MDMX 与 CK1α对 p53 抑癌蛋白调节机制的研究进展

作为抑癌蛋白, p53 参与了细胞内多种信号转导过程, 并在细胞周期调控、 细胞凋亡及衰老等过程中发挥了重要的作用。鼠双微基因 （murine double minute, MDM） 2 和 MDMX （又称 MDM4, murine double minute 4） 是 p53 两个重要的调控因子。其中, MDMX 能够通过与 p53 蛋白的相互作用以及转录后修饰来调节 p53 蛋白功能。虽然MDMX 与 MDM2 蛋白结构同源, 但是由于 MDMX 缺少 E3 连接酶, 因此无法介导 p53 蛋白的降解。然而, MDMX 本身能够通过分子内部结构的折叠与展开, 与 p53 蛋白相互作用后调节其活性。在该过程中, MDMX 的主要分子伴侣——CK1α（casein kinase 1 alpha）通过磷酸化 MDMX 并干扰其分子内部结合, 从而协同调节 p53 蛋白。因而,MDMX 及 CK1α对 p53 蛋白的调节是一个多步骤、 多因素参与的复杂过程。本文拟就 MDMX 以及CK1α对 p53蛋白的具体调节机制进行综述。     

MDM2/MDMX inhibitor peptide: WO2008106507

Background: The evidence that some human cancers show wild-type p53 and overexpressed levels of MDM2 and/or MDMX has fueled the search for new therapeutic agents that could rescue p53 from the inhibition of MDM2 and MDMX. Recent data, suggesting a distinct and complementary mode of action of MDM2 and MDMX in the regulation of the pro-apoptotic activity of p53, have raised the notion that the development of dual or combined inhibitors of the two oncogenic proteins may result in more effective antitumor strategies. Objective: The objective of the present patent concerns the disclosure by two researchers of the University of South Florida, reporting a dual MDM2/MDMX inhibitor peptide that selectively blocks neoplastic growth and induces apoptosis in tumor cells. Conclusion: Although the researchers' results provide the proof of concept of the feasibility of blocking both MDM2 and MDMX regulatory functions for the development of novel and more incisive p53-based anticancer strategies, their invention supplies medicinal chemists with a new interesting lead compound to aid the design of novel small-molecule inhibitors of the oncogenic proteins with drug-like properties.     

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with K_d values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy.     

靶向MDM2小分子抑制药:肿瘤治疗的新方法

癌蛋白MDM2可与p53蛋白结合形成复合物,抑制p53基因的反式激活,而p53的缺失可诱导肿瘤发生。设计针对MDM2与p53蛋白间相互作用的小分子抑制药(如Nutlins),可再激活p53基因,利于肿瘤治疗。本文综述了MDM2小分子抑制药的特点、作用机制与治疗潜力等最新进展。     

抗肿瘤药物研制的新靶点MDM2-p53

癌基因MDM2编码的蛋白可与抑癌蛋白p53结合并抑制p53的功能，促进p53的降解。MDM2的过度表达是肿瘤发生和发展的重要因素之一。本文简述了MDM2的结构与功能，MDM2与p53相互作用的机制与模式，以及根据MDM2－p53复合物结构研制开发抗肿瘤药物的进展与前景。     

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.     

Small molecule antagonists of the MDM2 oncoprotein as anticancer agents

In this early phase of the new era of molecularly targeted patient friendly cancer chemotherapy, there is a need for novel viable anticancer molecular targets. The MDM2 oncoprotein has been validated as a potential target for cancer drug development. MDM2 amplification and/or overexpression occur in a wide variety of human cancers, several of which can be treated experimentally with MDM2 antagonists. MDM2 interacts primarily with the p53 tumor suppressor protein in an autoregulatory negative feedback loop to attenuate p53's cell cycle arrest and apoptosis functions. Inhibition of the p53-MDM2 interaction has been shown to cause selective cancer cell death, as well as sensitize cancer cells to chemotherapy or radiation effects. Consequently, this interaction has been the main focus of anticancer drug discovery targeted to MDM2. The promotion of the proteasomal degradation of the p53 protein by MDM2 is central to its repression of the tumor suppressor functions of p53, and many proteins impinge upon this activity, either enhancing or inhibiting it. MDM2 also has oncogenic activity independent of its interaction with p53, but this has so far not been explored for drug discovery. Among the approaches for targeting MDM2 for cancer therapy, small molecule antagonists have recently featured as effective anticancer agents in experimental models, although the repertoire is currently limited and none has yet entered human clinical trials. Small molecules that have been reported to disrupt the p53-MDM2 binding, thereby enhancing p53 activity to elicit anticancer effects include the following: synthetic chalcones, norbornane derivatives, cis-imidazoline derivatives (Nutlins), a pyrazolidinedione sulfonamide and 1,4-benzodiazepine-2,5-diones, as well as tryptophan derivatives. In addition to compounds disrupting p53pMDM2 binding, three compounds have been discovered that are effective in inhibiting the E3 ligase activity of MDM2 towards p53, and should serve as leads for drug discovery targeting this aspect of the p53-MDM2 interaction as well. These compounds were discovered from library screening and/or structure-based rational drug design strategies.     

Small-molecule correctors and stabilizers to target p53

The tumor suppressor p53 is the most frequently mutated protein in human cancer and tops the list of high-value precision oncology targets. p53 prevents initiation and progression of cancer by inducing cell-cycle arrest and various forms of cell death. Tumors have thus evolved ways to inactivate p53, mainly by TP53 mutations or by hyperactive p53 degradation. This review focuses on two types of p53 targeting compounds, MDM2 antagonists and mutant p53 correctors. MDM2 inhibitors prevent p53 protein degradation, while correctors restore tumor suppressor activity of p53 mutants by enhancing thermodynamic stability. Herein we explore both novel and repurposed p53 targeting compounds, discuss their mode of action, and examine the challenges in advancing them to the clinic.     

An ultrahigh affinity d-peptide antagonist Of MDM2

The oncoprotein MDM2 negatively regulates the activity and stability of the p53 tumor suppressor and is an important molecular target for anticancer therapy. Aided by mirror image phage display and native chemical ligation, we have previously discovered several proteolysis-resistant duodecimal d-peptide antagonists of MDM2, termed (D)PMI-α, β, γ. The prototypic d-peptide inhibitor (D)PMI-α binds ((25-109))MDM2 at an affinity of 220 nM and kills tumor cells in vitro and inhibits tumor growth in vivo by reactivating the p53 pathway. Herein, we report the design of a superactive d-peptide antagonist of MDM2, termed (D)PMI-δ, of which the binding affinity for ((25-109))MDM2 has been improved over (D)PMI-α by 3 orders of magnitude (K(d) = 220 pM). X-ray crystallographic studies validate (D)PMI-δ as an exceedingly potent inhibitor of the p53-MDM2 interaction, promising to be a highly attractive lead drug candidate for anticancer therapeutic development.     

Actinomycin D synergistically enhances the cytotoxicity of CDDP on KB cells by activating P53 via decreasing P53–MDM2 complex

The aim of this study is to investigate the synergism of low dose of actinomycin D (LDActD) to the cytotoxicity of cisplatin (CDDP) on KB cells. The role of P53 reactivation by LDActD in the synergism and its mechanism were further studied. Cell viability was determined by MTT assay. Apoptosis was determined by AnnexinV-FITC/PI staining. Mitochondrial membrane potential (MMP) was detected by JC-1 staining. Expression of proteins was detected by Western blotting (WB) and/or immunofluorescence (IF). Molecular docking of actinomycin D (ACTD) to Mouse double minute 2 homolog (MDM2) and Mouse double minute 2 homolog X (MDMX). MDMX was analyzed by Discovery Studio. The content of P53–MDM2 complex was detected by ELISA assay. The cytotoxicity of CDDP was increased by the combination of LDActD in kinds of cancer cells. Molecular docking showed strong interaction between ACTD and MDM2/MDMX. Meanwhile, LDActD significantly decreased P53–MDM2 complex. Significant increase of the apoptotic activity by the combination therapy in KB cells is P53 upregulated modulator of apoptosis (PUMA) dependent. In addition to the decrease in MMP, LDActD increased P53 regulated protein and decreased BCL-XL in KB cells. LDActD efficiently enhanced the cytotoxicity of CDDP in cancer cells and induced P53-PUMA-dependent and mitochondria-mediated apoptosis in KB cells. The reactivation of P53 was probably achieved by disturbing the interaction of P53 and MDM2/MDMX.     

Small molecule inhibitors of p53/MDM2 interaction

The discovery of the key negative regulator MDM2 (mouse double minute 2, also termed HDM2 for its human equivalent) provided a great opportunity to manipulate the levels of the tumor suppressor p53 in cancer cells. Activation of p53 in tumor cells by inhibiting the interaction of MDM2 with p53 has therefore been the focus of a large effort in drug discovery. The modulation of protein-protein interactions, however, has historically been very difficult to achieve owing to the large surface area of interaction. In this article, we review the recent accomplishments in this area and our quest for a clinically viable MDM2 inhibitor.     

Identification of FDA-approved Drugs that Computationally Bind to MDM2

The integrity of the p53 tumor suppressor pathway is compromised in the majority of cancers. In 7% of cancers p53 is inactivated by abnormally high levels of MDM2 - an E3 ubiquitin ligase that polyubiquitinates p53, marking it for degradation. MDM2 engages p53 through its hydrophobic cleft, and blockage of that cleft by small molecules can re-establish p53 activity. Small molecule MDM2 inhibitors have been developed, but there is likely to be a high cost and long time period before effective drugs reach the market. An alternative is to repurpose FDA-approved drugs. This report describes a new approach, called Computational Conformer Selection, to screen for compounds that potentially inhibit MDM2. This screen was used to computationally generate up to 600 conformers of 3244 FDA-approved drugs. Drug conformer similarities to 41 computationally-generated conformers of MDM2 inhibitor nutlin 3a were ranked by shape and charge distribution. Quantification of similarities by Tanimoto combo scoring resulted in scores that ranged from 0.142 to 0.802. In silico docking of drugs to MDM2 was used to calculate binding energies and to visualize contacts between the top-ranking drugs and the MDM2 hydrophobic cleft. We present 15 FDA-approved drugs predicted to inhibit p53/MDM2 interaction.     

MDM2 splice variants and their therapeutic implications

MDM2 splice variants have now been identified in many different tumor types, and their expression has been associated with advanced disease. However, published data concerning their function is contradictory, and therefore their role in tumorigenesis and their potential as a therapeutic target are unclear. Expression of a specific splice variant, MDM2-B, in a transgenic mouse model results in tumor development; and expression of several splice variants has been shown to enhance tumor formation in Emu-myc transgenic mice. However, expression of similar variants in vitro results in growth inhibition, an observation inconsistent with a transformed phenotype. The observed growth inhibition is p53-dependent, resulting from the binding of splice variants with an intact C-terminal RING finger domain to full-length MDM2 protein. In doing so, p53 can no longer bind MDM2, and p53 activity is elevated. Subsequent inactivation of p53 or p53-mediated apoptosis could contribute to the MDM2 splice variant-mediated tumorigenesis observed in vivo. However, MDM2 splice variants, like full-length MDM2, probably display p53-independent activities. Therefore, the potential for MDM2 splice variants as therapeutic targets will be dependent upon their phenotype within specific tumor types.     

Probing the Origin of Structural Stability of Single and Double Stapled p53 Peptide Analogs Bound to MDM2

The stabilization of secondary structure is believed to play an important role in the peptide–protein binding interaction. In this study, the α‐helical conformation and structural stability of single and double stapled all‐hydrocarbon cross‐linked p53 peptides when bound and unbound to MDM2 are investigated. We determined the effects of the peptide sequence, the stereochemistry of the cross‐linker, the conformation of the double bond in the alkene bridge, and the length of the bridge, to the relative stability of the α‐helix structure. The binding affinity calculations by WaterMap provided over one hundred hydration sites in the MDM2 binding pocket where water density is greater than twice that of the bulk, and the relative value of free energy released by displacing these hydration sites. In agreement with the experimental data, potentials of mean force obtained by weighted histogram analysis methods indicated the order of peptides from lowest to highest binding affinity. Our study provides a comprehensive rationalization of the relationship between peptide stapling strategy, the secondary structural stability, and the binding affinity of p53/MDM2 complex. We hope our efforts can help to further the development of a new generation p53/MDM2 inhibitors that can reactivate the function of p53 as tumor suppressor gene.     

Peptides and peptidomimetics in the p53/MDM2/MDM4 circuitry - a patent review

Introduction: Restoration of the p53 tumor suppressor function is an attractive anticancer strategy. Despite the development of several therapeutics targeting the two main p53 negative regulators, MDM2 and MDM4, no one has yet reached clinical application. In the past, several efforts have been employed to develop more specific and efficient compounds that can improve and/or overcome some of the features related to small molecule compounds (SMC). Peptides and peptidomimetics are emerging as attractive molecules given their increased selectivity, reduced toxicity and reduced tendency to develop tumor-resistance compared to SMC.

Area covered: This article reviews publications and patents (publicly available up to April 2016) for peptides and derivatives aimed to reactivate the oncosuppressive function of p53, with a particular focus on inhibitors of MDM2/MDM4. Emphasis is placed on the efficacy of these compounds compared to the p53-reactivating small molecules developed so far.

Expert opinion: A number of promising peptides for p53 reactivation in cancer therapy have been developed. These compounds appear to possess improved features compared to SMC, especially for their ability to simultaneously target the MDM2/MDM4 inhibitors, and their increased specificity.