p53-MDM2 Inhibitors Research Articles

The Fluorescent Two-Hybrid Assay to Screen for Protein–Protein Interaction Inhibitors in Live Cells: Targeting the Interaction of p53 with Mdm2 and Mdm4

Protein–protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells.We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53–Mdm2 inhibitors. However, none exhibited intracellular activity on p53–Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53–Mdm2 interaction as well as p53–Mdm4 interaction.Here, we show that the F2H assays enable side-by-side analysis of substances’ dual Mdm2–Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.

Discovery of AMG 232, a Potent, Selective, and Orally Bioavailable MDM2–p53 Inhibitor in Clinical Development

We recently reported the discovery of AM-8553 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Continued research investigation of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface, led to the discovery of a one-carbon tethered sulfone which gave rise to substantial improvements in biochemical and cellular potency. Further investigation produced AMG 232 (2), which is currently being evaluated in human clinical trials for the treatment of cancer. Compound 2 is an extremely potent MDM2 inhibitor (SPR KD = 0.045 nM, SJSA-1 EdU IC50 = 9.1 nM), with remarkable pharmacokinetic properties and in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model (ED50 = 9.1 mg/kg).

Prospective virtual screening for novel p53–MDM2 inhibitors using ultrafast shape recognition

The p53 protein, known as the guardian of genome, is mutated or deleted in approximately 50% of human tumors. In the rest of the cancers, p53 is expressed in its wild-type form, but its function is inhibited by direct binding with the murine double minute 2 (MDM2) protein. Therefore, inhibition of the p53-MDM2 interaction, leading to the activation of tumor suppressor p53 protein presents a fundamentally novel therapeutic strategy against several types of cancers. The present study utilized ultrafast shape recognition (USR), a virtual screening technique based on ligand-receptor 3D shape complementarity, to screen DrugBank database for novel p53-MDM2 inhibitors. Specifically, using 3D shape of one of the most potent crystal ligands of MDM2, MI-63, as the query molecule, six compounds were identified as potential p53-MDM2 inhibitors. These six USR hits were then subjected to molecular modeling investigations through flexible receptor docking followed by comparative binding energy analysis. These studies suggested a potential role of the USR-selected molecules as p53-MDM2 inhibitors. This was further supported by experimental tests showing that the treatment of human colon tumor cells with the top USR hit, telmisartan, led to a dose-dependent cell growth inhibition in a p53-dependent manner. It is noteworthy that telmisartan has a long history of safe human use as an approved anti-hypertension drug and thus may present an immediate clinical potential as a cancer therapeutic. Furthermore, it could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53-MDM2 inhibitors against variety of cancers. Importantly, the present study demonstrates that the adopted USR-based virtual screening protocol is a useful tool for hit identification in the domain of small molecule p53-MDM2 inhibitors.

Discovery of Potent and Orally Active p53-MDM2 Inhibitors RO5353 and RO2468 for Potential Clinical Development.

The development of small-molecule MDM2 inhibitors to restore dysfunctional p53 activities represents a novel approach for cancer treatment. In a previous communication, the efforts leading to the identification of a non-imidazoline MDM2 inhibitor, RG7388, was disclosed and revealed the desirable in vitro and in vivo pharmacological properties that this class of pyrrolidine-based inhibitors possesses. Given this richness and the critical need for a wide variety of chemical structures to ensure success in the clinic, research was expanded to evaluate additional derivatives. Here we report two new potent, selective, and orally active p53-MDM2 antagonists, RO5353 and RO2468, as follow-ups with promising potential for clinical development.

Rational Design and Binding Mode Duality of MDM2–p53 Inhibitors

Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 μM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone 27 (IC50 = 0.10 μM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21(WAF1) mRNA induction (2.7-fold over vehicle) upon oral dosing of 27 at 300 mg/kg.

Discovery of a new small-molecule inhibitor of p53–MDM2 interaction using a yeast-based approach

The virtual screening of a library of xanthone derivatives led us to the identification of potential novel MDM2 ligands. The activity of these compounds as inhibitors of p53–MDM2 interaction was investigated using a yeast phenotypic assay, herein developed for the initial screening. Using this approach, in association with a yeast p53 transactivation assay, the pyranoxanthone (3,4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one) (1) was identified as a putative small-molecule inhibitor of p53–MDM2 interaction.The activity of the pyranoxanthone 1 as inhibitor of p53–MDM2 interaction was further investigated in human tumor cells with wild-type p53 and overexpressed MDM2. Notably, the pyranoxanthone 1 mimicked the activity of known p53 activators, leading to p53 stabilization and activation of p53-dependent transcriptional activity. Additionally, it led to increased protein levels of p21 and Bax, and to caspase-7 cleavage. By computational docking studies, it was predicted that, like nutlin-3a, a known small-molecule inhibitor of p53–MDM2 interaction, pyranoxanthone 1 binds to the p53-binding site of MDM2.Overall, in this work, a novel small-molecule inhibitor of p53–MDM2 interaction with a xanthone scaffold was identified for the first time. Besides its potential use as molecular probe and possible lead to develop anticancer agents, the pyranoxanthone 1 will pave the way for the structure-based design of a new class of p53–MDM2 inhibitors.

Diaryl- and triaryl-pyrrole derivatives: inhibitors of the MDM2–p53 and MDMX–p53 protein–protein interactions

Screening identified 2-(3-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile as an MDM2-p53 inhibitor (IC50 = 12.3 μM). MDM2-p53 and MDMX-p53 activity was seen for 5-((1-(4-chlorophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (MDM2 IC50 = 0.11 μM; MDMX IC50 = 4.2 μM) and 5-((1-(4-nitrophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (MDM2 IC50 = 0.15 μM; MDMX IC50 = 4.2 μM), and cellular activity consistent with p53 activation in MDM2 amplified cells. Further SAR studies demonstrated the requirement for the triarylpyrrole moiety for MDMX-p53 activity but not for MDM2-p53 inhibition.

Discovery, Synthesis, and Biological Evaluation of Orally Active Pyrrolidone Derivatives as Novel Inhibitors of p53–MDM2 Protein–Protein Interaction

The p53-MDM2 interaction has been proved to be a valuable target to develop effective antitumor agents. Novel p53-MDM2 inhibitors bearing pyrrolidone scaffolds were successfully identified by structure-based design. The nanomolar inhibitor 5 possessed good p53-MDM2 inhibitory activity (K(i) = 780 nM) due to its hydrophobic and hydrogen bonding interactions with MDM2. Further hit optimization led to the discovery of a number of highly potent pyrrolidone derivatives with improved p53-MDM2 inhibitory activity and in vitro antiproliferative potency. Compounds 41 (K(i) = 260.0 nM) and 60a (K(i) = 150.0 nM) showed good and selective activity against tumor cells with deleted p53. In addition, these two compounds also effectively inhibited the tumor growth in the A549 xenograft model. Interestingly, compound 41 was proved to be a potent MDM2/MDMX dual inhibitor. The novel pyrrolidone p53-MDM2 inhibitors represent promising lead structures for the development of novel antitumor agents.

1122P - Inherited Abnormalities in Genes That During the Apoptosis Process and Cutaneous Melanoma Risk

ABSTRACT Background P53 (guardian of the genome), MDM2 (p53 inhibitor), BCL2 (anti-apoptotic) and BAX (proapoptotic) genes remove cells damaged by ultraviolet (UV) rays of sunlight and, therefore, are related to the origin of cutaneous melanoma (CM). All these genes are polymorphic in humans. The Arg wild allele of the P53 Arg72Pro polymorphism is more efficient than the variant Pro allele at inducing apoptosis. The variant G and A alleles of MDM2 T309G and the BCL2 C (-948) A polymorphisms, and the variant A allele of the BAX G(-248)A polymorphism are related to higher and lower expressions of the encoded proteins, respectively, than their wild T, C and G alleles. This study aimed to clarify the roles of the above-mentioned genetic polymorphisms in the risk and clinical manifestation of the tumor. Material and methods In this case-control study, genomic DNA from peripheral blood of 150 consecutive CM patients (75 males and 75 females, 138 Caucasians and 12 non-Caucasians aged 20-89 years) and 150 healthy subjects, matched by age, gender and race, was analyzed by polymerase chain reaction followed by enzymatic digestion. Results The frequencies of the P53 ArgArg and the BCL2 AA genotypes were higher in patients than in controls (58.7% versus 44.7%, P= 0.01) and (28.0% versus 15.3%, P= 0.004), respectively. Carriers of these genotypes had a 1.86 and 2.87-fold increased risk for CM than those with the remaining genotypes, respectively. Excesses of the P53 ArgArg plus BCL2 AA and P53 ArgArg plus BAX AA were seen in patients when compared to controls (36.1% versus 16.9%, P= 0.002) and (29.5% versus 15.3%, P= 0.008). Carriers of these genotypes had a 3.43 and 2.71-fold increased risk for CM than others, respectively. Conclusions The data suggest that P53 Arg72Pro, BCL2 C(-248)A and BAX G(-248)A polymorphisms, alone or combined, alter the risk for CM in our region. We believe that carriers of specific genotypes of the above-mentioned genes should receive additional recommendation to avoid exposition to sunlight, in addition to frequently being evaluated by a dermatologist for early disease diagnosis. Financial support: (FAPESP) Disclosure All authors have declared no conflicts of interest.

P53–Mdm2 inhibitors: patent review (2009 – 2010)

Introduction: p53 plays a central role in protecting the integrity of the genome. Its activity is ubiquitously lost in cancers, either by inactivation of its protein (p53 pathway) or by mutation in the p53 gene, thereby indicating its importance in understanding cancer and as a therapeutic target. Activated p53 is known to induce cell cycle arrest thereby leading to apoptosis and has been the subject of intensive research in the area of medicinal chemistry. Efforts are in progress to synthesize a variety of scaffolds that could inhibit the p53–Mdm2 interaction by binding to Mdm2 in the region where p53 is likely to bind. These molecules have the potential to be developed as anticancer drug candidates and have been largely explored by both academia and industry. Interestingly, some of these molecules are in the early stage of clinical trials.Areas covered: Areas covered in this review include patents relating to p53–Mdm2 inhibitors during the time period 2009 – 2010. The focus of the review was on small-molecule inhibitors.Expert opinion: Inducing apoptosis in cancerous cells by the activation of p53 is an area that is being actively explored. There are strong indications that it could become a therapeutic method for the treatment of cancer. As a result, extensive research is being performed by both academia and industry. It is observed that small molecules that are present in early clinical trials are expected to be developed as potential drugs for cancer therapy.

Targeted delivery of a novel palmitylated D-peptide for antiglioblastoma molecular therapy

Effective glioblastoma treatment with low toxicity is one of the most difficult challenges in cancer therapy. The interaction between tumor suppressor protein p53 and its negative regulator murine double minute 2 (MDM2) provides a promising target for specific therapy because an important subtype of glioblastoma harbors wild-type p53 and overexpressed MDM2. Several D-peptides have been previously reported to effectively antagonize MDM2 for binding to p53 with high affinity and unsurpassed specificity. However, poor cell penetration and lack of efficient delivery method hampered the therapeutic applicability of the most potent D-peptide, D-PMIβ. In this study, a novel lipophilic derivate of D-PMIβ (pDP) was developed. Liposome was chosen as a carrier for pDP, and cyclic pentapeptide c(RGDyK) was used as a targeting moiety for the treatment of glioblastoma. D-PMIβ was N-terminally modified with palmitic acid and the resultant c(RGDyK) decorated liposomes (RGD-liposomal pDP) showed almost 100% encapsulation efficiency and 10% loading efficiency. The abilities of palmitylated D-peptide to antagonize MDM2 and reactivate p53 specifically were confirmed by the western blot assay. The IC50 ratio of RGD-liposomal pDP in treating human umbilical vascular endothelial normal cells vs. U87 tumor cells was 10 times higher than that of RGD-liposomal doxorubicin. After intravenous administration, the median survival time of intracranial U87 glioblastoma-bearing nude mice treated with RGD-liposomal pDP (29 days) was significant longer than that of mice treated with blank RGD-liposome (23 days) (p<0.001). These results indicated that palmitylated D-peptide inhibitor of p53-MDM2 combined with RGD modified liposomes provided a potential molecular therapy for glioblastoma.

Synthesis and biological evaluation of thio-benzodiazepines as novel small molecule inhibitors of the p53–MDM2 protein–protein interaction

A series of thio-benzodiazepine p53–MDM2 inhibitors were designed and synthesized based on the principle of bioisosterism. Most of the thio-benzodiazepines had nanomolar to micromolar affinity toward MDM2. Particularly, compounds 8a (Ki = 0.52μM) and 8f (Ki = 0.32μM) showed binding activity comparable to the positive drug nutlin-3a (Ki = 0.23μM). Meanwhile, compound 8j exhibited excellent antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 1.06μM, which was about 23 times higher than that of nutlin-3a. The docking model also successfully predicted that this class of compounds mimicked three p53 critical residues binding to MDM2. The thio-benzodiazepines represent a promising class of non-peptide inhibitors of the p53–MDM2 interaction.

A direct arylation-oxidation route to 3-arylisoindolinone inhibitors of MDM2-p53 interaction

A route to the MDM2-p53 inhibitor isoindolinone pharmacophore from a pre-formed phthalimide is detailed. The route involves treatment of 3-hydroxy-2-(n-propyl)isoindolinone with a substituted benzene in the presence of triflic acid. The resulting 3-aryl-2-(n-propyl)isoindolinones are then oxidized to the corresponding 3-hydroxy-3-aryl-2-(n-propyl) isoindolinones by treatment with 2,2’-bipyridinium chlorochromate. The benzylic oxidation represents a rather rare oxochromium (VI)-mediated reaction in which a selective C–H to C–OH transformation occurs.

E2F1-dependent oncogenic addiction of melanoma cells to MDM2

One of the defining features of aggressive melanomas is their complexity. Hundreds of mutations and an ever increasing list of changes in the transcriptome and proteome distinguish normal from malignant melanocytic cells. Yet, despite this altered genetic background, a long-known attribute of melanomas is a relatively low rate of mutations in the p53 gene. However, it is unclear whether p53 is maintained in melanoma cells because it is required for their survival, or because it is functionally disabled. More pressing from a translational perspective, is to define whether there is a tumor cell-selective wiring of p53 that offers a window for therapeutic intervention. Here we provide genetic and pharmacological evidence demonstrating that p53 represents a liability to melanoma cells which they thwart by assuming an oncogenic dependency on the E3 ligase MDM2. Specifically, we used a combination of RNA interference and two structurally independent small molecule inhibitors of the p53/MDM2 interaction to assess the relative requirement of both proteins for the viability of normal melanocytes and a broad panel of melanoma cell lines. We demonstrated in vitro and in vivo that MDM2 is selectively required to blunt latent pro-senescence signals in melanoma cells. Notably, the outcome of MDM2 inactivation depends not only on the mutational status of p53, but also on its ability to signal to the transcription factor E2F1. These data support MDM2 as a drug target in melanoma cells, and identify E2F1 as a biomarker to consider when stratifying putative candidates for clinical studies of p53/MDM2 inhibitors.

Isoindolinone Inhibitors of the Murine Double Minute 2 (MDM2)-p53 Protein−Protein Interaction: Structure−Activity Studies Leading to Improved Potency

Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC(50) = 0.23 ± 0.01 μM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC(50) = 0.17 ± 0.02 μM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.

Catalytic, enantioselective synthesis of stilbene cis-diamines: A concise preparation of (−)-Nutlin-3, a potent p53/MDM2 inhibitor

The first highly diastereo- and enantioselective additions of aryl nitromethane pronucleophiles to aryl aldimines are described. Identification of an electron rich chiral Bis(Amidine) catalyst for this aza-Henry variant was key to this development, leading ultimately to differentially protected cis-stilbene diamines in two steps. This method then became the lynchpin for an enantioselective synthesis of (-)-Nutlin-3 (Hoffmann-LaRoche), a potent cis-imidazoline small molecule inhibitor of p53-MDM2 used extensively as a probe of cell biology and currently in drug development.

Abstract 4529: Cellular pharmacokinetic and activity studies with the MDM2-p53 inhibitor Nutlin-3

Abstract The tumor suppressor p53 plays a central role in the cell cycle, apoptosis, DNA repair and senescence. Murine double minute 2 (MDM2) negatively regulates the activity of the tumor suppressor protein p53. Nutlin-3, a small molecule inhibitor of MDM2 which can block the interaction of p53 and MDM2, is under preclinical investigation as a nongenotoxic activator of the p53 pathway. In order to understand the cellular pharmacology of nutlin-3 in cells with different p53 status, Nutlin-3 activity and its cellular uptake were studied in p53 wild type and p53 non-functional cancer cell lines. Human colon cancer cell lines HCT116 (p53wt), HCT116 p53−/− and HCT116 N7 (p53 non-functional due to E6 expression), and the human ovarian cancer cell lines A2780, A2780CP70 (p53 mutated) were treated with Nutlin-3 alone for up to 96 hours. Nutlin-3 is stable in tissue culture medium for up to 120 hours and can be readily detected in both HCT116 and A2780 cells. Although intracellular concentrations do not exceed extracellular levels, at 10 µM Nutlin-3 96 hours cellular uptake in p53 wild type cells is two to three times higher (HCT116 25 µg/1×106 cells, A2780 12 µg/1×106 cells) than in p53 non-functional cells (HCT116 p53−/− 9.1 µg/1×106 cells, HCT116 N7 8.2 µg/1×106 cells, A2780 CP70 5.3 µg/1×106 cells). Up to a concentration of 50 μM Nutlin-3, there is no clear evidence of saturation of uptake in HCT116 cells; however, in A2780 cells the intracellular concentrations are the same following exposure to 30μM or 50μM Nutlin-3. Nutlin-3 is 10 to 20-fold more growth-inhibitory and 3 to 9-fold more cytotoxic (by SRB and clonogenic assay) in p53 wild type cells: HCT116 (GI50 = 2.4 ± 0.3 µM, LD50 = 6.8 ± 2.9 µM) and A2780 (GI50 = 0.9 ± 0.3 µM, LD50 = 5.5 ± 0.5 µM), versus matched p53-deficient cells: HCT116 p53−/− (GI50 = 24 ± 2.6 µM, LD50 = 48 ± 1.4 µM), HCT116 N7 (GI50 = 17 ± 2 µM, LD50 ≥ 50 µM) and A2780 CP70 (GI50 = 23 ± 1.7 µM, LD50 = 31 ± 13 µM). Following rernoval of extracellular Nutlin-3 differential cytotoxicity is lower in cells remaining after 24 or 96 hours treatment. Overall, these studies demonstrate that both Nutlin-3 cellular uptake and growth inhibition/cytoxicity is dependent upon p53 status. Our results suggest that functional p53 promotes the intracellular accumulation of Nutlin-3. Potential mechanisms are: a) Direct binding to p53 or MDM2, b) Increased uptake - possibly as a result of expression of a p53-regulated influx transporter, c) Decreased efflux - possibly due to the reduced activity of an efflux pump secondary to Nutlin-3 induced cellular toxicity. These experiments demonstrate that both the cellular uptake and growth inhibition/cytotoxicity of Nutlin-3 is more pronounced in p53 wild type cell lines than in p53 mutated or deficient cell lines. Furthermore, maximal differential activity of p53/MDM2 antagonists in p53 wild type and p53 non-functional cells requires continuous exposure. (This work was supported by the CRUK) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4529.

Differential binding of p53 and nutlin to MDM2 and MDMX: Computational studies

Half of human tumours have mutated p53 while in the other half, defective signalling pathways block its function. One such defect is the overexpression of the MDM2 and MDMX proteins. This has led to an intense effort to develop inhibitors of p53-MDM2/MDMX interactions. Nutlin is the first such compound described to block p53-MDM2 interactions. Molecular dynamics simulations have been used to explore the differences in binding of p53 and nutlin to MDM2/MDMX. Simulations reveal that p53 has a higher affinity for MDM2 than MDMX, driven by stronger electrostatic interactions. p53 is displaced from MDM2 by nutlin because it is more flexible, thus paying a larger entropic penalty upon sequestration by MDM2. The inherent plasticity of MDM2 is higher than that of MDMX, enabling it to bind both p53 and nutlin. The less flexible MDMX interacts with the more mobile p53 because the peptide can adapt conformationally to dock into MDMX, albeit with a reduced affinity; nutlin, however is rigid and hence can only interact with MDMX with low affinity. Evolutionarily, the higher affinity of MDM2 for p53 may enable MDM2 to bind p53 for longer periods as it shuttles it out of the nucleus; in contrast, MDMX only needs to mask the p53 TA domain. This study enables us to hypothesize gain of function mutations or those that have decreased affinity for nutlin. These conclusions provide insight into future drug design for dual inhibitors of MDM2 and MDMX, both of which are oncoproteins found overexpressed in many cancers.

Discovery and Optimization of Chromenotriazolopyrimidines as Potent Inhibitors of the Mouse Double Minute 2−Tumor Protein 53 Protein−Protein Interaction

Tumor protein 53 (p53) is a critical regulator of cell cycle and apoptosis that is frequently disabled in human tumors. In many tumor types, p53 is deleted or mutated, but in others p53 is inactivated by overexpression or amplification of its negative regulator mouse double minute 2 (MDM2). A high-throughput screening effort identified 6,7-bis(4-bromophenyl)-7,12-dihydro-6H-chromeno[4,3-d][1,2,4]triazolo[1,5-a]pyrimidine as a potent inhibitor of the MDM2-p53 protein-protein interaction. This screening hit was found to be chemically unstable and difficult to handle due to poor DMSO solubility. Co-crystallization with the target protein helped to direct further optimization and provided a tractable lead series of novel MDM2-p53 inhibitors. In cellular assays, these compounds were shown to upregulate p53 protein levels and p53 signaling and to cause p53-dependent inhibition of proliferation and apoptosis.