Activation of the p53 pathway by small-molecule-induced MDM2 and MDMX dimerization

Abstract

Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo-and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMXoverexpressing cancers.T he tumor suppressor p53 is a powerful growth-suppressive and proapoptotic protein tightly controlled by its negative regulators: murine double minute (MDM)2 and MDMX (1, 2). These proteins bind p53 with their structurally similar N-terminal domains and effectively inhibit p53 transcriptional activity (1, 3). They both possess a RING (really interesting new gene) domain in their C termini, but it is only functional in MDM2, which serves as a specific E3 ligase and main regulator of p53 stability (4, 5). Despite its RING domain, MDMX does not have an intrinsic ligase activity and does not affect directly p53 stability (6). However, MDMX can enhance ligase activity of MDM2 toward p53 by forming MDM2/MDMX heterodimers (7,8). It has been reported that the MDM2/MDMX complex is responsible for polyubiquitination of p53, whereas MDM2 alone primarily induces monoubiquitination (9). Targeted disruption of MDM2/MDMX heterocomplexes is embryonic-lethal in mice, suggesting that complex formation is essential for p53 regulation in vivo (10). On the other hand, MDM2 can also ubiquitinate MDMX and is, therefore, responsible for its stability as well (11,12). MDM2 is a transcriptional target of p53, and both proteins form an autoregulatory feedback loop by which they mutually control their cellular levels (13).The functional relationship between MDM2 and MDMX is still being refined at the molecular level, but it is well established that these two negative regulators play a critical role in controlling p53 tumor-suppressor function in normal cells (2,14). This is why they are frequently overproduced through gene amplification and/or overexpression in tumors that retain wildtype p53 (14). Therefore, antagonizing the binding of MDM2 and MDMX to p53 is expected to restore p53 function and may offer a strategy for cancer therapy (15). Recently identified small-molecule inhibitors of the p53-MDM2 interaction have validated this approach, and the first pharmacological MDM2 antagonists ar...

In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas

“…The overall results of this study provide some interesting new insights into the molecular profile of WD/DD LPS that not only help to explain their variable sensitivity to Nutlin-3A in vitro and in the clinical setting, but also support the development of dual MDM2/MDMX compounds 38 that are expected to improve clinical outcomes in non-resectable WD/DD patients.…”

“…The lower affinity of MDM2-B, which was further confirmed by MD simulations, is keeping with preclinical in vitro and in vivo data 37 and supports the idea that MDM2-B may contribute to the formation of cancer by means of a TP53-independent mechanism. 38 In particular, transduction of MDM2-B into a variety of cell types reveals that MDM2-B promotes TP53-independent cell growth, inhibits apoptosis, and upregulates the RelA subunit of NF k B. 38 and that is expected to correlate with a more aggressive behavior.…”

“…38 In particular, transduction of MDM2-B into a variety of cell types reveals that MDM2-B promotes TP53-independent cell growth, inhibits apoptosis, and upregulates the RelA subunit of NF k B. 38 and that is expected to correlate with a more aggressive behavior. In line with recent sarcoma genetic data, 22 all of the analyzed WD/DD LPS were disomic for MDMX.…”

Autoinhibition of MDMX by intramolecular p53 mimicry

“…Although the detailed mechanism of p53 inhibition by MDMX is still unclear, previous studies using p53-mimetic peptides and small molecule disruptors showed that MDMX-p53 binding is a critical step for p53 inactivation (18,19). MDMX-p53 binding is stimulated by CK1α, which is a major binding partner of MDMX.…”

Stabilization of protein–protein interactions by small molecules

“…However, to take full advantage of the strategy to stabilize p53 function, MDM2 and MDMX need to be targeted [86]. Such dual-activity inhibitors were identified by high-throughput screening by scientists at Roche and belonged to the class of indolyl-hydantoins [87]. Surprisingly, biochemical experiments and the co-crystal structure of MDMX or MDM2 in complex with one of these compounds, (5Z)-5-[(6-chloro-7-methyl-1H-indol-3-yl)methylidene]-3-(3,4-difluorobenzyl)imidazolidine-2,4-dione (RO2443; Table 1), revealed that RO2443 bound to the interface of the homodimers and stabilized their interaction.…”