In this study, we conducted a screen to identify inhibitors of Mdm2 E3 ligase activity. We found two closely related analogs out of 270,080 tested compounds—MEL23 and MEL24. Using full-length Mdm2 allowed us to take advantage of the presence of all domains of Mdm2 in order to improve the likelihood of discovering inhibitors. Additionally, the use of a cell-based assay enabled targeting of potential cellular co-factors necessary for ligase activity. The high-throughput screen we present here could be modified in order to discover inhibitors of other E3 ligases. The ease of this type of high-throughput screen and its adaptability may therefore be valuable in the identification of new small molecule inhibitors directed against different E3 ligases, which have previously been considered challenging targets for drug discovery.
Mdm2 inhibitors described to date include peptide inhibitors (35
) and small molecules such as Nutlin-3 (23
) and RITA (36
). Mdm2 E3 ligase inhibitors identified by in vitro
screens, such as the HLI series of compounds, have also been described (24
). Although the HLI compounds were able to induce p53-dependent apoptosis, they lack specificity towards Mdm2 (24
). Other Mdm2 E3 ligase inhibitors identified in in vitro
ubiquitination assays, such as sempervirine and lissochlinidine B, have not been shown to inhibit Mdm2-MdmX E3 ligase activity (37
). More recently, the first small-molecule inhibitor of MdmX binding to p53 was identified (39
). Unlike other Mdm2 and MdmX inhibitors identified, MEL23 and MEL24 are a unique class of Mdm2 inhibitors identified in a cell-based assay that are able to inhibit the E3 ligase activity of the Mdm2-MdmX complex.
These data show that MEL23 and MEL24 preferentially inhibit the E3 ligase activity of the Mdm2-MdmX complex, although they do have some activity against Mdm2 alone. The inhibition of the Mdm2-MdmX complex in vitro
and stabilization of p53 and Mdm2 in cells indicate that the Mdm2-MdmX complex is centrally involved in regulating the degradation of these proteins. These observations are in agreement with multiple studies that describe the importance and activity of the Mdm2-MdmX complex. While Mdm2 can catalyze p53 ubiquitination in vitro
), in cells MdmX is needed along with Mdm2 for efficient p53 degradation (19
). MdmX can lower the concentration of Mdm2 needed for both p53 ubiquitination and auto-ubiqutination, and the Mdm2-MdmX complex has been shown to be a better ligase for p53 than Mdm2 alone (41
). Additionally mouse studies have shown that the interaction between Mdm2 and MdmX is essential for regulating p53 during early embryogeneisis (42
). Enhancement of activity of RING domain hetero-oligomers has also been demonstrated for BRCA1-BARD1 complexes, where the presence of BARD1 in the complex increases the ligase activity of BRCA1 (44
). Therefore our results support studies suggesting that although Mdm2 is able to function as an E3 ligase on its own, MdmX augments the nature and activity of the ligase with profound functional consequences in cells.
One caveat in these mechanistic studies was that high concentrations of MEL compounds were necessary to see inhibition of ubiquitination in vitro
. It is possible that the enzymatic activity of the Mdm2-MdmX ligase is difficult to inhibit in vitro
; the well-known Nutlin-3 compound does not inhibit Mdm2 ubiquitination (45
). Perhaps the MEL compounds only partially inhibit E3 ligase activity in cells, and this is sufficient to cause p53 accumulation, consistent with mouse data showing that just a 20–30% reduction in Mdm2 can lead to p53 activation (46
). It is tempting to speculate that compounds that completely inhibited Mdm2 or Mdm2-MdmX E3 ligase activity would activate p53 in both cancer and normal cells to such a large extent that the compounds would not be tolerated and would therefore not be useful as therapeutics. The MEL compounds, on the other hand, have a small but consistent differential activity between tumor derived and non-transformed cell lines, decreasing the survival of the tumor-derived cells to a greater extent (Supplementary Fig. 20
Since Mdm2 can inhibit p53 through two independent mechanisms—E3 ligase-mediated degradation and binding-mediated functional inactivation—it is formally possible that inhibitors of Mdm2 E3 ligase activity would not be sufficient to activate p53. In this model, Mdm2 E3 ligase inhibitors would increase both p53 and Mdm2 levels, but would not prevent Mdm2 from binding p53 and thereby inhibiting its activity. Based on qPCR data ( and Supplementary Fig. 7
) and also co-immunoprecipitation data ( and Supplemental Fig. 16
), it is likely that Mdm2 and p53 are still interacting to some extent upon MEL compound treatment and that Mdm2 is partially inhibiting p53 activity. However, we observed increased p53 activity upon MEL treatment compared to proteasome inhibition, which also increases Mdm2 and p53 levels. In RKO cells, such increases in p53 activity caused by MEL compound treatment appear to be sufficient for p53-dependent cell death, albeit at higher concentrations of MEL23 than are needed for p53 stabilization. Indeed, HLI compounds, another class of Mdm2 E3 ligase inhibitors, also induce p53-dependent cell death, suggesting that complete disruption of the Mdm2-p53 interaction may not be necessary for activation of p53 (24
). Additionally, mutant knock-in mice studies have demonstrated that Mdm2-p53 binding, without Mdm2-mediate p53 ubiquitination, is not sufficient to control p53 activity (47
). Furthermore while MEL compounds do not induce p53 post-translational modifications, some cancer cells may already have such modifications in place as a consequence of oncogenic stress. Nevertheless, we predict that the combination of the MEL compounds and DNA-damaging agents would enhance their therapeutic potential. This could also allow for use of DNA-damaging agents at concentrations that do not cause some of their normal deleterious side effects.
MEL23 cooperated with DNA-damaging agents in p53-null cells to a small, yet reproducible, extent. Mdm2 has been shown to have p53-independent oncogenic effects; for example, (1) overexpression of Mdm2 in mice causes tumors independent of p53 status (33
) (2) splice variants of Mdm2 that cannot bind to p53 have been shown to be oncogenic (48
), (3) Mdm2 destabilization of Rb (49
) and p21 (51
) may contribute to tumor growth, and (4) Nutlin-3-mediated disruption of p73-Mdm2 binding enhances p73 function (52
). Therefore, the MEL compounds and other specific Mdm2 E3 ligase inhibitors may be beneficial in p53-null or p53-mutant tumors. This hypothesis, while intriguing, requires further study.
As Mdm2-MdmX ligase inhibitors, MEL compounds may provide further insight into the function of the Mdm2-MdmX E3 ligase and allow for investigation of the differences in activity between the hetero-complex and the Mdm2 homo-complex. It will be informative to ultimately determine the precise biophysical mechanism of action of the MEL compounds, as well as the binding site of the compounds. Additionally, the MEL compounds may be used as molecular tools to validate novel targets of Mdm2-MdmX. Finally, while these compounds were not suitable for testing in mice, we have demonstrated that targeting Mdm2-MdmX with small molecules is feasible and future studies could either optimize these compounds or use this high-throughput assay to discover additional drug-like scaffolds with similar activity. An improved understanding of the mechanism of action of the MEL compounds, and/or future inhibitors of this pathway, may lead to new ways to inhibit E3 ligases, which could be beneficial in diverse applications.