p53-MDM2 Inhibitors Research Articles

Abstract 1225: NVP-HDM201: cellular and in vivo profile of a novel highly potent and selective PPI inhibitor of p53-Mdm2

Abstract Stabilization of p53 protein by preventing its interaction with the negative regulator Mdm2 leads to selective induction of the p53 pathway, thus offering a promising cancer therapeutic strategy in p53 wild-type tumors. In the present study, we show the identification of NVP-HDM201, a novel, highly optimized, and selective inhibitor of the p53-Mdm2 interaction. NVP-HDM201 activates p53 in human cells and induces robust p53-dependent cell cycle arrest and apoptosis, selectively in p53 wild-type tumor cells. Its activity and selectivity has been tested and confirmed across a large panel of cancer cell lines from the Cancer Cell Line Encyclopedia. In vivo, NVP-HDM201 shows a dose-proportional pharmacokinetic (PK) profile and a clear PK/PD relationship, resulting in tumor growth inhibition and regression in SJSA-1 tumor-bearing rats at well-tolerated oral (p.o.) doses. The validation and understanding of its mechanism of action, the overall favorable drug-like properties and the characterization of its on-target toxicological profile in preclinical species strongly supported the initiation of Phase I clinical trials with NVP-HDM201 in pre-selected patients with p53 wild-type tumors. Citation Format: Sébastien Jeay, Patrick Chène, Stéphane Ferretti, Pascal Furet, Bjoern Gruenenfelder, Vito Guagnano, Nelson Guerreiro, Ensar Halilovic, Francesco Hofmann, Joerg Kallen, Michelle Léonard, Robert Mah, Keiichi Masuya, Rita Ramos, Caroline Rynn, Stephan Ruetz, Thérèse Stachyra-Valat, Stefan Stutz, Andrea Vaupel, Jens Wuerthner, Philipp Holzer. NVP-HDM201: cellular and in vivo profile of a novel highly potent and selective PPI inhibitor of p53-Mdm2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1225.

Abstract 1239: NVP-HDM201: Biochemical and biophysical profile of a novel highly potent and selective PPI inhibitor of p53-Mdm2

Abstract An effective strategy to restore p53 activity in cancer cells containing wild type p53 is to inhibit the Mdm2-p53 protein-protein interaction (PPI). NVP-HDM201 is a novel PPI inhibitor currently under evaluation in a Phase I clinical trial. It binds to the p53 binding-site of the Mdm2 protein, disrupting the interaction of the two proteins and leading to the activation of the p53 pathway. NVP-HDM201 belongs to a novel chemical series with a distinct biophysical and biochemical profile. Affinity constant of NVP-HDM201 for Mdm2 is in the picomolar range, with a selectivity ratio greater than a 10000-fold vs. Mdm4. Analysis of its binding mode provides evidence for a distinct set of critical interactions between the small molecule and its target, as compared with our other Mdm2 inhibitor NVP-CGM097, and explains as to why NVP-HDM201 binds equally to human, mouse, rat and dog Mdm2. Characterization of its binding kinetics indicates that the optimized interactions of NVP-HDM201 with Mdm2 protein are responsible for the increased stabilization of the complex resulting in high potency against Mdm2. This feature, together with favorable physicochemical and drug-like properties, supported the selection of NVP-HDM201 for clinical development. Citation Format: Thérèse Stachyra-Valat, Frédéric Baysang, Anne-Cécile D’Alessandro, Erdmann Dirk, Pascal Furet, Vito Guagnano, Joerg Kallen, Lukas Leder, Robert Mah, Keiichi Masuya, Stefan Stutz, Andrea Vaupel, Francesco Hofmann, Patrick Chène, Sébastien Jeay, Philipp Holzer. NVP-HDM201: Biochemical and biophysical profile of a novel highly potent and selective PPI inhibitor of p53-Mdm2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1239.

An Effective Virtual Screening Protocol To Identify Promising p53-MDM2 Inhibitors.

The p53-MDM2 interaction is a well-known protein-protein contact, and its disruption is a key event for p53 activation and induction of its oncosuppressor response. The design of small molecules that can block the p53-MDM2 interaction and reactivate the p53 function is a promising strategy for cancer therapy. To date, several compounds have been identified as p53-MDM2 inhibitors, and X-ray structures of MDM2 complexed with several ligands are available in the Brookhaven Protein Data Bank. These data have been exploited to compile a hierarchical virtual screening protocol. The first steps were aimed at selecting a focused library, which was submitted in parallel to docking and pharmacophore model alignment. Selected compounds were subjected to inhibition assays of both cellular vitality (MTT) and p53-MDM2 interaction (ELISA and co-immunoprecipitation), disclosing four nanomolar inhibitors.

Design and synthesis of new inhibitors of p53–MDM2 interaction with a chalcone scaffold

The virtual screening of a library of chalcone derivatives led us to the identification of potential new MDM2 ligands. The chalcones with the best docking scores obeying the Lipinski rule of five were subsequently prepared by base-catalyzed aldol reactions. The activity of these compounds as inhibitors of p53–MDM2 interaction was investigated using a yeast-based screening assay. Using this approach two chalcones (3 and 4) were identified as putative small molecule inhibitors of p53–MDM2 interaction. The activity of both chalcones was further investigated in a panel of human tumor cells. Chalcones 3 and 4 revealed a pronounced tumor cell growth inhibitory effect on tumor cell lines. Additionally, chalcone 4 caused alterations in the cell cycle profile, induced apoptosis and increased the levels of p53, p21 and PUMA proteins in NCI-H460 cells. Computational docking studies allowed to predict that, like nutlin-3A (a well-known small-molecule inhibitor of p53–MDM2 interaction), chalcones 3 and 4 bind to the p53-binding site of MDM2. The results here presented will be valuable for the structure-based design of novel and potent p53–MDM2 inhibitors.

Design, synthesis and biological evaluation of novel benzimidazole-2-substituted phenyl or pyridine propyl ketene derivatives as antitumour agents

A series of novel benzimidazole-2-subsituted phenyl or pyridine propyl ketene derivatives were designed and synthesized. The biological activities of these derivatives were then evaluated as potential antitumour agents. These compounds were assayed for growth-inhibitory activity against HCT116, MCF-7 and HepG2 cell lines in vitro. The IC50 values of compounds A1 and A7 against the cancer cells were 0.06–3.64 μM and 0.04–9.80 μM, respectively. Their antiproliferative activities were significantly better than that of 5-Fluorouracil (IC50: 56.96–174.50 μM) and were close to that of Paclitaxel (IC50: 0.026–1.53 μM). The activity of these derivatives was over 100 times more effective than other reported structures of chalcone analogues (licochalcone A). A preliminary mechanistic study suggested that these compounds inhibit p53-MDM2 binding. Compounds A1, A7 and A9 effectively inhibited tumour growth in BALB/c mice with colon carcinoma HCT116 cells. The group administered 200 mg/kg of compound A7 showed a 74.6% tumour growth inhibition with no signs of toxicity at high doses that was similar to the inhibition achieved with the 12.5 mg/kg irinotecan positive control (70.2%). Therefore, this class of benzimidazole-2-subsituted phenyl or pyridine propyl ketene derivatives represents a promising lead structure for the development of possible p53-MDM2 inhibitors as new antitumour agents.

Meeting Organocatalysis with Drug Discovery: Asymmetric Synthesis of 3,3'-Spirooxindoles Fused with Tetrahydrothiopyrans as Novel p53-MDM2 Inhibitors.

An organocatalytic enantioselective Michael-Michael cascade reaction is developed for the synthesis of chiral spirotetrahydrothiopyrans. This highly functionalized scaffold was assembled in moderate to good yield (55-74%) and excellent diastereo- and enantioselectivities (>30:1 dr, ≥ 99% ee) with the creation of four consecutive stereogenic centers. The novel spiro-oxindole scaffold is validated as a new class of p53-MDM2 protein-protein interaction inhibitors with good antitumor activity.

Abstract B45: Molecular modeling approach for exploring the MDM2 active site for the discovery of new P53-MDM2 inhibitors

Abstract MDM2 is a protein that regulates the action of tumor suppressor protein P53. Inhibiting the interaction between the two proteins is widely considered in anti-cancer drug discovery approach. Targeting p53-bindig site on the MDM2 protein can be useful for the development of new anticancer agents. Several drugs with selective activity have been introduced to the market and are used for treatment of several types of cancers. In this study we explored the inhibition of MDM2 activity through studying 32 crystal structures of MDM2-ligand complexes downloaded from protein data bank (PDB). MDM2 protein structures were analyzed using protein-ligand interaction fingerprint to determine key residues for ligand binding. A 3D pharmacophore set was created and validated with a set of features that include aromatic and hydrophobic properties. Furthermore libraries of potential small lead-like ligands were screened on the pharmacophore. Ligands which pose three pharmacophoric features were carried to the next step where receptor-based virtual screening was performed using GLIDE docking software. The top 10% obtained from this screening were then redocked into the MDM2 binding site using more extensive search and scoring technique (GLIDE_XP) in order to enhance screening accuracy. The resultant top list was visually examined and compounds with best binding mode were selected for experimental testing. This virtual technique for discovering new lead-like MDM2 inhibitors will take into consideration several features that current inhibitors may lack such as solubility and bioavailability. Citation Format: Noor Atatreh, Ramez A. Mansour, Mohammad A. Ghattas. Molecular modeling approach for exploring the MDM2 active site for the discovery of new P53-MDM2 inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B45.

Discovery of dihydroisoquinolinone derivatives as novel inhibitors of the p53-MDM2 interaction with a distinct binding mode.

Blocking the interaction between the p53 tumor suppressor and its regulatory protein MDM2 is a promising therapeutic concept under current investigation in oncology drug research. We report here the discovery of the first representatives of a new class of small molecule inhibitors of this protein-protein interaction: the dihydroisoquinolinones. Starting from an initial hit identified by virtual screening, a derivatization program has resulted in compound 11, a low nanomolar inhibitor of the p53-MDM2 interaction showing significant cellular activity. Initially based on a binding mode hypothesis, this effort was then guided by a X-ray co-crystal structure of MDM2 in complex with one of the synthesized analogs. The X-ray structure revealed an unprecedented binding mode for p53-MDM2 inhibitors.

Small-molecule MDM2-p53 inhibitors: recent advances.

Potent and selective small-molecule inhibitors of the p53-MDM2 interaction intended for the treatment of p53 wild-type tumors have been designed and optimized in a number of chemical series. This review details recent disclosures of compounds in advanced optimization and features key series that have given rise to clinical trial candidates. The structure-activity relationships for inhibitor classes are discussed with reference to x-ray structures, and common structural features are identified.

The Frequent Application of Multicomponent and Cycloaddition Reactions for the Synthesis of Potent MDM2-p53 Inhibitors

Multicomponent reactions (MCRs) and cycloaddition reactions can generate structurally complex molecules in a very efficient manner and are a valuable tool not only for the synthesis of compound libraries for screening campaigns, but also for the optimization of compounds during lead optimization. This article highlights the frequent application of these reactions for the synthesis of potent inhibitors of the MDM2-p53 protein–protein interaction.

Recent advances of p53-MDM2 small molecule inhibitors (2011-present).

P53 is an important transcriptional factor that plays a pivotal role in different biological process (cell cycle, apoptosis, DNA repair, angiogenesis and cellular metabolism). While p53 binds to the promoter and increases the gene expression of Mdm2, MDM2 protein directly binds to p53 and inhibits its activity. Therefore, inhibitor of p53 and MDM2 has been considered as a potential cancer therapeutic agent due to the critical inhibitory role of MDM2 on p53. Small-molecule inhibitor of p53-MDM2 has been designed to serve as an effective way to treat cancer. Several compounds have moved into different phase of clinical trials based on major advances in the development of small-molecule inhibitors in recent years. Since there are few reviews covering the structure- activity relationship analysis of recent p53-MDM2 inhibitors reported from 2011 to the present time, in this review, attentions are focused on the development of p53-MDM2 inhibitors published from 2011 to the present time.

Mechanistic study of NVP-CGM097: A potent, selective and species specific inhibitor of p53-Mdm2

Mechanistic study of NVP-CGM097: A potent, selective and species specific inhibitor of p53-Mdm2

Searching for Dual Inhibitors of the MDM2-p53 and MDMX-p53 Protein-Protein Interaction by a Scaffold-Hopping Approach.

Two libraries of substituted benzimidazoles were designed using a 'scaffold-hopping' approach based on reported MDM2-p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X-ray structure. Benzimidazole libraries were prepared using an efficient solution-phase approach and screened for inhibition of the MDM2-p53 and MDMX-p53 protein-protein interactions. Key examples showed inhibitory activity against both targets.

Identification of antipsychotic drug fluspirilene as a potential p53-MDM2 inhibitor: a combined computational and experimental study.

The inhibition of tumor suppressor p53 protein due to its direct interaction with oncogenic murine double minute 2 (MDM2) protein, plays a central role in almost 50% of all human tumor cells. Therefore, pharmacological inhibition of the p53-binding pocket on MDM2, leading to p53 activation, presents an important therapeutic target against these cancers expressing wild-type p53. In this context, the present study utilized an integrated virtual and experimental screening approach to screen a database of approved drugs for potential p53-MDM2 interaction inhibitors. Specifically, using an ensemble rigid-receptor docking approach with four MDM2 protein crystal structures, six drug molecules were identified as possible p53-MDM2 inhibitors. These drug molecules were then subjected to further molecular modeling investigation through flexible-receptor docking followed by Prime/MM-GBSA binding energy analysis. These studies identified fluspirilene, an approved antipsychotic drug, as a top hit with MDM2 binding mode and energy similar to that of a native MDM2 crystal ligand. The molecular dynamics simulations suggested stable binding of fluspirilene to the p53-binding pocket on MDM2 protein. The experimental testing of fluspirilene showed significant growth inhibition of human colon tumor cells in a p53-dependent manner. Fluspirilene also inhibited growth of several other human tumor cell lines in the NCI60 cell line panel. Taken together, these computational and experimental data suggest a potentially novel role of fluspirilene in inhibiting the p53-MDM2 interaction. It is noteworthy here that fluspirilene has a long history of safe human use, thus presenting immediate clinical potential as a cancer therapeutic. Furthermore, fluspirilene could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53-MDM2 inhibitors against several types of cancer. Importantly, the combined computational and experimental screening protocol presented in this study may also prove useful for screening other commercially-available compound databases for identification of novel, small molecule p53-MDM2 inhibitors.

Abstract 1798: Mechanistic study of NVP-CGM097: a potent, selective and species specific inhibitor of p53-Mdm2

Abstract An effective strategy to restore p53 activity in cancer cells containing wild type p53 is to inhibit the Mdm2-p53 protein-protein interaction (PPI). NVP-CGM097 is a novel PPI inhibitor under evaluation in a Phase I clinical trial. It binds to the p53 binding-site of the Mdm2 protein, disrupting the interaction between both proteins, leading to an activation of the p53 pathway. The main biophysical and biochemical inhibitory characteristics of NVP-CGM097 are presented here. These include an affinity constant for Mdm2 in the nanomolar range and a selectivity of 3 orders of magnitude vs. Mdm4. The binding kinetics of NVP-CGM097 to Mdm2 are characterized by a high association rate constant (Kon =37 x 106 M-1.s-1) and a moderate dissociation rate constant (Koff =0.071 s-1). Additionally, NVP-CGM097 exhibits an 8-fold greater affinity for Mdm2 over Nutlin-3 due to a longer residence time of the Mdm2-inhibitor complex. Moreover, biochemical studies have revealed the species specificity of NVP-CGM097 with human Mdm2 being inhibited more strongly than the dog, mouse or rat forms of the protein. This was confirmed in cellular assays where NVP-CGM097 treatment resulted in induction of p53 target gene expression (p21, PUMA and Mdm2) only in human, but not in dog, mouse or rat cell lines. Citation Format: Thérèse Valat, Keiichi Masuya, Frédéric Baysang, Geneviève Albrecht, Nicole Buschmann, Dirk Erdmann, Pascal Furet, Tobias Gabriel, François Gessier, Francesco Hofmann, Philipp Holzer, Joerg Kallen, Carole Pissot-Solderman, Stefan Stutz, Patrick Chène, Sébastien Jeay. Mechanistic study of NVP-CGM097: a potent, selective and species specific inhibitor of p53-Mdm2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1798. doi:10.1158/1538-7445.AM2014-1798

Design, synthesis and biological evaluation of sulfamide and triazole benzodiazepines as novel p53-MDM2 inhibitors.

A series of sulfamide and triazole benzodiazepines were obtained with the principle of bioisosterism. The p53-murine double minute 2 (MDM2) inhibitory activity and in vitro antitumor activity were evaluated. Most of the novel benzodiazepines exhibited moderate protein binding inhibitory activity. Particularly, triazole benzodiazepines showed good inhibitory activity and antitumor potency. Compound 16 had promising antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 4.17 μM, which was much better than that of nutlin-3. The molecular docking model also successfully predicted that this class of compounds mimicked the three critical residues of p53 binding to MDM2.

Polymorphisms in the Apoptosis Pathway in Cutaneous Melanoma: Recurrence and Survival

ABSTRACT Aim: Cutaneous melanoma (CM) is notorious for its poor prognosis and resistance to conventional chemotherapy. Genes involved in the apoptosis pathway, such as P53, (tumor suppressor gene), MDM2 (p53 inhibitor), BAX (proapoptotic) and BCL2 (anti-apoptotic) are important for CM growth and survival. The aim of this study was to evaluate whether P53 Arg72Pro, MDM2 T309G, BAX G(-248)A and BCL2 C(-938)A polymorphisms, involved with inherited variations in apoptosis, are associated with relapse free survival (RFS) and overall survival (OS) of CM patients. Methods: Our analysis included 234 consecutive CM patients at diagnosis seen at our University Hospital from December 1989 to November 2013. Genomic DNA from peripheral blood of patients was analyzed by polymerase chain reaction followed by enzymatic digestion for discrimination of pertinent genotypes. RFS and OS were calculated using the univariate Kaplan-Meier estimate probabilities, and differences between survival curves were analysed by the log-rank test. RFS was defined as the time to beginning of treatment until the date of the first relapse. OS was calculated from date of first diagnosis until the date of death or last follow-up. Results: The median period of observation of patients in study was 50 months (9-192) for RFS and 58 months (range: 9-285) for OS. We observed at 120 months of segment, that the RFS was higher in patients with BCL2 CC + CA plus MDM2 TT + TG than patients with BCL2 AA plus MDM2 GG genotypes (54% versus 37%, P = 0.01). The OS in CM patients with the BAX GA + AA was higher than in those with the GG genotypes (100% versus 80%, P = 0.01). Moreover, the OS was also higher in patients with BAX GA + AA plus BCL2 CC + CA combined genotype than in those with BAX GG plus BCL2 AA (100% versus 78%, P = 0.02) and BAX GA + AA plus MDM2 TT + TG than BAX GG plus MDM2 GG (100% versus 71%, P = 0.001). Conclusions: The data suggest, for the first time, that BAX G(-248)A polymorphism may independent or jointly with BCL2 C(-938)A and MDM2 T309G polymorphisms modulate RFS and OS in CM patients. Additional studies will provide some promising guidance for clinical management and tailored or personalized therapeutics in treating for CM. Disclosure: All authors have declared no conflicts of interest.

The orally bioavailable MDM2 antagonist RG7112 and pegylated interferon α 2a target JAK2V617F-positive progenitor and stem cells

The Philadelphia chromosomal-negative chronic myeloproliferative neoplasms (MPNs) originate at the level of the hematopoietic stem cell (HSC). The protracted clinical course of the MPNs has limited the use of potentially toxic treatment modalities, which may eliminate the responsible malignant clone. Treatment with low doses of RG7112, an orally available small-molecule inhibitor of p53-MDM2, both alone and combined with pegylated interferon α 2a (Peg-IFNα 2a), significantly decreased MPN colony-forming unit-granulocyte macrophage and burst-forming unit-erythroid numbers and preferentially eliminated the total number of JAKV617F(+) MPN hematopoietic progenitor cells. The effects of RG7112 and Peg-IFNα 2a on MPN progenitor cells were dependent on blocking p53-MDM2 interactions and activating the p53 pathway, thereby increasing MPN CD34(+) cell apoptosis. Treatment of polycythemia vera (PV) and primary myelofibrosis (PMF) CD34(+) cells with low doses of RG7112 and Peg-IFNα 2a before their transplantation into immune-deficient mice decreased the degree of donor-derived chimerism as well as the JAK2V617F allele burden, indicating that these drugs can each alone or in combination deplete MPN HSCs. These results provide a rationale for the use of combinations of low doses of RG7112 and Peg-IFNα 2a for the treatment of PV or PMF patients with the intent of altering their natural history.

Group-based QSAR and molecular dynamics mechanistic analysis revealing the mode of action of novel piperidinone derived protein–protein inhibitors of p53-MDM2

Tumour suppressor p53 is known to play a central role in prevention of tumour development, DNA repair, senescence and apoptosis which is in normal cells maintained by negative feedback regulator MDM2 (Murine Double Minute 2). In case of dysfunctioning of this regulatory loop, tumour development starts thus resulting in cancerous condition. Inhibition of p53-MDM2 binding would result in activation of the tumour suppressor. In this study, a novel robust fragment-based QSAR model has been developed for piperidinone derived compounds experimentally known to inhibit p53-MDM2 interaction. The QSAR model developed showed satisfactory statistical parameters for the experimentally reported dataset (r2=0.9415, q2=0.8958, pred_r2=0.8894 and F-test=112.7314), thus judging the robustness of the model. Low standard error values (r2_se=0.3003, q2_se=0.4009 and pred_r2_se=0.3315) confirmed the accuracy of the developed model. The regression equation obtained constituted three descriptors (R2-DeltaEpsilonA, R1-RotatableBondCount and R2-SssOCount), two of which had positive contribution while third showed negative correlation. Based on the developed QSAR model, a combinatorial library was generated and activities of the compounds were predicted. These compounds were docked with MDM2 and two top scoring compounds with binding affinities of −10.13 and −9.80kcal/mol were selected. The binding modes of actions of these complexes were analyzed using molecular dynamics simulations. Analysis of the developed fragment-based QSAR model revealed that addition of unsaturated electronegative groups at R2 site and groups with more rotatable bonds at R1 improved the inhibitory activity of these potent lead compounds. The detailed analysis carried out in this study provides a considerable basis for the design and development of novel piperidinone-based lead molecules against cancer and also provides mechanistic insights into their mode of actions.

Discovery of 1-arylpyrrolidone derivatives as potent p53–MDM2 inhibitors based on molecule fusing strategy

Introducing an aryl moiety to our previous pyrrolidone scaffold by molecule fusing strategy afforded two sets of isopropylether–pyrrolidone and α-phenylethylamine–pyrrolidone derivatives. Two novel compounds 8b and 8g of the latter serial showed potent p53–MDM2 inhibitory activities with Ki values of 90nM which were three-time higher than that of the parent compound. We also confirmed compound 8b can activate p53 proteins in lung cancer A549 cells. The results offered us valuable information for further lead optimization.