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 MDMX-overexpressing cancers.

The 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 wild-type 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 are now undergoing clinical evaluation (16, 17). MDM2 inhibitors have shown effective p53 activation followed by cell cycle arrest, induction of apoptosis, and tumor regression in cancer cells with mdm2 gene amplification (18, 19). However, their apoptotic activity has been found to be moderate to marginal in many tumor cell lines expressing normal levels of MDM2, suggesting that cancer uses other mechanisms to attenuate or disable p53 signaling (20), such as the overexpression of the other negative p53 regulator, MDMX. High levels of MDMX protein can make MDM2 antagonists, which have shown very low activity against p53-MDMX binding, ineffective in killing cancer cells (21–23). Thus, simultaneous inhibition of MDM2 and MDMX is needed to release the full activity of stabilized p53 (15, 17). Therefore, recent efforts have been focused on identification of dual MDM2/MDMX antagonists.

Because of distinct structural differences between MDM2 and MDMX in their p53-binding pockets (24–26), small molecules optimized for MDM2 have shown very low affinity for MDMX (27). For example, the first potent and selective small-molecule MDM2 antagonist, nutlin-3a, has ∼400-fold lower potency against MDMX than MDM2 (28). This trend has been followed by other MDM2 inhibitors (19). Efforts to identify MDMX-specific inhibitors have recently yielded a class of small molecules with in vitro binding activity in the high nanomolar range but relatively poor cellular potency and uncertain mechanism of cellular activity (29). Nearly equipotent MDM2/MDMX peptide inhibitors have been identified and characterized structurally but their activity has been detected only in cell-free systems (30). Recently, a cell-penetrating “stapled” peptide with good MDMX binding affinity has been identified and evaluated in cancer cells (31). Although cellular potency against p53-MDMX interaction has been found adequate, this peptide was unable to disrupt effectively p53-MDM2 binding, and it has been combined with the MDM2 antagonist, nutlin-3, to assess the antitumor potential of this emerging therapeutic modality.

Here, we identify a class of small molecules that can potently inhibit p53 interactions with both MDM2 and MDMX by induced protein dimerization and effectively restore p53 activity in MDMX-overexpressing cancer cells. We show that antagonizing both negative p53 regulators significantly improves the apoptotic outcome in cancer cells overproducing MDMX.

Results

Identification of Indolyl Hydantoins as MDM2/MDMX Antagonists.

A diverse library of small molecules was screened for suppression of p53-MDMX binding (Table S1). The hits were then tested for activity against the p53-MDM2 interaction. One series of indolyl hydantoin compounds emerged as potent, dual MDM2/MDMX antagonists. For example, RO-2443 (Fig. 1A) showed a remarkably similar inhibitory activity against both MDM2 (IC₅₀ = 33 nM) and MDMX (IC₅₀ = 41 nM) binding to p53. For its size, RO-2443 appeared highly potent (ligand efficiency, defined as binding energy per heavy atom, is 0.36) and likely to bind into, at most, two of the three subpockets on the surface of MDMX or MDM2. These pockets were defined by the original structure of a p53 peptide bound to MDM2 (32) which showed that there were three key residues from the p53 peptide, Phe¹⁹, Trp²³, and Leu²⁶. Throughout this report, these binding pockets on the surface of MDM2 and MDMX are referred to as the Phe, Trp, and Leu pockets.

Fig. 1.

RO-2443 inhibits the interaction of p53 with MDM2 and MDMX. (A) Chemical structure and in vitro activity of RO-2443. (B) [¹H-¹⁵N]HSQC NMR spectra of hzMDMX (His₆-15-106, L45V, V95L) in the absence (black contours) and presence (red contours) of RO-2443. The decrease in cross-peak intensities observed upon binding of RO-2443 is evidenced by fewer contours in the red spectrum. (C) Binding of RO-2443 to MDMX in solution changes its mobility consistent with protein dimerization. SLS detection of N-terminal zebrafish MDMX fragment eluting from a size-exclusion column shows the shift of the protein peak to a position consistent with that of a dimer. (D) Kinetic analysis of the change in Trp fluorescence intensity over a wide range of RO-2443 concentrations. Curves based on a binding model with the monomer or a dimer show that the data are consistent with the dimer model (K_dhigh = 0.4 μM and K_dlow = 57.2 μM). (E) Isothermal calorimetry shows the impact of binding RO-2443 to hMDMX and that the energy of the interaction is dominated by the entropy component.

To investigate the mechanism of action of RO-2443, we first used NMR spectroscopy. The [¹H-¹⁵N]HSQC spectrum of the MDMX N-terminal fragment (Fig. 1B) in the presence and absence of the small molecule indicated that: (i) the compound was binding to the p53 pocket; and (ii) there was a substantial up-field shift for Y63, which is consistent with shielding by an aromatic group. A similar shift had been observed upon binding of a p53 peptide to MDMX attributable to shielding by Phe¹⁹. In addition, there was an overall resonance broadening in the presence of the compound, which was manifested in the HSQC spectrum as a decrease in cross-peak intensities, suggesting formation of a higher-molecular-mass species. To determine the effect of RO-2443 on the state of MDMX in solution, size-exclusion chromatography with static light scattering (SEC-SLS) was performed (Fig. 1C). In the absence of RO-2443, the protein gave a SLS-calculated mass of 12.8 kDa, which agrees with the theoretical mass of a monomer (12.3 kDa). Addition of RO-2443 resulted in the protein eluting at an earlier elution volume, indicating that the shape and/or mass of the protein had changed. The SLS-calculated mass of the complex was 24.1 kDa, which suggests that MDMX forms a dimer when bound to RO-2443. Kinetic analysis of the binding of RO-2443 to MDMX (Fig. 1D) showed that the binding was in line with a two-molecule binding model. Isothermal calorimetry (ITC) confirmed a 1:1 ratio between compound and protein but could not distinguish between 1:1, 2:2, or higher-order species (Fig. 1E). It revealed a binding constant (K_d = 78 nM) that is in good agreement with the binding assay. The ITC also shows that the binding is completely dominated by the entropy component, consistent with binding interactions involving primarily hydrophobic effects. Thus, a consistent picture emerged that RO-2443 induces some sort of dimer formation of MDMX and MDM2.

Crystal Structures Reveal Tight MDMX Dimer Formation.

Crystals of MDMX bound to RO-2443 were grown that diffracted to relatively high resolution (1.8 Å) and molecular replacement with the structure of MDMX bound to a p53 peptide (33) was successful (Table S2). The structure is comprised of four monomers in the asymmetric unit arranged as a pair of dimers (Fig. 2A). These dimers show at their core two molecules of the inhibitor, each of which has binding interactions to both protein monomers. For each inhibitor molecule, the indolyl-hydantoin moiety occupies the Phe pocket of one protein monomer, whereas the di-fluoro-phenyl group reaches into the Trp pocket of the other. From a different viewpoint, Fig. 2B shows an overlay of the inhibitor dimer structure with that of the p53 peptide (33). Clearly, among the key interactions that the compounds form is an extensive aromatic stacking interaction between the two indolyl-hydantoin groups with interplanar distances ranging from 3.3 to 3.7 Å. The stacking interaction extends to include a tyrosine residue (Y63) from each of the protein monomers, resulting in a four-level π-sandwich. The small molecule–protein interactions were even slightly shorter, ranging from 3.1 to 3.5 Å. Another positive interaction between the small molecules is the σ-hole of the chlorine attached to the indole ring pointing at the di-fluoro-phenyl ring of the other molecule. There are multiple contacts at 4 Å between the chlorine atom and the phenyl ring, which are within the range described by Bissantz et al. (34).

Fig. 2.

RO-2443 binds to the p53 pocket of MDMX and induces protein dimerization. (A) Crystal structure of RO-2443 bound to MDMX. Close-up view of the p53 binding regions of two MDMX molecules forming a dimer. One MDMX molecule is shown as a surface rendition (carbon, white; oxygen, red; nitrogen, blue; and sulfur, yellow). The other MDMX molecule is shown as a stick figure with the same color scheme, but the carbon atoms are colored cyan. The two RO-2443 molecules are shown as stick figures. The molecule with cyan-colored carbon atoms is binding with the indole-hydantoin moiety in the Phe pocket of MDMX, shown as a stick figure and the di-fluoro-phenyl group in the Trp pocket of MDMX shown as a surface. The molecule with green-colored carbon atoms binds in the reverse mode. (B) View of the two inhibitor molecules (∼90° rotation) showing how they relate to the binding of a p53 peptide with carbon atoms colored magenta (32). The indolyl group of one inhibitor (green) overlays with Phe¹⁹ of the peptide, whereas the di-fluoro-phenyl group of the other inhibitor (cyan) overlays with Trp²³. It is worth noting that the chlorine atom of the 6-chloro-tryptophan of the peptide is nearly coincident with the parafluoro atom of the inhibitor. (C) Dimer model for binding of RO-2443 to MDMX. This rendition of the possible steps in the formation of the dimer indicates that monomeric interactions likely form first, followed by a pairing of monomers to form the dimer. This is based on the lack of any detection of dimer interactions by the small molecules alone. A binding model in which the formation of the monomer is followed by the addition of a second small molecule and subsequent addition of a vacant copy of MDMX cannot be ruled out.

Modeling studies indicated that MDM2 should be able to form the exact same homodimer and that MDMX and MDM2 could form heterodimers. Indeed, crystal structures of MDM2 bound to RO-2443 and other analogs confirm the expected MDM2 homodimer structure (Fig. S1A). Using both structures, a model of the potential heterodimer can be assembled showing no serious conflicts (Fig. S1B). Fig. 2C provides a conceptually clearer picture of the nature of the MDMX/RO-2443 dimer. As suggested by the model (also see Fig. S2A), the p53 binding pockets on MDMX (or MDM2) are nearly completely occluded. The most exposed part of the inhibitor is the methylene bridge between the hydantoin and phenyl groups, which provides a means for extending the inhibitor to reach into the Leu pocket (Fig. S2B). Partly for this reason, but also to impact the physicochemical properties of the compounds, this site was targeted for modification, resulting in analogs such as RO-5963.

RO-5963 Inhibited p53 Binding to MDM2 and MDMX in Cancer Cells.

RO-2443 showed potent MDM2/MDMX inhibitory activity in vitro, but poor water solubility did not allow for a meaningful assessment of its cellular activity. Further chemical optimization of RO-2443 yielded RO-5963 (Fig. 3A), a close analog with slightly increased potency but substantially improved solubility. In the same binding assay, RO-5963 showed p53-MDM2 inhibitory activity (IC₅₀, ∼17 nM) similar to that of nutlin-3a (IC₅₀, ∼19 nM) (Fig. 3A). Its p53-MDMX inhibitory activity (IC₅₀, ∼24 nM) was nearly equivalent to MDM2 activity but ∼400-fold better than the MDMX potency of nutlin-3a (IC₅₀, ∼9 μM). RO-5963 penetrated MDMX-overexpressing breast cancer cells (MCF7), stabilized p53, and elevated protein levels of its transcription targets, p21 and MDM2, in a dose-dependent manner (Fig. 3B). The increase in p21 and MDM2 protein levels was attributable to induction of their transcription as revealed by the dose-dependent increase of their mRNA and of two other p53 transcriptional targets, macrophage inhibitory cytokine-1 (MIC-1) and bcl-2 associated X protein (BAX), but not MDMX, which is not under p53 control (Fig. 3C). Stabilization and activation of p53 was induced by disrupting its interaction with MDM2 and MDMX in MCF7 cells as demonstrated by immunoprecipitation of either p53 or MDMX proteins from cells exposed to RO-5963 followed by Western analysis (Fig. 3D). At 20 μM, RO-5963 was equivalent to 10 μM nutlin in inhibiting p53-MDM2 binding and also effectively blocked p53-MDMX binding at both 10 and 20 μM concentration. As expected, the MDM2-specific inhibitor, nutlin-3a, showed no effect on the p53-MDMX interaction. A notable increase in MDMX and MDM2 proteins pulled down by immunoprecipitated MDMX (Fig. 3D, Right) was consistent with predicted RO-5963-induced formation of MDMX/MDMX and MDMX/MDM2 dimers in MCF7 cells.

Fig. 3.

RO-5963 stabilizes p53 and activates the p53 pathway in cancer cells. (A) Chemical structure and in vitro inhibitory activity of RO-5963 and the MDM2 antagonist, nutlin-3a. (B) RO-5963 stabilizes p53 and elevates protein levels of p53 targets, p21 and MDM2. Log-phase MCF7 cells were incubated with RO-5953 for 24 h, and cell lysates were analyzed by Western blotting. (C) Dose-dependent induction of p53 target genes in MCF7 cells 24 h post RO-5963 addition. (D) RO-5963 inhibits p53-MDM2 and p53-MDMX binding in cancer cells. MCF7 cells were incubated with 10 μM nutlin-3a and 10 or 20 μM RO-5963 for 4 h, and the levels of p53, MDM2, and MDMX were determined in protein complexes immunoprecipitated with anti-MDMX or anti-p53 antibodies by Western blotting.

RO-5963 Activates p53 Pathway in Cancer Cells Expressing Wild-Type p53.

Inhibition of p53-MDM2 binding should induce p53 signaling only if the cells express wild-type but not mutant p53, which generally loses its transcriptional activity. Indeed, the EC₅₀ of RO-5963 was ∼10-fold lower in cancer cells expressing wild-type p53 compared with mutant p53 cells (Fig. 4A) and only activated the p53 transcriptional targets p21 and MDM2 in p53 wild-type but not p53 mutant cancer cells (Fig. S3). RO-5963 affected the viability of HCT116 cells but not their nutlin-resistant clone HCT116R1, which has lost ability to induce p53 response (Fig. S4). Treatment of four cancer cell lines with RO-5963 did not increase the levels of p53^Ser15 phosphorylation, suggesting that p53 activation is not caused by genotoxic stress induced by the compound (Fig. 4B).

Fig. 4.

RO-5963 activates p53 signaling in diverse cellular context by a nongenotoxic mechanism. (A) Antitumor activity of RO-5963 depends on the p53 status. Viability of three wild-type p53 (MCF7, HCT116, RKO) and two mutant p53 (SW480, MDA-MB-435) cancer cell lines was determined by the CellTiter-Glo assay after 5 d of incubation with RO-5963 and expressed as percentage of controls ± SD. (B) RO-5963 does not induce genotoxic response in cancer cells. Cells were incubated with 10 μM RO-5963 or 1 μM doxorubicin for 24 h, and the levels of total and Ser¹⁵-phosphorylated p53 were determined by Western blotting. (C) Binding of RO-5963 prevents MDM2-mediated degradation of MDMX. G401 and H460 cells were incubated with the indicated concentrations of RO-5963 with or without 10 μM nutlin-3a, and relative levels of p53, p21, MDM2, and MDMX were determined by Western blotting. Blots representative of three independent experiments are shown. (D) RO-5963 activates p53 signaling in multiple cancer cell lines with wild-type p53. Exponentially growing cancer cell were incubated with 10 μM RO-5963 for 24 h, and the relative levels of p53, p21, and MDM2 were determined by Western blotting.

By disrupting p53-MDM2 binding and the autoregulatory feedback loop, small-molecule antagonists (e.g., nutlins) can elevate MDM2 protein levels and, thus, facilitate MDMX ubiquitination and degradation. As a result, MDMX protein levels were found reduced in many cancer cell lines in the presence of nutlin-3 (28). If RO-5963 induces homo- or heterodimerization of MDM2 and MDMX inside living cells, that might interfere with MDM2 ligase activity and/or its ability to ubiquitinate MDMX. To investigate this possibility, we incubated G401 cancer cells with increasing concentrations of RO-5963 in the presence or absence of 10 μM nutlin-3a. As expected, nutlin substantially reduced MDMX protein levels (Fig. 4C). RO-5963 dose-dependently increased p53, MDM2, and p21 levels but only slightly reduced MDMX. However, it protected MDMX from nutlin-induced degradation by MDM2 in both G401 and H460 cells (Fig. 4C). These results suggest that RO-5963-induced dimerization of MDM2 and MDMX is the likely cause for protection of MDMX from MDM2-mediated degradation. However, despite increased MDMX levels in cells exposed to RO-5963, compared with nutlin, MDMX was prevented from binding to and inhibiting p53 in the presence of the dual inhibitor.

Next, we tested RO-5963 in a wider panel of 11 solid tumor cell lines expressing wild-type p53 and representing diverse tumor types: breast (MCF7), prostate (LNCaP, 22Rv1), colon (HCT116, RKO), lung (H460, A549), kidney (A498), osteosarcoma (U2OS), melanoma (LOX). The dual inhibitor was able to effectively activate p53 and elevate p21 and MDM2 levels, suggesting that it penetrates well cultured cells and can be used in diverse cellular context (Fig. 4D).

RO-5963 Effectively Activates Main Functions of the p53 Pathway in Cancer Cells.

One of the main functions of activated p53 is induction of cell cycle arrest. As previously demonstrated by the specific MDM2 antagonist, nutlin-3 (20), RO-5963 potently arrested cell cycle progression in exponentially growing cancer cells in G₁ and G₂ phase, effectively depleting the S phase compartment (Fig. S5A). Induction of apoptosis is another major p53 function that is frequently altered in cancers expressing wild-type p53 (20). Similar to MDM2, MDMX overexpression has been shown to effectively disable this function by inhibiting p53 transcriptional activity (6, 14). Therefore, MDMX could be a barrier to p53 apoptotic activity even in the presence of MDM2 antagonists (21–23). The SJSA1 osteosarcoma line, which expresses very high levels of MDM2 protein, is presumed to be free of other defects in the p53 pathway and has shown a robust apoptotic response to the specific MDM2 antagonists (20). However, its engineered clone, SJSA-X, is nearly completely resistant to nutlin because of the high levels of exogenously expressed MDMX from a CMV promoter (23). Therefore, the SJSA-X clone offers an excellent mechanistic model for assessing the cellular activity of p53-MDMX inhibitors. The high levels of both MDM2 and MDMX proteins in SJSA-X cells represent a fairly high hurdle to dual inhibitors. When RO-5963 was tested for apoptotic activity on the parental cell line, SJSA-V, it showed slightly lower but still strong Annexin V signal compared with nutlin (Fig. S5B). As expected, similar apoptotic activity was measured also in the SJSA-X clone in which nutlin was practically inactive. The apoptotic activity of RO-5963 was dose-dependent and was enhanced when both molecules were combined. Western blotting of SJSA-X cell lysates revealed that 20 μM RO-5963 induced p53 accumulation comparable to 10 μM nutlin-3a because p53 stabilization is caused by disruption of the p53-MDM2 interaction (Fig. 5C). However, p53 transcriptional activity, indicated by p21 and MDM2 levels, was higher in the presence of RO-5963, presumably because of blocking not only p53-MDM2 but also p53-MDMX binding and liberating the elevated p53 protein from both inhibitors. Protein levels of p21 and MDM2 were increased further by combining RO-5963 with nutlin, reflecting the increased transcriptional activity of p53 (Fig. S5C) that explains enhanced apoptotic activity of the combination (Fig. S5B). These results indicate that RO-5963 can effectively inhibit both p53-MDM2 and p53-MDMX binding and can induce apoptosis in a cancer cell model overexpressing high levels of both negative p53 regulators.

Fig. 5.

Apoptotic activity of RO-5963 and MDMX status. (A) Breast cancer cells respond to RO-5963 depending on the levels of MDM2 and MDMX. Three cell lines with variable ratios of MDMX/MDM2 proteins were exposed to nutlin-3a (10 μM) and RO-5963 (10 or 20 μM) for 48 h, and the percentage of apoptotic cells (± SD) was determined by the Annexin V assay. (B) Cytotoxicity of RO-5963 on MCF7 cells. Phase-contrast images were taken 48 h after addition of 20 μM RO5963. (C) Relative protein levels of MDM2 and MDMX in a panel of solid tumor cell lines. Cells lines at subconfluent stage of growth were analyzed for protein levels by Western blotting. (D) Apoptotic response to RO-5963 and nutlin in a panel of cancer cell lines with wild-type and mutant p53. Log-phase cells were incubated with nutlin-3a and RO-5963 for 48 h and analyzed for apoptosis as in A. Two mutant p53 cell lines, MDA-MB-435 and SW480, were included as controls.

RO-5963 Overcomes the Resistance of MDMX-Overexpressing Cancer Cells to MDM2 Antagonists.

Cancer cells overexpressing MDMX have substantially reduced apoptotic response to small-molecule MDM2 antagonists (e.g., nutlins) (21–23). This resistance may be partly attributable to the inability of nutlin to inhibit p53-MDMX binding, leading to incomplete restoration of p53 activity. MCF7 breast cancer cell line represents such a MDMX-dependent model. These cells have high levels of MDMX protein and are fairly insensitive to nutlin-induced apoptosis. Therefore, we first tested the apoptotic activity of RO-5963 in MCF7 and two other breast cancer cell lines with low (ZR75-1) and intermediate (ZR75-30) MDMX levels (35). As an MDM2-only inhibitor (20, 28), nutlin-3a had relatively low apoptotic activity in MCF7 cells and practically no activity in ZR75-30 cells (Fig. 5A). ZR75-30 cells express moderate levels of MDMX but very low levels of MDM2, suggesting that MDMX plays an important role in p53 regulation in these cells. RO-5963 (20 μM) showed much higher apoptotic activity than nutlin in both MCF7 and ZR75-30 cell lines. These results suggest that RO-5963 is capable of releasing p53 from MDMX inhibition and restoring its activity. Although this led to efficient apoptosis and cell death in the MCF7 line (Fig. 5 A and B), ZR75-30 cells had an enhanced but incomplete apoptotic response. This is likely attributable to other abnormalities in cancer signaling that can attenuate p53 apoptotic pathways as seen in many epithelial cancer cell lines with normal MDM2 and relatively low MDMX levels (20, 28). Interestingly, the dual antagonist was less active than nutlin in the ZR75-1 cell line expressing barely detectable MDMX levels. This may result from the fact that nutlin is slightly more effective than RO-5963 at inhibiting p53-MDM2 binding in cells.

Next, we tested the apoptotic activity of RO-5963 and nutlin against a randomly selected panel of nine cancer cell lines expressing wild-type p53 and two with mutant p53. As expected, no apoptotic activity was detected in the mutant p53 lines, and variable levels of apoptosis were measured in the wild-type lines (Fig. 5D). RO-5963 showed better apoptotic activity than nutlin in 4/9 (H460, RKO, LS174T, AGS) and similar or slightly lower in five of nine wild-type p53 cell lines. Three out of the four lines with enhanced apoptosis had relatively high levels of MDMX. Overall, MDMX protein levels correlated with the enhanced apoptotic response to the dual MDM2/MDMX antagonists (Fig. 5 C and D). These data suggest that inhibition of p53-MDM2 interaction is critical for stabilization of p53 and that simultaneous inhibition of p53-MDMX binding can enhance p53 apoptotic activity in cancer cells with MDMX overexpression. However, inhibition of both MDM2 and MDMX does not substantially enhance p53-dependent apoptotic response in cancer cells with normal or low levels of MDMX that is likely affected by other factors than MDMX.

Discussion

The role of MDMX in the fine regulation of p53 is still emerging, but it is an established fact that MDMX overexpression can block p53 function and render cancer cells resistant to MDM2 antagonists (6, 15). Even normal levels of MDMX could partially silence activated p53 because all known inhibitors of the p53-MDM2 interaction are unable to liberate p53 from the remaining MDMX, suggesting that dual MDM2/MDMX inhibitors may substantially improve the outcome of this p53 activation strategy. However, recent efforts to develop inhibitors of p53 binding with both MDM2 and MDMX have been hindered by the structural differences in the p53 pockets of the proteins (26). Using high-throughput screening and a diverse small-molecule library, we identified a class of indolyl-hydantoin compounds that are roughly equipotent. The series demonstrated clear SAR and the better analogs showed remarkable potency given their small size and the ability to occupy only two of the three surface subpockets in the p53 binding region. The presence of the 6-chloro-indole group provided the expectation that the binding mode could be predicted because this same group is used in some of the known MDM2 antagonists (36).

The predicted binding mode of the dual MDM2/MDMX antagonists placed the chloro-indole moiety in the Trp pocket with the remainder of the compound extending into the Phe pocket. This was, in general, consistent with the available information. First, the fact that the compounds were equipotent against MDM2 and MDMX made it less likely that they bound in the Leu pocket, where the two protein structures diverge the most. Secondly, the large up-field shift in the NMR spectrum for Y63 meant that this residue was likely interacting with an aromatic group from the inhibitor, the di-fluoro-phenyl group, in this model. However, the actual structure turned these predictions completely upside down (Fig. 2A). The indole and hydantoin moieties remain essentially coplanar and occupy an extended Phe pocket. In this orientation, it is the indolyl-hydantoin that is providing the shielding for the Y63 residue. It also forms a stacking interaction with the phenolic ring of Y63 (Fig. S1A). Most surprisingly, the di-fluoro-phenyl group of one molecule bound to one protein monomer reaches over to bind into the Trp pocket of the other protein monomer (Fig. 2B). Although unusual, the structure nicely explained the preference for fluorine in the paraposition for binding to MDMX. It also provided an explanation for the preference for chlorine at the 6-position of the indole.

Although MDM2 and MDMX naturally form homodimers and heterodimers, these are driven by the C-terminal RING domains (6, 37). Thus, the dimerization of the N-terminal p53-binding domains in this fashion has not been observed previously. It represents a significant advantage in that small molecules are able to achieve potent binding and inhibitory effects and to do it in a way that avoids addressing the Leu subpocket, where the two protein structures are the most different. As a result, these compounds are equipotent against MDM2 and MDMX. Nevertheless, it is possible to modify these compounds to enable them to address the Leu pocket and RO-5963 (Fig. 3A) represents one such approach.

RO-5963 penetrated cancer cells and inhibited the binding of p53 to MDM2 and MDMX. Although nearly equipotent in vitro, RO-5963 was more effective in disrupting the p53-MDMX interaction than p53-MDM2 interaction in cultured cancer cells. One possible reason for this is the increasing cellular levels of MDM2 in the presence of MDM2 inhibitors. Whereas MDMX levels are generally stable, MDM2 protein levels rise as a result of inducing its expression by the accumulating p53. Increasing MDM2 protein may change the balance between homo- and heterodimers induced by RO-5963 and make MDM2 inhibitory activity a limiting factor in the course of exposure to the dual inhibitor. This is a likely reason why addition of nutlin further enhanced the apoptotic outcome of RO-5963 treatment especially in the MDM2 amplified SJSA-X line (Fig. S5B).

Forced or natural overexpression of MDMX can significantly impede the activity of MDM2 antagonists (19, 21–23). These agents have been optimized for binding to the p53 pocket of MDM2 and are practically inactive against the p53-MDMX interaction (18, 19, 28). Unobstructed binding of MDMX to p53 could silence its transcriptional activity and may alter other properties, leading to inadequate activation of p53 functions. Therefore, the effectiveness of MDM2 antagonists in tumors with high levels of MDMX will be diminished. Our experimental results with the dual MDM2/MDMX inhibitor are in full agreement with this prediction. RO-5963 restored p53 transcriptional activity and overcame the apoptotic resistance of MDMX-overexpressing cell line, SJSA-X, to nutlin-3 (Fig. 5C). The same is true in the “naturally” MDMX-overexpressing breast cancer cell lines MCF7 and ZR75-30 (Fig. 5). Both cell lines exhibit higher MDMX to MDM2 ratios, suggesting that MDMX may play a role in their resistance to nutlin-induced apoptosis. The relatively incomplete apoptotic response in ZR75-30 line is likely attributable to factors other than MDMX attenuating p53-dependent apoptosis signaling. In MCF7 cells, used routinely as a cellular model of MDMX-overexpressing breast cancer, RO-5963 showed massive cell death with nearly all cells losing their viability within 72 h of treatment (Fig. 5B). These results are in agreement with previously published data using RNA interference to address the role of MDMX in the response to nutlin (23, 28, 35).

Nearly one-fifth of breast, colon, and lung cancers overexpress MDMX because of gene amplification (38), and that could be the only abnormality in their p53 signaling as proposed for MDM2-amplified tumors (20). Our data suggest that patients with this signature may greatly benefit from treatment with dual MDM2/MDMX antagonists. The identification of the indolyl hydantoin class of dual inhibitors is the first step toward development of MDM2/MDMX-targeted cancer therapy. Further optimization of the potency and pharmacological properties of this chemical class should allow the extension of our observations to in vivo cancer models and eventually the clinic.

Materials and Methods

Materials and detailed methods are available in SI Materials and Methods.

Cell proliferation/viability was evaluated by the methyl-thiazolyl-tetrazolium (MTT) and CellTiter-Glo (Promega) assays. Cell cycle analysis, Annexin V assays, and Western blotting were performed as described previously (20). MDM2-p53 and MDMX-p53 binding was assessed by time-resolved (TR)-FRET binding and fluorescence quenching assays. NMR spectra were collected using ¹⁵N-labeled humanized-zebrafish (hz)MDMX. For cocrystallization with RO-2443, a 1.3:1 stoichiometric excess of compound was added to HDMX protein. The protein construct (14-111,C17S) was as described in ref. 33. Crystallization conditions, data collection, molecular replacement, and refinement were performed as detailed in SI Materials and Methods. Statistics for the refined model are in Table S2.