干扰 p53-MDM2 蛋白相互作用的小分子抑制剂的研究进展

p53-MDM2 蛋白相互作用是抗肿瘤药物研究的重要作用靶点,抑制 p53-MDM2 结合是激活p53 的有效途径。大量的药物筛选方法和手段已经用于寻找 p53-MDM2 相互作用的新型抑制剂,其中小分子抑制剂是最具前景的研究领域之一。该文总结了近年来国内外p53-MDM2 蛋白结合小分子抑制剂的研究进展。     

Chemosensitization by antisense oligonucleotides targeting MDM2

The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.     

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.     

小分子p53-MDM2抑制剂先导化合物苄普地尔的研究

目的 采用老药新用药物设计方法,探寻p53-MDM2蛋白结合小分子抑制剂的先导化合物.方法 通过荧光偏振(FP)法和蛋白印迹试验法,分别测定化合物的p53-MDM2蛋白结合抑制活性和相关蛋白的表达变化,采用四甲基偶氮唑盐微量酶反应比色法(MTT法)测试其体外抗肿瘤活性,并且测定人肝微粒体中代谢产物.结果 发现苄普地尔具...     

Pharmacologic activation of p53 by small-molecule MDM2 antagonists

Restoring p53 activity by inhibiting the interaction between p53 and MDM2 represents an attractive approach for cancer therapy. To this end, a number of small-molecule p53-MDM2 binding inhibitors have been developed during the past several years. Nutlin-3 is a potent and selective small-molecule MDM2 antagonist that has shown considerable promise in pre-clinical studies. This review will highlight recent advances in the development of small-molecule MDM2 antagonists as potential cancer therapeutics, with special emphasis on Nutlin-3.     

Small-molecule inhibitors of p53-MDM2 interaction: the 2006-2010 update

Increasing knowledge of the relationship between p53 and MDM2 has led to development of potential small molecule inhibitors useful for clinical studies. Herein, we discuss the patented (2006-2010) inhibitors of p53-MDM2 interaction. The anticancer agents discussed in this review belong to several different chemical classes including benzodiazepinediones, cis-imidazolines, oxindoles, spiro-oxindoles, and numerous miscellaneous groups. This review also provides comprehensive information on inhibitors of p53-MDM2 interaction that are currently being tested in clinical trials. It is important to note that many of the disclosed inhibitors need further validation to be considered as bona fide inhibitors of p53-MDM2 interaction and some will not be further considered for future studies. On the other hand, JNJ-26854165, a novel tryptamine derivative and RG7112, a cis-imidazoline representative have shown promising results in early phases of trials in cancer patients. AT-219, a spiroindolinone in late stage preclinical studies is a likely candidate to proceed into clinical trials. It remains to be seen how these inhibitors will perform in future clinical studies as single agents and in combination with the currently approved chemotherapeutic agents.     

A facile method to screen inhibitors of protein-protein interactions including MDM2-p53 displayed on T7 phage

Protein-protein interactions are essential in many biological processes including cell cycle and apoptosis. It is currently of great medical interest to inhibit specific protein-protein interactions in order to treat a variety of disease states. Here, we describe a facile multiwell plate assay method using T7 phage display to screen for candidate inhibitors of protein-protein interactions. Because T7 phage display is an effective method for detecting protein-protein interactions, we aimed to utilize this technique to screen for small-molecule inhibitors that disrupt these types of interaction. We used the well-characterized interaction between p53 and MDM2 and an inhibitor of this interaction, nutlin 3, as a model system to establish a new screening method. Phage particles displaying p53 interacted with GST-MDM2 immobilized on 96-well plates, and the interaction was inhibited by nutlin 3. Multiwell plate assay was then performed using a natural product library, which identified dehydroaltenusin as a candidate inhibitor of the p53-MDM2 interaction. We discuss the potential applications of this novel T7 phage display methodology, which we propose to call 'reverse phage display'.     

Small molecule protein-protein inhibitors for the p53-MDM2 interaction

This article describes recent progress in the development of small molecule protein-protein inhibitors of the p53-MDM2 (purine double minute 2, or HDM2 for the human congener) protein-protein interaction, with special attention to the diversity of chemotypes reported to disrupt this protein-protein interaction. In >50% of all human cancers, the tumor suppressor 53 KDa phospho-protein p53 is either mutated or deleted. The discovery that MDM2 (HDM2) negatively regulates p53 and therefore inhibits the tumor-suppressor activity of p53 has instigated numerous drug discovery campaigns aimed at disrupting this protein-protein interaction as a potential cancer therapy. Once regarded as intractable targets disrupted by only large macromolecules, protein-protein interactions (PPI) are now mainstream targets due in large part to the intensive effort applied to the study of p53 and the surprising diversity of small molecules (peptides, natural products, terphenyl and other alpha-helix mimetics, chalcones, piperidines, piperazines, fused indoles, isoindolinones, spiro-oxindoles, cis-imidazolines (nutlins), quinolinol and benzodiazepines) capable of disrupting the p53-HDM2 PPI. In addition, drug discovery researchers have employed a number of screening approaches and technologies to identify SMPPIs of the p53-HDM2 interaction, and these discovery paradigms will be discussed. This review will detail the biology of the p53-MDM2 interaction, the major classes of SMPPIs and key medicinal chemistry and in vitro/in vivo biological data reported through October 2006.     

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.     

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.     

An antibody‐free strategy for screening putative HDM2 inhibitors using crude bacterial lysates expressing GST‐HDM2 recombinant protein

Targeting the interaction of p53 with its natural inhibitor MDM2 by the use of small synthetic molecules has emerged as a promising pharmacological approach to restore p53 oncosuppressor function in cancers retaining wild‐type p53. The first critical step in the experimental validation of newly synthesized small molecules developed to inhibit MDM2‐p53 interaction is represented by the evaluation of their efficacy in preventing the formation of the MDM2‐p53 complex. This can be achieved using the in vitro reconstructed recombinant MDM2‐p53 complex in cell‐free assays. A number of possible approaches have been proposed, which are however not suitable for screening large chemical libraries, due to the high costs of reagents and instrumentations, or the need of large amounts of highly pure recombinant proteins. Here we describe a rapid and cheap method for high‐throughput screening of putative inhibitors of MDM2‐p53 complex formation – based on the use of GST‐recombinant proteins – that does not require antibodies and recombinant protein purification steps from bacterial cell lysates. Copyright © 2013 John Wiley & Sons, Ltd.     

P53-MDM2界面的肽类及拟肽类抑制剂的研究进展

当前,肿瘤疾病以日益增高的发病率越来越受到人们的重视。抑制P53-MDM2的相互作用已经成为治疗癌症药物设计的重要靶标,通过各种药物筛选手段,研究人员发现了许多肽类及小分子抑制剂。综述近年来国内外关于肽类及拟肽类的P53-MDM2抑制剂的研究进展。     

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.     

Discovery of a nanomolar inhibitor of the human murine double minute 2 (MDM2)-p53 interaction through an integrated, virtual database screening strategy

An integrated, virtual database screening strategy has led to 7-[anilino(phenyl)methyl]-2-methyl-8-quinolinol (4, NSC 66811) as a novel inhibitor of the murine double minute 2 (MDM2)-p53 interaction. This quinolinol binds to MDM2 with a Ki of 120 nM and activates p53 in cancer cells with a mechanism of action consistent with targeting the MDM2-p53 interaction. It mimics three p53 residues critical in the binding to MDM2 and represents a promising new class of non-peptide inhibitors of the MDM2-p53 interaction.     

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

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Small-molecule MDM2/X inhibitors and PROTAC degraders for cancer therapy: advances and perspectives

Blocking the MDM2/X–P53 protein–protein interaction has been widely recognized as an attractive therapeutic strategy for the treatment of cancers. Numerous small-molecule MDM2 inhibitors have been reported since the release of the structure of the MDM2–P53 interaction in 1996, SAR405838, NVP-CGM097, MK-8242, RG7112, RG7388, DS-3032b, and AMG232 currently undergo clinical evaluation for cancer therapy. This review is intended to provide a comprehensive and updated overview of MDM2 inhibitors and proteolysis targeting chimera (PROTAC) degraders with a particular focus on how these inhibitors or degraders are identified from starting points, strategies employed, structure–activity relationship (SAR) studies, binding modes or co-crystal structures, biochemical data, mechanistic studies, and preclinical/clinical studies. Moreover, we briefly discuss the challenges of designing MDM2/X inhibitors for cancer therapy such as dual MDM2/X inhibition, acquired resistance and toxicity of P53 activation as well as future directions.