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REVIEWS INHIBITING THE p53–MDM2 INTERACTION: AN IMPORTANT TARGET FOR CANCER THERAPY Patrick Chène p53 is an attractive therapeutic target in oncology because its tumour-suppressor activity can be stimulated to eradicate tumour cells. Inhibiting the p53–MDM2 interaction is a promising approach for activating p53, because this association is well characterized at the structural and biological levels. MDM2 inhibits p53 transcriptional activity, favours its nuclear export and stimulates its degradation, so inhibiting the p53–MDM2 interaction with synthetic molecules should lead to p53-mediated cell-cycle arrest or apoptosis in p53-positive stressed cells. Novartis, K125 443, CH-4002 Basel, Switzerland. e-mail: [email protected] doi:10.1038/nrc991 102 The p53 tumour suppressor is present at a low concentration in normal cells. Stress, such as hypoxia or DNA damage, causes p53 to accumulate in the nucleus, where it is active1. Depending on the cellular stress and cell type, the activation of p53 can lead to various responses (FIG. 1). For example, DNA damage might result in growth arrest to allow for repair of the damage, or apoptosis — both of these responses aim to prevent damaged cells from proliferating and passing mutations on to the next generation2. p53 acts as a transcription factor, and is able to activate many genes to induce these specific functions. As cells that lack functional p53 are unable to respond appropriately to stress, they can accumulate mutations that favour the development of cancer. The influence of p53 loss in cancer has been shown in vivo by inactivating the Trp53 gene (which encodes p53) in mice 3. These mice develop normally, but are tumour prone. By 6 months of age, 74% of the animals have developed tumours, and, within 10 months, all of them are dead. In humans, about 50% of tumours are thought to possess a mutated form of TP53 (REF. 4) and, in some tumour types, these mutations are associated with poor prognosis and treatment failure5. For these reasons, many pharmaceutical companies and academic laboratories are engaged in drug discovery activities to target tumours with defective p53 (REFS 6,7). In many other tumours, however, p53 is present in its wild-type form, which offers the possibility of stimulating its tumour-suppressor activity to eradicate | FEBRUARY 2003 | VOLUME 3 the tumours. The involvement of p53 in effective cancer treatment is shown by the fact that radiotherapy induces various proteins that recognize damage and transmit this information to p53, which, in turn, induces cell death8. A new approach consists of stimulating p53 by inhibiting its interaction with MDM2. MDM2 and p53 are part of an auto-regulatory feedback loop9,10 (FIG. 2). MDM2 is transcriptionally activated by p53 (REF. 11) and MDM2, in turn, inhibits p53 activity in several ways. MDM2 binds to the p53 transactivation domain12 and thereby inhibits p53-mediated transactivation13. MDM2 also contains a signal sequence that is similar to the nuclear export signal of various viral proteins14 and, after binding to p53, it induces its nuclear export15. As p53 is a transcription factor, it needs to be in the nucleus to be able to access the DNA; its transport to the cytoplasm by MDM2 prevents this. Finally, MDM2 is a ubiquitin ligase16, so is able to target p53 for degradation by the proteasome17,18. In normal cellular conditions, p53 is constantly degraded by MDM2, and is therefore present at low levels. The importance of MDM2 in the control of p53 activity is demonstrated with Mdm2 gene-knockout mice19,20. Their embryos die very early during gestation, but additional deletion of Trp53 rescues them from death. This indicates that, during development, MDM2’s ability to control p53 is essential. It has also been shown that MDM2 overexpression blocks p53-mediated cell-cycle www.nature.com/reviews/cancer © 2003 Nature Publishing Group REVIEWS Summary • The tumour suppressor p53 induces cell death by apoptosis in response to various stress conditions, such as oncogene activation or DNA damage. • The loss of p53 tumour-suppressor activity — either by mutation/deletion of the TP53 gene or by inhibition of the p53 protein — favours the development of cancer. • The MDM2 protein is a negative regulator of p53. After binding to p53, it inhibits its transcriptional activity, favours its nuclear export and stimulates its degradation. • The overexpression of MDM2 in various tumours inhibits p53, therefore favouring uncontrolled cell proliferation. • The inhibition of the p53–MDM2 interaction is an attractive strategy to activate p53mediated apoptosis in tumours with overexpressed MDM2, but wild-type p53. • Several low-molecular-weight compounds and peptides that inhibit the p53–MDM2 interaction have been obtained. The peptidic inhibitors show an antiproliferative effect in tumour cells overexpressing MDM2. Cellular stress DNA damage Activated oncogenes Hypoxia Ribonucleotide depletion Telomere erosion Cytoplasm p53 Properties of the p53–MDM2 complex Cellular responses Apoptosis Cell-cycle arrest DNA repair Differentiation Senescence p53 targets Nucleus Figure 1 | The p53-mediated response. p53 exists in nonstressed cells at a very low concentration. Under stress conditions, the p53 protein accumulates in the cell, binds in its tetrameric form to p53-response elements and induces the transcription of various genes that are involved in cell-cycle control, apoptosis, DNA repair, differentiation and senescence. The loss of p53 tumour-suppressor activity by mutation/deletion of TP53 or inhibition of p53 allows the proliferation of the cells that are damaged under the stress conditions. This uncontrolled proliferation can lead to tumour development. NUCLEAR MAGNETIC RESONANCE (NMR). A technique that uses the magnetic properties of certain atomic nuclei (such as 1 H, 13C and 15N) to determine the structure of the proteins. X-RAY CRYSTALLOGRAPHY A technique that uses the diffraction of the X-rays to determine the structure of the proteins. samples shows that MDM2 is amplified in 7% of these tissues30. The highest frequency of MDM2 amplification is observed in soft-tissue tumours, osteosarcomas and oesophageal carcinomas. Furthermore, many reports describe the overexpression of MDM2 in different types of tumour 31–33. The presence of high levels of MDM2 in these tumours might be an important element for their survival, because it decreases their ability to activate p53. The design of compounds that prevent the interaction between p53 and MDM2 is therefore an attractive strategy for activating p53 tumour-suppressor activity in tumours. Two different approaches have so far been described. The first one, which consists of designing MDM2 antisense oligodeoxynucleotides, has already been reviewed34 and will not be covered here. The second one consists of synthesizing lowmolecular-weight compounds that, after binding at the interface between the p53 and the MDM2 proteins, prevent their association. So, what progress has been made in this area of research? arrest and apoptosis21. The activation of p53 tumoursuppressor activity therefore depends on its association with MDM2. Several pathways activate p53 via the control of its interaction with MDM2. For example, DNA damage induces the phosphorylation of different p53 residues (Ser15, Thr18 or Ser20), which prevents them from binding to MDM2 (REFS 22–27). Alternatively, the activation of oncogenes such as c-MYC or RAS prevents MDM2-mediated degradation of p53 via expression of ARF, which — after binding to MDM2 — abolishes MDM2-mediated degradation of p53 (REFS 28,29). It is important to note that ARF does not bind at the p53–MDM2 interface and, therefore, is not a competitive inhibitor. Overexpression of the MDM2 protein should have negative consequences for the cell, because it diminishes its ability to activate the p53 pathway under stress conditions. The analysis of more than 3,000 tumour NATURE REVIEWS | C ANCER The regions involved in the interaction between p53 and MDM2 were first identified in a yeast two-hybrid screen35 and in immunoprecipitation experiments12. The MDM2-binding domain on p53 was localized at the amino terminus, from residues 1–41 (REF. 35) or 1–52 (REF. 12); the p53-binding domain on MDM2 was also localized at the amino terminus, from residues 1–118 (REF. 35) or 19–102 (REF. 12). Site-directed experiments have subsequently shown the importance of a few key p53 residues — Leu14, Phe19, Leu22 and Trp23 (REF. 36) — and a minimal MDM2-binding site on the p53 protein was mapped with p53-derived peptides from residues 18–23 (REF. 37). The strength of the interaction between p53 peptides and MDM2 fragments has been determined with several methods22,25,38,39. The experimental values of Kd (apparent dissociation constant) range from 60 to 700 nM depending on the length of the p53-derived peptides. NUCLEAR MAGNETIC RESONANCE (NMR) studies of p53derived peptides show that they are loosely folded in solution40–42. These measurements, together with the X-ray data (see below), indicate that the peptide probably adopts a helical conformation upon binding to MDM2. The regions that correspond to residues 13–119 of Xenopus laevis MDM2 and residues 17–125 of human MDM2 have been crystallized in complex with a peptide that corresponds to amino acids 15–29 of p53 (p53 15–29) and their structures have been determined by X-RAY CRYSTALLOGRAPHY39. p5315–29 binds into a large cleft that is present at the surface of MDM2 (FIG. 3a). The residues 19–25 form an α-helix and residues 17, 18 and 26–29 take a more extended conformation. Thr18 is particularly important for the stability of the helix39 and the regulation of the p53–MDM2 interaction by phosphorylation22,23. A detailed structural analysis of the interface between p53 and MDM2 reveals many factors that must be considered when aiming to inhibit this interaction. Only one of the two partners (MDM2) has a structurally VOLUME 3 | FEBRUARY 2003 | 1 0 3 © 2003 Nature Publishing Group REVIEWS DNA damage p53 Ubiquitylation Cytoplasm Inhibitors of the p53–MDM2 interaction Induces p53 degradation MDM2 Favours nuclear export Proteasome Activated oncogenes ? Nucleus p53 targets ARF Inhibitors of MDM2 Tumour-suppressor activity Blocks transactivation p53-independent activities MDM2 Figure 2 | Regulation of p53 by MDM2. p53 and MDM2 form an auto-regulatory feedback loop. p53 stimulates the expression of MDM2; MDM2 inhibits p53 activity because it blocks its transcriptional activity, favours its nuclear export and stimulates its degradation. Different cellular signals, such as DNA-damage or oncogene activation, induce p53 activation. DNA damage favours p53 phosphorylation, preventing its association with MDM2. Activated oncogenes activate the ARF protein, which prevents the MDM2-mediated degradation of p53. Similarly, inhibitors of the p53–MDM2 interaction should activate p53 tumour-suppressor activity in tumour cells that express wild-type p53. These compounds, because they bind to MDM2, could also affect the p53independent activities of MDM2. well-defined binding site. The inhibitors should therefore aim to mimic the other partner (p53). One of the two interfaces (p53) is formed by only one segment of contiguous amino acids, allowing the design of peptidic inhibitors (p53 mimics). Three residues — Phe19, Trp23 and Leu26 (FIG. 3b) — contribute to a large extent to the interaction and consequently to the binding energy of the p53 peptides43. The inhibitors of the p53–MDM2 interaction will have to contain mimics of these amino acids. There are only three hydrogen bonds connecting p53 to MDM2, and at least the most buried one (via p53 Trp23) will have to be preserved to ensure sufficient affinity of the inhibitors. The interface between both proteins is rather small (the calculated accessible surface area44 that is buried at the interface on MDM225–109 and p5317–29 is about 660 Å2 and 809 Å2, respectively), indicating that it might be possible to design relatively small inhibitors. This is important because molecules with molecular weights that are higher than 500 Da usually have a lower oral bioavailability. The interface is twisted (the planarity 44 of the MDM2 interface is 3.1). The less flat the interface between two partners, the greater the tendency of one of the two partners to be buried (here, p53). The burial of an inhibitor is usually linked to its partial desolvation, which leads to a favourable entropic contribution in the binding energy. The p53–MDM2 interface is hydrophobic (70% of the atoms at the interface are non-polar), and therefore inhibitors of the p53–MDM2 interaction will have to contain lipophilic groups. The presence of lipophilic groups usually improves the binding energy because of the favourable contribution of entropy. However, highly lipophilic inhibitors will show a decrease in bioavailability. 104 | FEBRUARY 2003 | VOLUME 3 p53 and MDM2 family members Recently, two TP53-related genes (TP63 and TP73) and one MDM2-related gene (MDMX) have been identified45–47. Several splice variants of TP63, TP73 and MDM2 are expressed in the cell48–50 — for example, more than 40 different splice variants of MDM2 have been identified — which further increases the number of p53 and MDM2 family members. These proteins share many properties with p53 or MDM2, but they also have distinct cellular functions50,51. Several lines of evidence indicate that p53 is more involved in tumour suppression, whereas p73 and p63 are more important during development and differentiation52. This is particularly well exemplified with the different phenotypes that are observed with the Trp53-, Trp63- and Trp73knockout mice53. p53 has an overall primary sequence similarity of 69% and 62% with p73 and p63, respectively, and MDM2 has an overall primary sequence similarity of 37% with MDMX. This homology indicates that p53 and MDM2 might associate with other family members. Indeed, MDMX has been shown to bind to p53 (REF. 47) and, not surprisingly, inhibitors of the p53–MDM2 interaction also disrupt the p53–MDMX interaction54. However, because small structural differences exist between MDM2 and MDMX, it might be possible in the future to identify compounds that more specifically inhibit one of these two interactions. p73 and p63 also associate with MDM2 (REFS 55,56), as would be expected from their strong homology with p53. p73derived peptides have a similar affinity for MDM2 as do p53-derived peptides22,57. p73 and p63 also interact with MDMX47,56. Altogether, the available data indicate that www.nature.com/reviews/cancer © 2003 Nature Publishing Group REVIEWS a Side negative contribution of entropy in the binding energy. To minimize this effect, the non-natural amino acids α-amino isobutyric acid (AIB) and 1-amino-cyclopropanecarboxylic acid (AC3C) were introduced in the peptides to enhance their pre-organization in solution. These modifications led to more potent peptides61,62 (4 and 5, TABLE 1). Circular dichroism and NMR measurements confirm an increased pre-organization of peptides 4 and 5 in solution61,62. However, because both peptides are still flexible in solution, their cyclization could further enhance their pre-organization and, therefore, their potency. Two complementary strategies have been adopted to further improve the affinity (enthalpic contribution) of peptide 5. The first one was to replace the residue corresponding to p53 Tyr22 with a phosphonomethylphenylalanine (PMP) to favour the formation of a salt bridge with MDM2 (REF. 62). The second one was to substitute the residue corresponding to p53 Trp23 for 6-chloro-tryptophan (6CLTRP), to fill a small hydrophobic cavity left unoccupied by wild-type p53 in the MDM2 cleft62. The replacement of the residue corresponding to p53 Tyr22 led to an ~7-fold increase in potency (6, TABLE 1) and the second strategy led to a further 60-fold increase in potency (7, TABLE 1). Altogether, blocking the conformation and enhancing the affinity of peptide 3, leads to an increase of potency of about 1,780-fold. Top N N C C b Phe19 Trp23 Leu26 PHAGE DISPLAY A technology that is used for displaying a protein (or peptide) on the surface of a bacteriophage, which contains the gene(s) that encodes the displayed protein(s), thereby physically linking the genotype and phenotype. IC50 The concentration of an inhibitor that is required to inhibit 50% of the p53–MDM2 interaction. AIB (α-amino isobutyric acid). A non-natural amino acid that is used to favour helical conformations in peptides. AC3C (1-aminocyclopropanecarboxylic acid). A non-natural amino acid that is used to stabilize 310-helix conformations in peptides. PMP (Phosphonomethylphenylalanine). A tyrosine substituted at its hydroxyl group by a phosphonomethyl moiety. Figure 3 | Structure of the p53–MDM2 complex. a | The surface of MDM225–109 is in white and the backbone of p5317–29 is in green. Two different views of the complex are presented, and the amino (N) and carboxyl (C) termini of the p53 peptide are indicated. b | The p5317–29 backbone is in grey and the side chains of Phe19, Trp23 and Leu26 are represented. The surface of MDM225–109 is in yellow. inhibitors of the p53–MDM2 interaction might also affect the other p53 and MDM2 family members. For example, it has been shown that MDM2 and MDMX inactivate the transcriptional activity and the apoptotic function of two splice variants of p73, p73α and p73β58. Therefore, inhibitors of the p53–MDM2 interaction might also activate the p73-dependent pathway. Inhibitors of the p53–MDM2 interaction Peptide inhibitors. Early experiments have shown that p53-derived peptides inhibit the p53–MDM2 interaction35 and that the minimal MDM2 binding site on the p53 molecule can be reduced to p5318–23 (REF. 37). This indicates that peptides could be used as starting points in the design of inhibitors of the p53–MDM2 interaction. An initial effort to obtain more potent peptides was to display different peptide libraries on PHAGES59. This approach allowed the identification of a 12-mer peptide (2, TABLE 1) that is 29 times more potent in vitro than the corresponding p53 wild-type peptide (1, TABLE 1)60. Truncations of peptide 2 show that its size can be reduced to eight residues (3, TABLE 1), keeping an IC value in the micromolar range60. The introduction of non-natural amino acids can further enhance binding because of two factors — they can help to organize the structural conformation of the peptide in solution (entropy) before binding and can alter chemical characteristics that directly affect binding (enthalpy). The p53 peptides take a helical structure when bound to MDM2, but adopt several conformations in solution40,41, probably leading to a 50 6CLTRP (6-chloro-tryptophan). Tryptophan with a chlorine at position-6 (corresponds to Cη2). ELISA (Enzyme-linked immunosorbent assay). A solidphase immunoassay that detects the interaction between proteins and specific antibodies. NATURE REVIEWS | C ANCER Non-peptidic or natural inhibitors. Little has been reported on non-peptidic or natural inhibitors of the p53–MDM2 interaction. Majeux et al. have developed a computational approach for the evaluation of electrostatic desolvation energies of receptor and ligand following binding 63. They docked into MDM2, using computing tools, a library of rigid fragments representing building blocks that are frequently found in drugs. This analysis indicates that 1,4-benzodiazepine2-one (8, TABLE 1) can mimic Phe19 and Trp23 and that C-3 substituted derivatives of this compound could also mimic Leu26. The screening of microbial extracts that were generated from the fermentation of a diverse collection of microorganisms led to the identification of the fungal metabolite, chlorofusin64 (9, TABLE 1). Chlorofusin inhibits the p53–MDM2 interaction with an IC50 of 4.6 µM in an enzyme-linked immunosorbent assay (ELISA). The binding mode of chlorofusin is not known and the published data do not show whether this natural compound binds at the interface between p53 and MDM2. Recently, Zao and collaborators have reported the synthesis of polycyclic compounds65. These molecules are built on the same building block, which is used as a scaffold for substitutions that mimic Phe19 and Trp23 (10, TABLE 1). One of these compounds induces p53 accumulation/activation and apoptosis in cell lines that express wild-type p53. Finally, it has been shown that chalcone derivatives inhibit the p53–MDM2 interaction66 (11, TABLE 1). On ELISA and gel-shift assays, these compounds showed an ability to inhibit the p53–MDM2 interaction. The VOLUME 3 | FEBRUARY 2003 | 1 0 5 © 2003 Nature Publishing Group REVIEWS Table 1 | Inhibitors of the p53–MDM2 interaction Compound Structure IC50 (µM) Type of References compound 1 Ac-Gln-Glu-Thr-Phe19-Ser-Asp-Leu-Trp23-Lys-Leu-Leu26-Pro-NH2 8.7 Wild-type p53 2 Ac-Met-Pro-Arg-Phe19-Met-Asp-Tyr-Trp23-Glu-Gly-Leu26-Asn-NH2 0.3 Phage-derived peptide 62 3 Ac-Phe19-Met-Asp-Tyr-Trp23-Glu-Gly-Leu26-Asn-NH2 8.9 Truncated phagederived peptide 62 4 Ac-Glu-Thr-Phe19-Aib-Asp-Aib-Trp23-Lys-Aib-Leu26-Aib-Glu-NH2 5.2 Constrained wild-type peptide 61 5 Ac-Phe19-Met-Aib-Tyr-Trp23-Glu-Ac3c-Leu26-Asn-NH2 2.2 Constrained peptide 3 62 6 Ac-Phe19-Met-Aib-Pmp-Trp23-Glu-Ac3c-Leu26-Asn-NH2 0.3 Peptide 5 with a PMP at position 22 62 7 Ac-Phe19-Met-Aib-Pmp-6ClTrp23-Glu-Ac3c-Leu26-Asn-NH2 0.005 Peptide 6 with a 6ClTrp at position 22 62 ND 1,4 benzodiazepine2-1 63 4.6 Chlorofusin 64 ND Polycyclic compound 65 117 Chalcone derivative 66 8 N NH O 9 OH O H N O O O NH O H2N O O HN OH O HN Cl O HN H N O O N N H HN H2N O O O O O N H HO O 10 OH N H O 11 O O Cl Cl O O OH Compounds 1 to 7 are peptides. Compounds 8, 10 and 11 are synthetic molecules. Compound 9 is a natural compound. Aib, α-amino isobutyric acid; Ac3c, 1-amino-cyclopropanecarboxylic acid; PMP, phosphonomethylphenylalanine; 6ClTrp, 6-chloro-tryptophan. binding mode of these compounds was determined by NMR spectroscopy. They bind to the p53 binding site on the MDM2 protein. However, they do not fully occupy the cleft and only interact with the region that is involved in the interaction with Trp23, which might explain their low potency (between 50 µM and 250 µM in ELISA). peptides to the leader sequence of the antennapedia protein (referred to below as tagged peptides) 69. Finally, it has been shown that peptide 7 can be used directly in cellular experiments70,71. However, data are not yet available on the biological properties of the non-peptidic inhibitors. Biological properties of the inhibitors Induction of p53 activity. The experimental results essentially agree with what would be expected, given our current knowledge of the p53–MDM2 interaction. Once transfected in MDM2-overexpressing osteosarcoma cells (SA1), the GST inhibitors bind to MDM2, preventing its interaction with p53 as shown in immunoprecipitation experiments68. This induces a redistribution of cellular p53 — so that it is found predominantly in the nucleus68 — and its accumulation67,68,70,71. This would be a predicted response following administration of inhibitors of the p53–MDM2 interaction, as these molecules should Various strategies have been used to study the biological effect of inhibitors of the p53–MDM2 interaction in cellular systems. Böttger and collaborators have inserted peptide 2 or the corresponding wild-type peptide into a loop region of the Escherichia coli thioredoxin protein (referred to below as TIP)67. Wasylyk and collaborators have fused the same peptide (or its dimer variant) to the glutathione S-transferase protein (referred to below as GST)68. Kanovsky and collaborators have coupled wild-type p53-derived 106 | FEBRUARY 2003 | VOLUME 3 www.nature.com/reviews/cancer © 2003 Nature Publishing Group REVIEWS prevent MDM2-mediated degradation of p53 (REFS 17,18). However it has not been shown directly that these inhibitors actually block MDM2-mediated ubiquitylation of p53. This nuclear accumulation of p53 also induces its transcriptional activation — transfection or microinjection of TIP inhibitors in various tumour cell lines induces the activation of a p53-responsive reporter gene67. Similar results are obtained with GST inhibitors68. Peptide 7 and GST inhibitors also stimulate the transcription of the endogenous MDM2 and CDKN1A genes, and expression of the corresponding proteins (MDM2 and WAF1, also known as p21 and CIP1)68,70,71. By contrast, peptide 7 does not induce the expression of WAF1 in the p53-null Saos-2 osteosarcoma cells, or in p53-mutant-expressing SK-BR-3 cells71. Similarly, GST inhibitors have no effect in head and neck carcinoma HSC2 cells that lack p53 (REF. 68). FACS (Fluorescence-activated cell sorting). A technique that is used in flow cytometry to detect cells that are labelled with fluorescent dyes. TUNEL (TdT-mediated dUTP-X nickend labelling). A method that is used to measure DNA strand breaks during apoptosis. E6 PROTEIN A viral oncoprotein that is derived from certain human papillomavirus types that are associated with increased risk of cervical cancer. E6 binds to and targets p53 for ubiquitinmediated degradation. Inhibition of cell proliferation. Inhibitors of the p53–MDM2 interaction also affect the proliferation of tumour cell lines. TIP inhibitors induce a decrease in the number of S-phase bromodeoxyuridine-positive T22 cells, indicating a cell-cycle arrest 67. In SA1 cells, the GST inhibitors decrease colony formation in a colony-forming assay, induce an increase in the number of cells with sub-G1 and G0/G1 contents of DNA, as measured by FACS, and, finally, stimulate cell death from apoptosis, as determined in a TUNEL assay 68. The effect of these inhibitors is abolished in the presence of the human papillomavirus 16 (HPV16) E6 PROTEIN, showing that they stimulate the p53-dependent pathway. Peptide 7 induces a cell-cycle arrest in the HCT116 colon carcinoma cell line, which contains low amounts of MDM2, whereas it induces apoptosis in SA1 and JAR (choriocarcinoma) cells, which have a higher MDM2 concentration70. In SA1 cells, peptide 7 induces apoptosis via the release of cytochrome c from the mitochondria, and the activation of caspase-9 and caspase-3. The absence of apoptosis in HCT116 cells is not the consequence of an alteration in the p53-dependent apoptotic pathway, as ectopic expression of p53 stimulates apoptosis in HCT116 cells with deleted CDKN1A (REF. 72). This shows that, in some cell lines, inhibition of the p53–MDM2 interaction is not sufficient for the induction of apoptosis. Toxicity to normal tissues? Blaydes and WynfordThomas reported that inhibition of the p53–MDM2 interaction prevents the proliferation of normal human fibroblasts, raising the possibility that inhibitors of the p53–MDM2 interaction could be toxic for healthy tissues73. Preliminary evidence shows that inhibitors of the p53–MDM2 interaction are more toxic for tumour cells than for normal cells70. The effect of peptide 7 in two non-tumour cell lines — NHDF710 (normal human dermal fibroblasts) and HMEC2595 (human mammary epithelial cells) — was compared in a proliferation assay with its effect in SA1 cells. The peptide, which activates p53 in all three cell lines, has a greater effect on the proliferation of SA1 cells than on that of the two non-tumour cell lines. NATURE REVIEWS | C ANCER The results obtained with the tagged peptides are different69. These peptides are toxic to various tumour cell lines that express wild-type p53, but not to normal cells. However, they are also toxic to tumour cells that lack p53. This, together with the fact that no p53dependent apoptotic markers have been identified after treatment with the tagged peptides, indicates that they induce cell death via a p53-independent mechanism. As the three tagged peptides — p5312–20 (this peptide does not contain Trp23 and Leu26), p5317–26 and p5312–26 — are equally cytotoxic, but a p53-unrelated tagged peptide is not, the authors explain the observed cytotoxicity by the fact that the peptides contain the same common sequence: Glu-Thr-Phe-Ser. Conclusions and perspectives The growth or the death of cells depends to a large extent on the fine-tuning of the p53–MDM2 interaction. As MDM2 controls p53 activity at the post-translational level, inhibition of the p53–MDM2 interaction allows an immediate p53-mediated response. Compounds that prevent this interaction might be useful as new anticancer agents that activate the p53 pathway in tumours. The design of inhibitors of protein–protein interaction is, today, a challenge in medicinal chemistry74. The feasibility of such an approach depends on the structure of the protein–protein interface. A flat, large and polar interface is more difficult to target than a twisted, small and apolar interface. The p53–MDM2 interface corresponds in many aspects to the second type of interface. The p53-binding site on MDM2 is a deep, rather small and hydrophobic cleft. The p53–MDM2 interface therefore contains several structural features that are favourable to the design of inhibitors. It has been shown that it is possible to obtain very potent peptidic inhibitors, but these molecules are by no means drugs. It is now very important to identify potent low-molecular-weight compounds that block this interaction. Some pharmaceutical companies have run random screens with their compound libraries, but the results are, so far, disappointing: only weak inhibitors have been identified. This can be explained by a variety of factors. The correct compounds are not present in the screened libraries because the pharmaceutical companies, which have mainly targeted enzymes in the past, do not have libraries with the structural diversity that is required to target protein–protein interactions. Another factor is the high complementarity of the p53–MDM2 interface. It has been shown that an optimal ratio of receptor volume to ligand volume exists for good molecular recognition75. It might be difficult to find molecules that fill the Phe19, Trp23 and Leu26 sub-pockets with an optimal ratio by random screening. De novo design might therefore be the best way to generate high-affinity compounds. In such an approach, a first sub-pocket (for example, Trp23) will be filled with the correct pharmacophore mimic and, using rigid spacers (for entropic reasons), the two other sub-pockets will be filled in a stepwise VOLUME 3 | FEBRUARY 2003 | 1 0 7 © 2003 Nature Publishing Group REVIEWS manner. This approach, which is obviously more time-consuming in chemistry, might ultimately be the most productive. On the biological side, it has been shown that inhibition of the p53–MDM2 interaction with synthetic inhibitors such as peptides leads to activation of p53. However, all of the data available today come from in vitro experiments. It is therefore of utmost importance to show that inhibitors of the p53–MDM2 interaction have an antiproliferative effect in vivo. Unfortunately, the potency and/or the physico-chemical properties of the inhibitors that are available at present do not allow us to carry out such experiments. Several other points also remain to be examined. Inhibitors of the p53–MDM2 interaction will probably affect other p53 and MDM2-family members. It is therefore important to determine the biological consequences of the interaction between the inhibitors and these proteins. This might be a promising area for drug discovery, as p53 is deleted or mutated in many tumours. Another aspect is that the inhibitors of the p53–MDM2 interactions could also interfere with other MDM2-dependent pathways. It has been shown that MDM2 does more than just keep p53 under control. For example, it interacts with various cellular proteins, such as TAFII250 (REF. 76), Numb77 and MTBP78. The influence of the inhibitors of the p53–MDM2 interaction on these interactions has not been studied and because the inhibitors bind to MDM2, they could also affect some of the p53-independent functions of MDM2. Are inhibitors of the p53–MDM2 interaction 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 108 Oren, M. Regulation of the p53 tumor suppressor protein. J. Biol. Chem. 274, 36031–36034 (1999). Vousden, K. H. & Lu, X. Live or let die: the cell’s response to p53. Nature Rev. Cancer 2, 594–604 (2002). Donehower, L. A. et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215–221 (1992). Hainaut, P. & Hollstein, M. p53 and human cancer: the first ten thousand mutations. Adv. Cancer Res. 77, 81–137 (2000). Kirsch, D. G. & Kastan, M. B. Tumor-suppressor p53: implications for tumor development and prognosis. J. Clin. Oncol. 16, 3158–3168 (1998). Blagosklonny, M. V. p53: an ubiquitous target for anticancer drugs. Int. J. 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Online links DATABASES The following terms in this article are linked online to: LocusLink: CDKN1A | Mdm2 | MDM2 | MDMX | MYC | p53 | RAS | TP63 | TP73 | Trp53 | Trp63 | Trp73 FURTHER INFORMATION MDM2 database: http://www.infosci.coh.org/mdm2 p53 home page: http://p53.curie.fr Structure of p53–MDM2: http://www.rcsb.org/pdb/cgi/explore.cgi?pid=13806103734896 1&pdbId=1YCR Access to this interactive links box is free online. VOLUME 3 | FEBRUARY 2003 | 1 0 9 © 2003 Nature Publishing Group