Discovery of potent and selective spiroindolinone MDM2 inhibitor,RO8994, for cancer therapy

The field of small-molecule inhibitors of protein–protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.     

Small-molecule antagonists of p53-MDM2 binding: research tools and potential therapeutics

p53 regulates a key pathway which protects normal tissues from tumor development that may result from diverse forms of stress. In the absence of stress, growth suppressive and proapoptotic activity of p53 is inhibited by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 antagonists could activate p53 and may offer a novel therapeutic approach to cancer. Recently, we identified the first potent and selective low molecular weight inhibitors of MDM2-p53 binding, the Nutlins. These molecules activate the p53 pathway and suppress tumor growth in vitro and in vivo. They represent valuable new tools for studying the p53 pathway and its defects in cancer. Nutlins induce p53-dependent apoptosis in human cancer cells but appear cytostatic to proliferating normal cells. Their potent activity against osteosarcoma xenografts suggests that MDM2 antagonists may have clinical utility in the treatment of tumors with wild-type p53.     

MDM2-P53蛋白质相互作用的小分子抑制物

p53作为重要的抑癌基因已经成为一个治疗癌症重点的突破目标之一。直接调节p53基因或调节P53和MDM2蛋白质相互作用是再激活p53基因的两种重要机制。对于表达野生型P53的癌症设计小分子阻断剂阻断MDM2与P53蛋白相互作用是一个很有前景的治疗癌症的方向。文章主要总结了作为治疗癌症的新方法-MDM2-P53蛋白相互作用小分子抑制物的最新研究进展,其中最新的是人工合成化合物Nutlin-3和MI-219。     

Small-Molecule Antagonists of p53-MDM2 Binding: Research Tools and Potential Therapeutics

p53 regulates a key pathway which protects normal tissues from tumor development that may result from diverse forms of stress. In the absence of stress, growth suppressive and proapoptotic activity of p53 is inhibited by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 antagonists could activate p53 and may offer a novel therapeutic approach to cancer. Recently, we identified the first potent and selective low molecular weight inhibitors of MDM2-p53 binding, the Nutlins. These molecules activate the p53 pathway and suppress tumor growth in vitro and in vivo. Nutlins represent valuable new tools for studying the p53 pathway and its defects in cancer. Their potent activity against osteosarcoma xenogrfts suggests that MDM2 antagonists may have a clinical utility in the treatment of tumors with wild-type p53.     

ATM and MET kinases are synthetic lethal with nongenotoxic activation of p53

The p53 tumor suppressor orchestrates alternative stress responses including cell cycle arrest and apoptosis, but the mechanisms defining cell fate upon p53 activation are poorly understood. Several small-molecule activators of p53 have been developed, including Nutlin-3, but their therapeutic potential is limited by the fact that they induce reversible cell cycle arrest in most cancer cell types. We report here the results of a genome-wide short hairpin RNA screen for genes that are lethal in combination with p53 activation by Nutlin-3, which showed that the ATM and MET kinases govern cell fate choice upon p53 activation. Genetic or pharmacological interference with ATM or MET activity converts the cellular response from cell cycle arrest into apoptosis in diverse cancer cell types without affecting expression of key p53 target genes. ATM and MET inhibitors also enable Nutlin-3 to kill tumor spheroids. These results identify new pathways controlling the cellular response to p53 activation and aid in the design of p53-based therapies.     

Mechanism of p53 stabilization by ATM after DNA damage

p53 suppresses tumor development by responding to unauthorized cell proliferation, growth factor or nutrient deprivation, and DNA damage. Distinct pathways have been identified that cause p53 activation, including ARF-dependent response to oncogene activation, ribosomal protein-mediated response to abnormal rRNA synthesis, and ATM-dependent response to DNA damage. Elucidating the mechanisms of these signaling events are critical for understanding tumor suppression by p53 and development of novel cancer therapeutics. More than a decade of research has established the ATM kinase as a key molecule that activates p53 after DNA damage. Our recent study revealed that ATM phosphorylation of MDM2 is likely to be the key step in causing p53 stabilization. Upon activation by ionizing irradiation, ATM phosphorylates MDM2 on multiple sites near its RING domain. These modifications inhibit the ability of MDM2 to poly-ubiquitinate p53, thus leading to its stabilization. MDM2 phosphorylation does not inactivate its E3 ligase activity per se, since MDM2 self-ubiquitination and MDMX ubiquitination functions are retained. The selective inhibition of p53 poly-ubiquitination is accomplished through disrupting MDM2 oligomerization that may provide a scaffold for processive elongation of poly ubiquitin chains. These findings suggest a novel model of p53 activation and a general mechanism of E3 ligase regulation by phosphorylation.     

MDM2 gene amplification is correlated to tumor progression but not to the presence of SNP309 or TP53 mutational status in primary colorectal cancers

Mdm2 is the main regulator of p53 and is amplified in approximately 7% of all human cancers. MDM2 gene amplification as well as expression has been correlated to an increased tumorigenic potential. We have analyzed the prevalence of MDM2 gene amplifications and SNP309 in 284 colorectal tumors using a relatively new highly sensitive PCR/ligase detection reaction method in relation to TP53 mutational status and genomic instability. We found MDM2 to be amplified in 9% of the 284 colorectal cancers analyzed and a significantly higher proportion of tumors with high MDM2 gene amplification retained a wild-type p53 gene (P = 0.058). MDM2 gene amplification was significantly correlated to advanced tumor stage. Several small-molecule MDM2 antagonists have already been identified that either physically inhibit the p53-MDM2 binding or the E3 ligase function of MDM2. Our results suggest that MDM2 is a promising target for this type of cancer therapy in a substantial subgroup of colorectal cancers.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding

Critical to the inhibitory action of the oncogene product, MDM2, on the tumour suppressor, p53, is association of the N-terminal domain of MDM2 (MDM2N) with the transactivation domain of p53. The structure of MDM2N was previously solved with a p53-derived peptide, or small-molecule ligands, occupying its binding cleft, but no structure of the non-liganded MDM2N (i.e. the apo-form) has been reported. Here, we describe the solution structure and dynamics of apo-MDM2N and thus reveal the nature of the conformational changes in MDM2N that accompany binding of p53. The new structure suggests that p53 effects displacement of an N-terminal segment of apo-MDM2N that occludes access to the shallow end of the p53-binding cleft. MDM2N must also undergo an expansion upon binding, achieved through a rearrangement of its two pseudosymetrically related sub-domains resulting in outward displacements of the secondary structural elements that comprise the walls and floor of the p53-binding cleft. MDM2N becomes more rigid and stable upon binding p53. Conformational plasticity of the binding cleft of apo-MDM2N could allow the parent protein to bind specifically to several different partners, although, to date, all the known liganded structures of MDM2N are highly similar to one another. The results indicate that the more open conformation of the binding cleft of MDM2N observed in structures of complexes with small molecules and peptides is a more suitable one for ligand discovery and optimisation.     

Synthesis and evaluation of an imidazole derivative–fluorescein conjugate

The murine double minute (MDM2) oncogene a negative regulator of protein 53 (p53) tumor suppressor, is found overexpressed in many different types of cancer and the interaction between MDM2 and p53 has become the target of intensive research. MDM2 inhibitors represent a promising class of p53 activating compounds that may be effective in cancer treatment and diagnostic imaging. Nutlins, a family of cis-imidazoline analogues and small-molecule MDM2 antagonists, have the potential use in cancer therapies. We have synthesized an imidazole derivative (Nutlin–Glycine) conjugated to the commonly used fluorophore, 6-carboxyfluorescein (FAM) and evaluated its possible use as an imaging agent. Cellular uptake studies demonstrated that the fluorescence intensity in human osteosarcoma (SJSA-1) and colon carcinoma (HCT116) cells were significantly increased with the treatment of Nutlin–Glycine–FAM when compared with FAM (control). Blocking studies also confirmed that our imidazole–fluorescein conjugate may be a good candidate for imaging tumors, suggesting the need for further in vivo evaluation by positron emission tomography.     

Enhanced self-renewal of hematopoietic stem/progenitor cells mediated by the stem cell gene Sall4

We reviewed preclinical data and clinical development of MDM2 (murine double minute 2), ALK (anaplastic lymphoma kinase) and PARP (poly [ADP-ribose] polymerase) inhibitors. MDM2 binds to p53, and promotes degradation of p53 through ubiquitin-proteasome degradation. JNJ-26854165 and RO5045337 are 2 small-molecule inhibitors of MDM2 in clinical development. ALK is a transmembrane protein and a member of the insulin receptor tyrosine kinases. EML4-ALK fusion gene is identified in approximately 3-13% of non-small cell lung cancer (NSCLC). Early-phase clinical studies with Crizotinib, an ALK inhibitor, in NSCLC harboring EML4-ALK have demonstrated promising activity with high response rate and prolonged progression-free survival. PARPs are a family of nuclear enzymes that regulates the repair of DNA single-strand breaks through the base excision repair pathway. Randomized phase II study has shown adding PARP-1 inhibitor BSI-201 to cytotoxic chemotherapy improves clinical outcome in patients with triple-negative breast cancer. Olaparib, another oral small-molecule PARP inhibitor, demonstrated encouraging single-agent activity in patients with advanced breast or ovarian cancer. There are 5 other PARP inhibitors currently under active clinical investigation.     

MDM2 and promyelocytic leukemia antagonize each other through their direct interaction with p53

p53 can be regulated through post-translational modifications and through interactions with positive and negative regulatory factors. MDM2 binding inhibits p53 and promotes its degradation by the proteasome, whereas promyelocytic leukemia (PML) activates p53 by recruiting it to multiprotein complexes termed PML-nuclear bodies. We reported previously an in vivo and in vitro interaction between PML and MDM2 that is independent of p53. In the current study, we investigated whether interaction between MDM2 and PML can indirectly affect p53 activity. Increasing amounts of MDM2 inhibited p53 activation by PML but could not inhibit PML-mediated activation of a p53 fusion protein that lacked the MDM2-binding domain. Conversely, increasing amounts of PML could overcome p53 inhibition by MDM2 but could not overcome MDM2-mediated inhibition of a p53 fusion protein that lacked the PML-binding domain. These results demonstrate that MDM2 and PML can antagonize each other through their direct interaction with p53 and suggest the combined effects of MDM2 and PML on p53 function are determined by the relative level of each protein. Furthermore, these results imply that interactions between MDM2 and PML by themselves have little or no effect on p53 activity.