The development of molecules that prevent the p53-Mdm2 interaction has garnered a significant amount of interest. Recent data suggest that such molecules may also be useful in preventing other Mdm2 interactions, and thus may even have efficacy in tumors lacking p53. The first inhibition of the p53-Mdm2 protein-protein interaction utilized an antibody through cellular microinjection that recognized the p53 binding motif in Mdm2 and increased the transcriptional activities of p53 (98
). The therapeutic value of such an antibody is less attractive, as large macromolecules are incapable of penetrating the cell membrane without conjugation that increases lipid permeability. Development of peptidomimetic inhibitors that successfully imitated the p53 alpha-helical region binding to Mdm2 has been performed, yet their structure and size render their cellular permeability rather low (99
Naturally-derived molecules have been found to inhibit the p53-Mdm2 interaction (). Chlorofusin, derived from fermentations of Microdochium caespitosum
, was shown to bind the amino terminus of Mdm2 and prevent the formation of the p53-Mdm2 complex (100
). A chalcone derivative, demonstrated by ELISA to interfere with p53-Mdm2 complex formation, bound an unexpected area of Mdm2, at residues Phe55 and Tyr56, which reside downstream of the p53 binding pocket. Interestingly, two of the chalcone derivatives also influenced p53 in such a way that rendered it incapable of binding DNA (101
). These natural product molecules were modified in hopes of enhancing their efficacy, but were met with limited success.
Small molecule inhibitors of MDM2
The first representative of SMIs, Syc-7, was shown to disrupt the p53-Mdm2 interaction and affect the viability of several tumor cell lines, all of which expressed wild-type p53 (102
). However, the ability of these groups to remain fixed in space was seen as negative for Syc-7, as linkages to the benzyl functional groups were rather flexible. A 3-dimensional search program of the NCI chemical database revealed a sulfonamide-based compound that was capable of hindering p53-Mdm2 complex formation. This compound was also shown to modestly increase transcriptional p53 activity in cells overexpressing Mdm2 (103
). This may have been due to a low cell permeability, which could be redesigned by using different functional groups to increase solubility.
Nutlins were the first small molecules developed at Roche that could selectively inhibit the p53-Mdm2 complex formation (104
). Initial high-throughput screens identified several structures and eventually Nutlin-3a was developed. NMR spectroscopy demonstrated that Nutlin-3 () could prevent the formation of the p53-Mdm2 (105
). Recent findings have implicated this compound in several anti-cancer pathways. Nutlin-3a was found to decrease the rate of tumor growth in nude mice xenografts (104
). Although some have suggested that Nutlin-3a has no efficacy on tumors expressing mutant p53, it has recently been shown to suppress angiogenesis independent of p53 (76
). Furthermore, it was recently demonstrated that Nutlin-3a can upregulate apoptotic genes independent of p53. It was shown to do so by blocking the p73-Mdm2 interaction (106
). This compound warrants further investigation, as it has been the first Mdm2 inhibitor to show substantial effects independent of wild-type p53.
In 2005 a group from Johnson & Johnson Pharmaceuticals reported a co-crystal structure of a benzodiazepinedione Mdm2 antagonist (). This molecule successfully mimicked the p53 alpha-helix that projects into the amino-terminus of Mdm2 (107
). These potent compounds, TDP521252 and TDP665759, were shown to decrease proliferation in JAR cells with wild-type p53, but were shown to have little effect on a breast cancer line MDA-MB-231 with mutant p53. These compounds modestly induced p53 target genes, but were shown to be most potent when used in conjunction with the chemosensitizing agent, doxorubicin (108
). This brought about an interesting question with regard to Mdm2 inhibitors. Even though these compounds were protecting p53 from Mdm2 mediated degradation, there appeared to be a requirement for a stimulus to activate p53. Some have suggested that the ability of these compounds to induce apoptosis in a p53-dependent manner suggests that phosphorylation of key serines on p53 may not be entirely necessary for apoptosis (109
Structure-based methods examining the p53-Mdm2 co-crystal led to spiro-oxindole compounds (). MI-219 was shown to be selectively toxic to tumor cells (110
). Analysis showed that MI-219 caused the upregulation of p21 and Mdm2, but only caused transcription-independent accumulation of p53. MI-219 was shown to cause apoptosis in cells with wild-type p53 to a greater extent than Nutlin-3, yet did not have good efficacy in mutant p53 lines.
An isoindolinone compound was developed and shown to inhibit the formation of the p53-Mdm2 complex (111
). This compound was shown to disrupt binding in Mdm2 overexpressing SJSA cells that contain wild-type p53, as well as, cause induction of p21 and Mdm2 (112
). Although this drug shows promise, due to its induction of apoptotic proteins, further investigations are necessary to show the gross biological effect this compound has on tumors. Through in silico
modeling and NMR, several iso-1-quinolinones () were designed that successfully interfered with Mdm2 binding to p53 (113
). One specific compound (NXN-11) induced Noxa, an apoptotic effector protein induced by p53 and p73, to a greater extent than Nutlin-3, and overall the compounds were effective in apoptotic induction in cells wild-type for p53.
An inhibitor of Mdm2 ubiquitin ligase activity was discovered using high-throughput screening. The HLI98 compounds () showed some specificity towards Mdm2 in vitro
, however, at higher concentrations the drug was also shown to interact with other RING and HECT domains of E3 ubiquitin ligases. These drugs were shown to stabilize p53 and Mdm2 in cells and induce the p53 target genes p21 and PUMA. According to the authors, the drugs would be of little therapeutic use due to their lack of solubility (114
). A recent report has revealed critical details on how p53 translation is regulated by Mdm2. The RING domain of Mdm2 was found to bind to the p53 mRNA region where Mdm2 is known to bind. This causes a reduction in Mdm2 ubiquitination activity which leads to enhanced p53 mRNA translation (115
). This may represent a point of argument to why an inhibitor specific for the RING domain of Mdm2 would not be ideal in cells with wild-type p53.
An alternative approach to preventing the p53-Mdm2 protein-protein interaction would be to develop a compound that binds p53 in such a way that prevents its association with Mdm2. A compound termed, RITA for (r
eactivation of p53 and i
nduction of t
umor cell a
poptosis) was discovered through a cell-based assay that measured proliferation in response to various compounds. RITA promoted the induction of several p53 target genes in cell lines with wild-type p53 status (116
). NMR studies using a portion of the Mdm2 protein have demonstrated that RITA can not block the p53-Mdm2 interaction (117
). However, a two-site ELISA and pulldown assay have demonstrated that the p53-Mdm2 interaction is blocked by RITA (116
). Collectively, small molecules that target Mdm2 are an evolving field and new series of compounds will surely continue to emerge.