p53-MDM2 Inhibitors Research Articles

Expanding the Utility of Midostaurin in Acute Myeloid Leukemia - Predictive Mutational Signatures in Patient Samples without FLT3 mutations and Clinically Applicable Synergistic Drug Combinations

BackgroundFollowing the positive outcome of the RATIFY phase 3 clinical trial, the multi-kinase inhibitor midostaurin was approved for the treatment of adult patients with newly diagnosed FLT3-mutated acute myeloid leukemia (AML). However, we and others have observed that single agent midostaurin yields responses also in a substantial portion of patients not carrying FLT3 mutations. The molecular basis and the kinase targets mediating these responses are poorly understood and no biomarkers predictive of response in FLT3 wildtype (wt) AML patients exist. To identify markers distinguishing the FLT3 wt responding subset of patients, we trained machine learning multi-marker models using AML patient baseline transcriptomic and mutational data to predict ex vivo responders vs. non-responders. Further, to better understand the molecular basis of midostaurin responses and to explore the unique signaling networks modulated by midostaurin, we profiled the sensitivities of AML patient samples to midostaurin in comparison to, and in combination with, several clinically relevant oncological targeted agents of diverse mechanistic classes.ResultsMidostaurin target space is unique and it retains anti-leukemic potency under cytoprotective conditions. We have previously established that single agent midostaurin is effective ex vivo in about 25% of FLT3 wt AML patient samples and retains potency in a cytoprotective medium that masks the effects of more selective FLT3 inhibitors such as quizartinib, crenolanib and sorafenib (Karjalainen et al, Blood 2017). To further investigate the unique pathways that midostaurin, but not other FLT3 inhibitors targets, we correlated the response patterns of 87 AML patient samples in cytoprotective medium to midostaurin and 261 other kinase inhibitors in our oncology compound collection. In unsupervised cluster analysis, midostaurin showed highly similar response patterns to AZD7762, OTS167, milciclib, pacritinib, ENMD-2076 and fostamatinib. Publicly available in vitro kinase profiling (Tang et al, Cell Chem. Biol. 2018) suggested that midostaurin does not inhibit most of the primary targets of these other inhibitors, with only aurora kinases, JAK kinases and SYK appearing to be shared potent targets.Midostaurin anti-leukemic potency is determined by the mutational background. Several multi-marker, supervised machine learning models were compared to extract biomarker signatures from either baseline transcriptomic or mutational data, in the task of predicting ex vivo midostaurin response in samples cultured in cytoprotective medium. In the full cohort (N=81), the presence of FLT3 mutations (both internal tandem repeat and tyrosine kinase domain mutations) was the strongest predictor of response. In the FLT3 wt cases (N=49), our results revealed that other select mutations correlated well with either response or non-response upon Bayesian Linear Regression analysis with cross-validation (Ammad-Ud-Din et al, Bioinformatics, 2017). Mutations in PTPN11, U2AF1, SRSF2, RUNX1, JAK2 and BCOR predicted midostaurin responders, while mutations in GATA2, WT1, NPM1 and IDH2 were enriched in non-responders (Figure 1). Baseline transcriptomic profiles, however, did not provide added value for the predictive power.Midostaurin efficacy can be enhanced by combination with other targeted agents. Combinatorial drug screening of midostaurin in cytoprotective medium revealed several synergizing drug classes, including BCL-2 and MDM2-p53 inhibitors. Further analysis of synergizing agents in broader AML patient sample cohorts is ongoing.ConclusionsOur results show that midostaurin may reach its biological effects through inhibition of additional kinases than just FLT3. In both FLT3 mutant and wt cases, midostaurin responses are influenced by the overall mutational background. Furthermore, our data indicates that midostaurin efficacy can be enhanced through combination with other agents. Together, we have significantly expanded the understanding of molecular determinants of midostaurin response in primary AML cells, supporting predictive biomarker discovery efforts and development of synergistic drug combinations. The emerging hypotheses from this work will have to be tested in clinical studies. [Display omitted] DisclosuresPorkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Marques Ramos:Novartis: Employment. Pallaud:Novartis: Employment. Aittokallio:Novartis: Research Funding. Wennerberg:Novartis: Research Funding.

P53-Mdm2 Interaction Inhibitors as Novel Nongenotoxic Anticancer Agents.

Cancer is a major global health problem with high mortality rate. Most of the clinically used anticancer agents induce apoptosis through genotoxic stress at various stages of cell cycle and activation of p53. Acting as a tumor suppressor, p53 plays a vital role in preventing tumor development. Tumor suppressor function of p53 is effectively antagonized by its direct interaction with murine double minute 2 (Mdm2) proteins via multiple mechanisms. Thus, p53-Mdm2 interaction has been found to be an important target for the development of novel anticancer agents. Currently, nutlin, spirooxindole, isoquilinone and piperidinone analogues inhibiting p53-Mdm2 interaction are found to be promising in the treatment of cancer. The current review focused to scrutinize the structural aspects of p53-Mdm2 interaction inhibitors. The present study provides a detailed collection of published information on different classes of inhibitors of p53-Mdm2 interaction as potential anticancer agents. The review highlighted the structural aspects of various reported p53-Mdm2 inhibitors for optimization. In the last few years, different classes of inhibitors of p53-Mdm2 have been designed and developed, and seven such compounds are being evaluated in clinical trials as new anticancer drugs. Further, to explore the role of p53 protein as a potential target for anticancer drug development, in this review, the mechanism of Mdm2 mediated inactivation of p53 and recent developments on p53- Mdm2 interactions inhibitors are discussed. Agents designed to block the p53-Mdm2 interaction may have a therapeutic potential for the treatment of a subset of human cancers retaining wild-type p53. We review herein the recent advances in the design and development of potent small molecules as p53-Mdm2 inhibitors.

Abstract 314: A novel MDM2-p53 antagonist APG-115 induces p53-mediated apoptosis and enhances radiosensitivity in colorectal cancer

Abstract Background: Colorectal carcinoma is the third leading cause of cancer-related mortality in China. MDM2-p53 inhibitor APG-115 is a novel potential anticancer agent. We aim to investigate the anticancer effect and radiosensitivity enhancement of APG-115 in colorectal cancer. Materials and methods: Human colorectal cancer cell lines (LOVO, HCT116, RKO, HT29, SW480 and SW620) were treated with the APG-115 alone or with radiation (0-8Gy). Transwell, clonogenic survival assays, CCK-8, flow cytometry, RT-qPCR, Western blot were used to assay cell invasion, cell survival, cell cycle progression, quantification of mRNA expression and DNA damage repair. In vivo, xenograft mouse models (RKO Cell line) were established to analyze the anticancer effect and radiosensitivity enhancement of APG-115. Results: In vitro, APG-115 not only inhibited invasion and proliferation of colorectal cell lines, but also induced cell-cycle arrest in G1/S phase. Meanwhile, APG-115 increased the mRNA and protein expression of p53 downstream targets dependent on MDM2-p53 signal pathway. The combination of APG-115 and radiation caused cells to accumulate in G2/M and resulted in the accumulation of H2AX-related DNA damage, indicating an increasing of radiosensitivity. In xenograft mouse models, single therapy with either radiation or APG-115 was equivalent in delaying tumor growth, although APG-115 slightly outperforming therapy with radiation alone. Combination therapy delayed tumor growth more significantly compared with either single agent therapy. By analyzing proliferation marker Ki67 in xenograft tumor specimens, we found that the number of proliferating cells significantly decreased in tumors receiving combination treatment of APG-115 and radiation compared with tumors receiving single agent treatment. These results strongly confirmed the enhanced antitumor capacity of APG-115 when combined with radiation. Conclusions: Our data strongly suggest that MDM2-p53 inhibitor APG-115 may serve not only as a stand-alone therapy, but also as an effective adjunct to current radiotherapeutic regimens for treating colorectal cancer dependent on MDM2-p53 axis. Citation Format: Han-Jie Yi, Xiang-Lei Yan, Qiu-Yun Luo, Luping Yuan, Baoxia Li, Wentao Pan, Lin Zhang, Miao-Zhen Qiu, Guangfeng Wang, Yifan Zhai, Da-Jun Yang. A novel MDM2-p53 antagonist APG-115 induces p53-mediated apoptosis and enhances radiosensitivity in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 314.

Abstract 1652: Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction

Abstract In response to cellular stress, the p53 tumor suppressor is activated to modulate cell cycle progression, DNA repair, and cell death. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 can therefore reactivate p53 and elicit an anti-cancer effect. Small molecule inhibitors of the MDM2-p53 interaction remains a promising strategy for cancer therapy and a number of these compounds are in clinical development. An isoindolinone series, identified by the Northern Institute for Cancer Research (NICR), has been used as a starting point for the development of potent MDM2-p53 inhibitors. Structure based drug design was applied during lead optimisation to gain potency whilst also focusing on stabilizing the main metabolically labile position and reducing lipophilicity. This approach led to potent compounds with EC50 <1 nM against MDM2 in cell-free ELISA assays and EC50 <30 nM for p53 induction in SJSA-1 osteosarcoma cells. Further analyses of the compounds demonstrated an increase in the levels of p53 and p53 transcriptional targets as a result of inhibiting the MDM2-p53 interaction. Using three pairs of isogenic cell lines, the compounds were shown to be specific for cell lines with wild-type p53. Key compounds were also characterized in pharmacokinetic and pharmacodynamic studies in mice bearing the SJSA-1 tumor xenograft where they displayed strong induction of p53, 3 hours post oral administration, together with an increase in the expression of p53 target genes p21 and MDM2. These potent MDM2-p53 inhibitors have also shown significant in vivo efficacy in the SJSA-1 xenograft model at well tolerated oral doses. Thus, promising lead compounds were identified, meriting further optimization of the series. Citation Format: Lynsey Fazal, Maria Ahn, Luke Bevan, Ildiko Buck, Juan Castro, Gianni Chessari, Ben Cons, Keisha Hearn, Steven Howard, Chris Johnson, Judith Reeks, Emiliano Tamanini, Neil Thompson, Hugh Walton, Pamela Williams, Ruth H. Bawn, Tim J. Blackburn, Celine Cano, Sarah J. Cully, Bernard Golding, Roger Griffin, Karen Haggerty, Ian Hardcastle, Herbie Newell, Martin Noble, Huw Thomas, Elaine Willmore, Yan Zhao, Steve Wedge. Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1652.

A novel small molecule inhibitor of MDM2-p53 (APG-115) enhances radiosensitivity of gastric adenocarcinoma

BackgroundGastric cancer is the leading cause of cancer related death worldwide. Radiation alone or combined with chemotherapy plays important role in locally advanced and metastatic gastric adenocarcinoma. MDM2–p53 interaction and downstream signaling affect cellular response to DNA damage which leads to cell cycle arrest and apoptosis. Therefore, restoring p53 function by inhibiting its interaction with MDM2 is a promising therapeutic strategy for cancer. APG-115 is a novel small molecule inhibitor which blocks the interaction of MDM2 and p53. In this study, we investigated that the radiosensitivity of APG-115 in gastric adenocarcinoma in vitro and in vivo.MethodsThe role of APG-115 in six gastric cancer cells viability in vitro was determined by CCK-8 assay. The expression level of MDM2, p21, PUMA and BAX in AGS and MKN45 cell lines was measured via real-time PCR (RT-PCR). The function of treatment groups on cell cycle and cell apoptosis were detected through Flow Cytometry assay. Clonogenic assays were used to measure the radiosensitivity of APG-115 in p53 wild type gastric cancer cell lines. Western blot was conducted to detect the protein expressions of mdm2-p53 signal pathway. Xenograft models in nude mice were established to explore the radiosensitivity role of APG-115 in gastric cancer cells in vivo.ResultsWe found that radiosensitization by APG-115 occurred in p53 wild-type gastric cancer cells. Increasing apoptosis and cell cycle arrest was observed after administration of APG-115 and radiation. Radiosensitivity of APG-115 was mainly dependent on MDM2-p53 signal pathway. In vivo, APG-115 combined with radiation decreased xenograft tumor growth much more significantly than either single treatment. Moreover, the number of proliferating cells (Ki-67) significantly decreased in combination group compared with single treatment group.ConclusionsIn summary, we found that combination of MDM2-p53 inhibitor (APG-115) and radiotherapy can enhance antitumor effect both in vitro and in vivo. This is the first report on radiosensitivity of APG-115 which shed light on clinical trial of the combination therapy of radiation with APG-115 in gastric adenocarcinoma.

Methyl elaidate: A major compound of potential anticancer extract of Moringa oleifera seeds binds with bax and MDM2 (p53 inhibitor) In silico

Background: Apoptosis is an energy-required programmed cell death process that is vital for normal homeostasis. This is because aged, malfunctioning, and genetically deficient cells such as cancer cells are eliminated through this process to avoid any abnormality in the function of the body system. Earlier, we have reported that the Lipophilic hexane fraction of crude ethanolic extract (HF-CEE) of Moringa oleifera seeds inhibited MCF7 breast cancer cell growth. Objectives: The aims of this study are to identify the esters responsible for HF-CEE cytotoxicity and to determine the ability of the compound to trigger Bax and p53 apoptotic proteins. Materials and Methods: Gas chromatography–mass spectrometry (GC-MS) analysis of the HF-CEE was performed. Methyl elaidate, the major compound identified, was molecularly docked with Bax and MDM2 (p53 inhibitor) using AutoDock 4.2. Drug suitability of methyl elaidate was determined by the rule of “5.” Results: Eleven different esters were identified in HF-CEE by GC-MS. Methyl elaidate was the major anticancer agent among the esters. Methyl elaidate bound with Bax and MDM2 at their active sites that can trigger apoptosis in cancer cells. Finally, methyl elaidate has potential drug properties according to the rule of “5.” Conclusion: Methyl elaidate of HF-CEE could be responsible for its anticancer activity and it is a potential apoptotic agent. Abbreviations used: HF-CEE: Hexane fraction of crude ethanolic extract of Moringa oleifera seeds; GC-MS: Gas chromatography mass spectrometry; MDM2: Mouse double minute 2 homolog.

Synthesis of spiro-tetrahydrothiopyran-oxindoles by Michael-aldol cascade reactions: discovery of potential P53-MDM2 inhibitors with good antitumor activity.

Using proline as the catalyst, an organocatalytic Michael-aldol cascade reaction was developed for the synthesis of spiro-tetrahydrothiopyran oxindoles. The highly functionalized scaffold was assembled in moderate to good yields (51-78%) and excellent diastereoselectivities (>20 : 1 dr). Interestingly, the oxindoles displayed moderate to good in vitro antitumor activities and were validated as p53-MDM2 inhibitors, which represented promising lead compounds for antitumor drug discovery.

A p53-MDM2 Interaction Inhibitor, DS-5272, Inhibits the Development of MLL-Fusion Leukemia with the Assistance of Tumor Immunity

MLL-fusion leukemia is an aggressive form of leukemia carrying chimeric fusion of the MLL gene. Adverse therapy response of MLL-fusion leukemia is partly associated with the attenuated p53 response. The principal cellular antagonist of p53 is an E3 ubiquitin ligase MDM2. MDM2 binds to p53 and induces proteasomal degradation to downregulate p53 protein level. Therefore, targeting of p53-MDM2 interaction to reactivate p53 function is an attractive therapeutic approach for MLL-fusion leukemia. In this study, we assessed the effect of an orally active inhibitor of p53-MDM2 interaction, DS-5272, in a mouse leukemia model driven by MLL-AF9. DS-5272 inhibited in vitro growth of mouse leukemia cells transformed by MLL-AF9 with the IC50 value in the nanomolar range. A single administration of DS-5272 in vivo upregulated expression of p53 protein as well as its target genes, and induced cell cycle arrest, apoptosis, and differentiation of MLL-AF9 cells. Multiple oral doseadministration of DS-5272 (3 times per week) caused nearly complete tumor regressions of MLL-AF9 leukemia cells that were sustained well beyond the drug administration period with tolerable toxicity. In contrast, DS-5272 showed little effect on p53-deficient bone marrow cells transformed by MLL-AF9 both in vitro and in vivo, confirming that the antileukemic effect of DS-5272 is mediated by p53 activation.Despite the remarkable effect of DS-5272 against MLL-AF9 leukemia with wild-type p53, all mice eventually developed leukemia after a long latency, indicating the existence of leukemia stem cells (LSCs) that are resistant to p53 activation. The therapy-resistant LSCs were relatively enriched within the bone marrow (BM) endosteal region where osteoblasts reside. In contrast, LSCs in the BM central portion were eliminated almost completely by the DS-5272 treatment. Thus, LSCs within the osteoblast-rich area appear to be protected from p53-induced cell death. RNA-Seq analyses revealed the upregulation of inflammation- and interferon-associated genes in MLL-AF9 leukemia cells treated with DS-5272. Furthermore, DS-5272 treatment induced upregulation of PD-L1, a well-known suppressor of tumor immunity, in MLL-AF9 cells. These expression changes suggest that p53 activation triggered an immune-inflammatory response that led to leukemia regression. Interestingly, LSCs reside in the BM endosteal region expressed higher level of PD-L1 compared with those in the BM central portion, which may account for their low sensitivity to DS-5272.To examine the potential contribution of tumor immunity to the DS-5272-mediated suppression of leukemic growth, we next assessed the in vivo effect of DS-5272 on MLL-AF9 leukemia using immunodeficient NOD.Cg- Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice as recipients. Strikingly, the antileukemia effect of DS-5272 was markedly attenuated in NSG mice, indicating the important role of tumor immunity to enhance the efficacy of DS-5272. We then depleted PD-L1 in MLL-AF9 cells using the CRISPR/Cas9 system, and found that PD-L1-depleted MLL-AF9 cells become more sensitive to DS-5272 treatment. These data suggest that DS-5272 inhibits the development of MLL-AF9 leukemia with the assistance of tumor immunity, and its therapeutic efficacy is enhanced by PD-L1 inhibition in MLL-AF9 cells.In summary, our study demonstrated the potent antitumor activity of a p53-MDM2 interaction inhibitor, DS-5272, against MLL-fusion leukemia with intact p53. LSCs reside within the BM endosteal region express high level of PD-L1 and are relatively resistant to DS-5272 treatment. Combining p53-MDM2 inhibitors with the immune checkpoint inhibitors may result in synergistic enhancement of cell death and improved efficacy in the treatment of myeloid leukemia. DisclosuresKitamura:Daiichi Sankyo: Research Funding.

Proapoptotic modification of substituted isoindolinones as MDM2-p53 inhibitors

A series of novel amino acid ester derivatives of 2,3-substituted isoindolinones was synthesized and evaluated for p53-mediated apoptotic activity. The rationale for augmentation of the target activity of 2,3-substituted isoindolinones was based on the introduction of new fragments in the structure of the inhibitor that would provide additional binding sites in the hydrophobic cavity of MDM2. To select for the anticipated modifications we employed molecular docking. Synthesized molecules were evaluated for their ability to induce apoptosis in two cancer cell lines and their derivatives with different status of p53 (colorectal HCT116 and osteosarcoma U2OS cells) by Annexin V staining. The target activity was estimated using high-content imaging system Operetta. Valine and phenylglycine ester derivatives were identified as potentially active MDM2-p53 inhibitors.

Artificial Macrocycles as Potent p53-MDM2 Inhibitors.

Based on a combination of an Ugi four component reaction and a ring closing metathesis, a library of novel artificial macrocyclic inhibitors of the p53–MDM2 interaction was designed and synthesized. These macrocycles, alternatively to stapled peptides, target for the first time the large hydrophobic surface area formed by Tyr67, Gln72, His73, Val93, and Lys94 yielding derivatives with affinity to MDM2 in the nanomolar range. Their binding affinity with MDM2 was evaluated using fluorescence polarization (FP) assay and 1H–15N two-dimensional HSQC nuclear magnetic resonance experiments.

D-Amino acid mutation of PMI as potent dual peptide inhibitors of p53-MDM2/MDMX interactions

According to the previously reported potent dual l-peptide PMI of p53-MDM2/MDMX interactions, a series of d-amino acid mutational PMI analogues, PMI-1-4, with enhanced proteolytic resistence and in vitro tumor cell inhibitory activities were reported, of which Liposome-PMI-1 showed a stronger inhibitory activity against the U87 cell lines than Nutlin-3. This d-amino acid mutation strategy may give a hand for enhancing the potential of peptide drugs.

Design, synthesis and biological evaluation of novel antitumor spirotetrahydrothiopyran–oxindole derivatives as potent p53-MDM2 inhibitors

p53–MDM2 protein-protein interaction is a promising target for novel antitumor drug development. Previously, we identified a new class of spirotetrahydrothiopyran–oxindole p53–MDM2 inhibitors by novel organocatalytic enantioselective cascade reactions. Herein, a series of new derivatives were designed, synthesized and assayed to investigate the structure-activity relationships. Among them, compound B14 bearing a novel spiroindole–thiopyranopyridone scaffold exhibited potent MDM2 inhibitory activity as well as antitumor activity, which could effectively induce the apoptosis of A549 cancer cells. It represents a promising lead compound for the development of novel antitumor agents.

DIMP53-1: a novel small-molecule dual inhibitor of p53-MDM2/X interactions with multifunctional p53-dependent anticancer properties.

The transcription factor p53 plays a crucial role in cancer development and dissemination, and thus, p53‐targeted therapies are among the most encouraging anticancer strategies. In human cancers with wild‐type (wt) p53, its inactivation by interaction with murine double minute (MDM)2 and MDMX is a common event. Simultaneous inhibition of the p53 interaction with both MDMs is crucial to restore the tumor suppressor activity of p53. Here, we describe the synthesis of the new tryptophanol‐derived oxazoloisoindolinone DIMP53‐1 and identify its activity as a dual inhibitor of the p53–MDM2/X interactions using a yeast‐based assay. DIMP53‐1 caused growth inhibition, mediated by p53 stabilization and upregulation of p53 transcriptional targets involved in cell cycle arrest and apoptosis, in wt p53‐expressing tumor cells, including MDM2‐ or MDMX‐overexpressing cells. Importantly, DIMP53‐1 inhibits the p53–MDM2/X interactions by potentially binding to p53, in human colon adenocarcinoma HCT116 cells. DIMP53‐1 also inhibited the migration and invasion of HCT116 cells, and the migration and tube formation of HMVEC‐D endothelial cells. Notably, in human tumor xenograft mice models, DIMP53‐1 showed a p53‐dependent antitumor activity through induction of apoptosis and inhibition of proliferation and angiogenesis. Finally, no genotoxicity or undesirable toxic effects were observed with DIMP53‐1. In conclusion, DIMP53‐1 is a novel p53 activator, which potentially binds to p53 inhibiting its interaction with MDM2 and MDMX. Although target‐directed, DIMP53‐1 has a multifunctional activity, targeting major hallmarks of cancer through its antiproliferative, proapoptotic, antiangiogenic, anti‐invasive, and antimigratory properties. DIMP53‐1 is a promising anticancer drug candidate and an encouraging starting point to develop improved derivatives for clinical application.

Structure based virtual screening, docking and molecular dynamic simulation studies to identify potent mdm2-p53 inhibitors: Future implications for cancer therapy

Objective: To identify the p53 binding pocket as well as active residues in mdm2 protein, and search for similar (or) better compounds to inhibit mdm2-p53 interactions in comparison to FDA approved drug (Nutlin) by ligand and structure based virtual screening methods, docking, and molecular dynamic simulation studies. Methods: A structure and ligand based virtual screening for mdm2 protein; targeting the key residues involved in their active binding of p53 peptide was conducted after obtaining structurally suitable compounds similar to Nutlin from ZINC database. These compounds are virtually screened onto the mdm2 protein targeting its active binding site where p53 binds. The best compound with highest binding affinity was taken up for further analysis using molecular dynamic simulations for further validating the docking studies and to reveal interactions during the conformational changes Results: We discovered several compounds that are potentially able to block the interaction between active residues of mdm2 and p53 complex, suggesting their capability to act as anti-cancer agents. As proven by our structure based virtual screening studies coupled with semi-flexible and flexible docking studies, compound ZINC59256947 was found to be the strongest inhibitors for mdm2 protein amongst all of those isolates from ZINC database. Conclusion: Our results suggest that, a simple, selective and reliable inhibition assay can be performed to search for novel inhibitors of p53-mdm2 interaction. Therefore this study provides a rationalization to the ability of a ZINC59256947, an isolate from ZINC database with strong binding affinity towards mdm2 protein, for future implications as anti-cancer agent.

Bioorthogonal Probes for the Study of MDM2-p53 Inhibitors in Cells and Development of High-Content Screening Assays for Drug Discovery.

To study the behavior of MDM2-p53 inhibitors in a disease-relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high-content screening assays. By using automated image analysis with single-cell resolution, we could visualize the intracellular target binding of compounds by co-localization and quantify target upregulation upon MDM2-p53 inhibition in an osteosarcoma model. Additionally, we developed a high-throughput assay to quantify target occupancy of non-tagged MDM2-p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications.

Discovery of Novel Spiro[3H-indole-3,2′-pyrrolidin]-2(1H)-one Compounds as Chemically Stable and Orally Active Inhibitors of the MDM2–p53 Interaction

Scaffold modification based on Wang's pioneering MDM2-p53 inhibitors led to novel, chemically stable spiro-oxindole compounds bearing a spiro[3H-indole-3,2'-pyrrolidin]-2(1H)-one scaffold that are not prone to epimerization as observed for the initial spiro[3H-indole-3,3'-pyrrolidin]-2(1H)-one scaffold. Further structure-based optimization inspired by natural product architectures led to a complex fused ring system ideally suited to bind to the MDM2 protein and to interrupt its protein-protein interaction (PPI) with TP53. The compounds are highly selective and show in vivo efficacy in a SJSA-1 xenograft model even when given as a single dose as demonstrated for 4-[(3S,3'S,3'aS,5'R,6'aS)-6-chloro-3'-(3-chloro-2-fluorophenyl)-1'-(cyclopropylmethyl)-2-oxo-1,2,3',3'a,4',5',6',6'a-octahydro-1'H-spiro[indole-3,2'-pyrrolo[3,2-b]pyrrole]-5'-yl]benzoic acid (BI-0252).

A Unique Mdm2-Binding Mode of the 3-Pyrrolin-2-one- and 2-Furanone-Based Antagonists of the p53-Mdm2 Interaction.

The p53 pathway is inactivated in almost all types of cancer by mutations in the p53 encoding gene or overexpression of the p53 negative regulators, Mdm2 and/or Mdmx. Restoration of the p53 function by inhibition of the p53-Mdm2/Mdmx interaction opens up a prospect for a nongenotoxic anticancer therapy. Here, we present the syntheses, activities, and crystal structures of two novel classes of Mdm2-p53 inhibitors that are based on the 3-pyrrolin-2-one and 2-furanone scaffolds. The structures of the complexes formed by these inhibitors and Mdm2 reveal the dimeric protein molecular organization that has not been observed in the small-molecule/Mdm2 complexes described until now. In particular, the 6-chloroindole group does not occupy the usual Trp-23 pocket of Mdm2 but instead is engaged in dimerization. This entirely unique binding mode of the compounds opens new possibilities for optimization of the Mdm2-p53 interaction inhibitors.

Design, synthesis and structure–activity relationship of 4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-ones as potent p53-MDM2 inhibitors

In the past decade, the p53-MDM2 protein–protein interaction by small molecules has been confirmed as a successful strategy for cancer therapy. In our previous work, pyrrolo[3,4-c]pyrazol-6(1H)-ones were found to be potent p53-MDM2 inhibitors. Further optimization and structure–activity relationship studies were described in the present work. The result revealed that benzyl group on position N1 of imidazole and bromine on C4-phenyl of pyrrolidone showed higher inhibitory activities. In vitro antiproliferative assay demonstrated the potent p53-MDM2 inhibitor 5c with 4-fold selectivity for U2 OS and Saos-2 cells. These data indicated that 4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one moiety is a valuable scaffold for further development of p53-MDM2 inhibitors.

Discovery of a novel class of highly potent inhibitors of the p53–MDM2 interaction by structure-based design starting from a conformational argument

The p53–MDM2 interaction is an anticancer drug target under investigation in the clinic. Our compound NVP-CGM097 is one of the small molecule inhibitors of this protein–protein interaction currently evaluated in cancer patients. As part of our effort to identify new classes of p53–MDM2 inhibitors that could lead to additional clinical candidates, we report here the design of highly potent inhibitors having a pyrazolopyrrolidinone core structure. The conception of these new inhibitors originated in a consideration on the MDM2 bound conformation of the dihydroisoquinolinone class of inhibitors to which NVP-CGM097 belongs. This work forms the foundation of the discovery of HDM201, a second generation p53–MDM2 inhibitor that recently entered phase I clinical trial.

Abstract 3027: Dual inhibition of PKC and p53-MDM2 or PKC and mTORC1 are novel efficient therapeutic approaches for uveal melanoma

Abstract Uveal melanoma (UM), although rare in incidence, is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified and therefore novel therapeutic approaches are urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway. This has led to the use of PKC inhibitors as a rational targeting strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models, several PKC inhibitor-based combination studies were performed using the PKC inhibitor AEB071 (Sotrastaurin). When combined with AEB071, the targeted agents CGM097 (p53-MDM2 inhibitor), RAD001 (Everolimus, mTORC1 inhibitor) and MEK162 (Binimetinib, a MEK inhibitor) demonstrated greater activity in the UM patient-derived xenograft models than their activity as single agents. Importantly, tumor regressions were observed in several UM models with AEB071 + RAD001 and AEB071 + CGM097 co-treatments. Follow-up in vitro studies in UM cell lines using AEB071 combined with either CGM097 or RAD001 provided a more detailed mechanistic understanding of their combination activity and confirmed their ability to induce cell death. Together, these preclinical studies reveal that combining PKC and p53-MDM2 inhibitors or PKC and mTORC1 inhibitors may provide significant clinical benefit for patients with UM. Citation Format: Guillaume Carita, Estelle Frisch Dit Leitz, Ahmed Dahmani, Chloe Raymondie, Nathalie Cassoux, Sophie Piperno-Neumann, Fariba Némati, Ensar Halilovic, Sebastien Jeay, Andrew Wylie, Caroline Emery, Sergio Roman-Roman, Marie Schoumacher, Didier Decaudin. Dual inhibition of PKC and p53-MDM2 or PKC and mTORC1 are novel efficient therapeutic approaches for uveal melanoma. [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 3027.