of PYR-41, Yang and coworkers12
also suggested that PYR-41 could potentially link to the E1 through its active site cysteine, as the addition of excess reduced glutathione eliminated restored E1 activity. Given the structure of PYR-41, it is tempting to invoke inhibition by hetero-conjugate addition of an E1 cysteine residue to the α,β-unsaturated pyrazolidinedione.
Our experimental results suggest that NSC624206 is not simply a thiol-reacting reagent. There is considerable selectivity of this compound in targeting the ubiquitin E1 enzyme. NSC624206 does not significantly inhibit ubiquitin conjugating enzymes (E2s), which also have catalytic Cys residues that accept activated ubiquitin via a thioester bond. However, not all E2s are insensitive to NSC624206. For example, Cdc34 is modestly inhibited by NSC624206. There are four classes of E2 enzymes. Classification of E2s is based on whether there are additional extensions to the catalytic core.31
UbcH5a and UBE2G2 (UBC7) belong to class I, which contains only the catalytic domain. Cdc34 is a class III E2 with C-terminal extensions. Based on the structure of yeast ubiquitin E1 and E1-E2 interfaces of other ubiquitin-like systems,32–34
it has been predicted that E1 (Uba1) will undergo a distinct ubiquitin-fold domain (UFD) movement and conformation change during the E1-E2 handoff.33
The C-terminus of Cdc34 has been shown to interact with ubiquitin.35
It is tempting to speculate that ubiquitin E1 transfer between E1 and Cdc34 involves distinct conformation changes. NSC624206 may perturb this transition. It will be interesting to test the effect of NSC624206 on E1-E2 ubiquitin transfer with all classes of E2s in future studies to determine if NSC624206 can functionally discriminate between different classes of E2s.
Mechanistically, NSC624206 had an in vitro inhibitory activity against the ubiquitin E1/E2 pathway that resulted in the reduction of p27 and TRF1 polyubiquitination in vitro. Moreover, NSC624206 stabilized endogenous p27 in HepG2 cells without having an effect on transcriptional activity. It is therefore possible that NSC624206 might operate via the inhibition of E1-Cdc34 inside of cells; however, further studies are necessary to better delineate whether the observed increase of cellular p27 is due to cellular E1/E2 inhibition.