Affinity-based screening of MDM2/MDMX–p53 interaction inhibitors by chemical array: Identification of novel peptidic inhibitors

MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein–protein interactions (PPIs) of MDM2–p53 and MDMX–p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2–p53 and MDMX–p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2–p53 and MDMX–p53 interactions.     

Negative regulation of HDM2 to attenuate p53 degradation by ribosomal protein L26

HDM2 is a p53-specific E3 ubiquitin ligase. Its overexpression leads to excessive inactivation of tumor protein p53, diminishing its tumor suppressor function. HDM2 also affects the cell cycle, apoptosis and tumorigenesis through interacting with other molecules, including several ribosomal proteins. To identify novel HDM2 regulators, we performed a yeast two-hybrid screening using HDM2 as bait. Among the candidates, ribosomal protein L26 (RPL26) was characterized as a novel HDM2-interactor. The interaction between HDM2 and RPL26 was further validated by in vivo and in vitro assays. RPL26 modulates the HDM2–p53 interaction by forming a ternary complex among RPL26, HDM2 and p53, which stabilize p53 through inhibiting the ubiquitin ligase activity of HDM2. The ribosomal stress caused by a low dose of Act D enhances RPL26–HDM2 interaction and activates p53. Overexpression of RPL26 results in activating of p53, inhibits cell proliferation and induces a p53-dependent cell cycle arrest. These results provide a novel regulatory mechanism of RPL26 to activate p53 by inhibiting HDM2.     

Peptide, Peptidomimetic, and Small-molecule Antagonists of the p53–HDM2 Protein–Protein Interaction

Modulation of intracellular protein–protein interactions has been – and remains – a challenging goal for the discovery and development of small-molecule therapeutic agents. Progress in the pharmacological targeting and understanding at the molecular level of one such interaction that is relevant to cancer drug research, viz. that between the tumour suppressor protein p53 and its negative regulator HDM2, is reviewed here. The first X-ray crystal structure of a complex between a small peptide from the trans-activation domain of p53 and the N-terminal domain of HDM2 was reported almost 10 years ago. The nature of this interaction, which involves just three residue side chains in the p53 peptide ligand and a compact hydrophobic binding pocket in the HDM2 receptor, together with the attractive concept of reactivating the anti-proliferative functions of p53 in tumour cells, has spurned a great deal of effort aimed at finding drug-like antagonists of this interaction. A variety of approaches, including both structure-guided peptidomimetic and de novo design, as well as high through-put screening campaigns, have provided a wealth of leads that might be turned into actual drugs. There is still some way to go as far as optimisation and preclinical development of such leads is concerned, but it is clear already now that antagonists of the p53–HDM2 protein–protein interaction have a good chance of ultimately being successful in providing a new anti-cancer therapy modality, both in monotherapy and to potentiate the effectiveness of existing chemotherapies.     

8-Triazolylpurines: Towards Fluorescent Inhibitors of the MDM2/p53 Interaction

Small molecule nonpeptidic mimics of α-helices are widely recognised as protein-protein interaction (PPIs) inhibitors. Protein-protein interactions mediate virtually all important regulatory pathways in a cell, and the ability to control and modulate PPIs is therefore of great significance to basic biology, where controlled disruption of protein networks is key to understanding network connectivity and function. We have designed and synthesised two series of 2,6,9-substituted 8-triazolylpurines as α-helix mimetics. The first series was designed based on low energy conformations but did not display any biological activity in a biochemical fluorescence polarisation assay targeting MDM2/p53. Although solution NMR conformation studies demonstrated that such molecules could mimic the topography of an α-helix, docking studies indicated that the same compounds were not optimal as inhibitors for the MDM2/p53 interaction. A new series of 8-triazolylpurines was designed based on a combination of docking studies and analysis of recently published inhibitors. The best compound displayed low micromolar inhibitory activity towards MDM2/p53 in a biochemical fluorescence polarisation assay. In order to evaluate the applicability of these compounds as biologically active and intrinsically fluorescent probes, their absorption/emission properties were measured. The compounds display fluorescent properties with quantum yields up to 50%.     

N-Acylpolyamine inhibitors of HDM2 and HDMX binding to p53

Selective inhibition of protein–protein interactions important for cellular processes could lead to the development of new therapies against disease. In the area of cancer, overexpression of the proteins human double minute 2 (HDM2) and its homolog HDMX has been linked to tumor aggressiveness. Both HDM2 and HDMX bind to p53 and prevent cell cycle arrest or apoptosis in damaged cells. Developing a strategy to simultaneously prevent the binding of both HDM2 and HDMX to p53 is an essential feature of inhibitors to restore p53 activity in a number of different cancers. Inhibition of protein–protein interactions with synthetic molecules is an emerging area of research that requires new inhibitors tailored to mimic the types of interfaces between proteins. Our strategy to create inhibitors of protein–protein interactions is to develop a non-natural scaffold that may be used as a starting point to identify important molecular components necessary for inhibition. In this study, we report an N-acylpolyamine (NAPA) scaffold that supports numerous sidechains in a compact atomic arrangement. NAPAs were constructed by a series of reductive aminations between amino acid derivatives followed by acylation at the resulting secondary amine. An optimized NAPA was able to equally inhibit the association of both HDM2 and HDMX with p53. Our results demonstrate some of the challenges associated with targeting multiple protein–protein interactions involved in overlapping cellular processes.     

Binding of Translationally Controlled Tumour Protein to the N-Terminal Domain of HDM2 Is Inhibited by Nutlin-3

Translationally Controlled Tumour Protein (TCTP), a highly conserved protein present in all eukaryotic organisms, has a number of intracellular and extracellular functions including an anti-apoptotic role. TCTP was recently shown to interact with both p53 and HDM2, inhibiting auto-ubiquitination of the latter and thereby promoting p53 degradation. In this study, we further investigated the interaction between TCTP and HDM2, mapping the reciprocal binding sites of TCTP and HDM2. TCTP primarily interacts with the N-terminal, p53-binding region of HDM2 through its highly basic domain 2. Furthermore, we discovered that Nutlin-3, a small molecule known to promote apoptosis and cell cycle arrest by blocking binding between HDM2 and p53, has a similar inhibitory effect on the interaction of HDM2 and TCTP. This result may provide an additional explanation of how Nutlin-derived compounds currently in clinical trials function to promote apoptosis in cancer cells.     

HDM2-binding partners: interaction with translation elongation factor EF1alpha

To understand the cellular functions of HDM2, we attempted to identify novel HDM2-interacting proteins by proteomic analysis. Along with previously identified interactions with the ribosomal proteins, our analysis reveals interactions of HDM2 with the ribosomal translation elongation factor EF1alpha, 40S ribosomal protein S20, tubulins, glyceraldehyde 3-phosphate dehydrogenase, and a proteolysis-inducing factor dermicidin in the absence of tumor suppressor p53. Because a CTCL tumor antigen HD-CL-08 has high degree of homology with EF1alpha, we confirmed interaction of HDM2 with EF1alpha by immunoprecipitation and Western blot analysis in transformed as well as near normal diploid cells. Endogenous HDM2- EF1alpha complex was detected in cancer cells overexpressing HDM2, suggesting a possible role of this interaction in HDM2-mediated oncogenesis. Consistent with their interaction, colocalization of HDM2 and EF1alpha can be detected in the cytoplasm of normal or transformed cells. Amino acid residues 1-58 and 221-325 of HDM2 were found to be essential for its interaction with EF1alpha, suggesting that the interaction is independent of its other ribosomal interacting proteins L5, L11, and L23. Overexpression of HDM2 did not affect translation. Because EF1alpha has been implicated in DNA replication and severing of microtubules, interaction of HDM2 with EF1alpha may signify a p53-independent cell growth regulatory role of HDM2.     

In Vitro Selection of Mutant HDM2 Resistant to Nutlin Inhibition

HDM2 binds to the p53 tumour suppressor and targets it for proteosomal degradation. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2 and abrogates its repressive function. Using a novel in vitro selection methodology, we simulated the emergence of resistance by evolving HDM2 mutants capable of binding p53 in the presence of Nutlin concentrations that inhibit the wild-type HDM2-p53 interaction. The in vitro phenotypes were recapitulated in ex vivo assays measuring both p53 transactivation function and the direct p53-HDM2 interaction in the presence of Nutlin. Mutations conferring drug resistance were not confined to the N-terminal p53/Nutlin–binding domain, and were additionally seen in the acidic, zinc finger and RING domains. Mechanistic insights gleaned from this broad spectrum of mutations will aid in future drug design and further our understanding of the complex p53-HDM2 interaction.     

A DIGE-based approach to study interacting proteins

A full spectrum of high-throughput protein identification and characterization approaches has been developed for protein profiling. However, the most demanding field to better understanding protein interactions known as the “interactome” is still of a perpetual need for modern proteomics. Recently developed DIGE (difference in-gel electrophoresis) system may be of potential use when studying interacting proteins. In this work we applied DIGE technique on native gel electrophoresis to study protein–protein interactions. As a proof of principle, we utilized an in vitro interaction model between p53 and HDM2 proteins. In parallel, we also showed interaction of these proteins using fluorescently labelled p53- or HDM2-immunoprecipitation pellets. Thus, we believe this study shows a good potential for investigating various interacting partners and benefits towards creation of interactome.     

A DIGE-based approach to study interacting proteins

A full spectrum of high-throughput protein identification and characterization approaches has been developed for protein profiling. However, the most demanding field to better understanding protein interactions known as the "interactome" is still of a perpetual need for modern proteomics. Recently developed DIGE (difference in-gel electrophoresis) system may be of potential use when studying interacting proteins. In this work we applied DIGE technique on native gel electrophoresis to study protein-protein interactions. As a proof of principle, we utilized an in vitro interaction model between p53 and HDM2 proteins. In parallel, we also showed interaction of these proteins using fluorescently labelled p53- or HDM2-immunoprecipitation pellets. Thus, we believe this study shows a good potential for investigating various interacting partners and benefits towards creation of interactome.     

Expression of the p53 Gene and Activation of p53-Dependent Transcription in Melanoma Cell Lines

Malignant melanoma has poor prognosis because of its high metastatic potential and resistance to chemotherapy. A possible approach to more effective therapy is induction of p53-dependent apoptosis. This approach is promising, since the wild-type _p53 is expressed in most melanomas. An attempt was made to estimate the functional activity of p53 in several malignant melanoma cell lines. Most lines were characterized by a high protein level and nuclear localization of p53. All cell lines expressing the wild-type p53 showed stabilization of p53, its translocation into the nucleus, and activation of several target genes in response to DNA-damaging agents, suggesting that p53 was functionally active. A high-molecular-weight protein localized in the cytoplasm and mimicking a p53 epitope was found in several cell lines. It was shown that the DO-1 epitope is not derived from p53, ruling out cytoplasmic retention of p53 in melanoma cell lines. A mechanism of camptothecin-induced stabilization of p53 by decreasing the level of the HDM2 mRNA was described for melanoma cells but not for normal melanocytes, suggesting a differential effect of camptothecin on tumor-derived and primary cells.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 445–456.Original Russian Text Copyright © 2005 by Razorenova, Agapova, Chumakov.     

Inhibition of HDM2 and activation of p53 by ribosomal protein L23

The importance of coordinating cell growth with proliferation has been recognized for a long time. The molecular basis of this relationship, however, is poorly understood. Here we show that the ribosomal protein L23 interacts with HDM2. The interaction involves the central acidic domain of HDM2 and an N-terminal domain of L23. L23 and L11, another HDM2-interacting ribosomal protein, can simultaneously yet distinctly interact with HDM2 together to form a ternary complex. We show that, when overexpressed, L23 inhibits HDM2-induced p53 polyubiquitination and degradation and causes a p53-dependent cell cycle arrest. On the other hand, knocking down L23 causes nucleolar stress and triggers translocation of B23 from the nucleolus to the nucleoplasm, leading to stabilization and activation of p53. Our data suggest that cells may maintain a steady-state level of L23 during normal growth; alternating the levels of L23 in response to changing growth conditions could impinge on the HDM2-p53 pathway by interrupting the integrity of the nucleolus.     

A Spiroligomer α-Helix Mimic That Binds HDM2, Penetrates Human Cells and Stabilizes HDM2 in Cell Culture

We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers [1]. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.     

Regulation of the HDM2-p53 pathway by ribosomal protein L6 in response to ribosomal stress

The HDM2-p53 loop is crucial for monitoring p53 level and human pathologies. Therefore, identification of novel molecules involved in this regulatory loop is necessary for understanding the dynamic regulation of p53 and treatment of human diseases. Here, we characterized that the ribosomal protein L6 binds to and suppresses the E3 ubiquitin ligase activity of HDM2, and subsequently attenuates HDM2-mediated p53 polyubiquitination and degradation. The enhanced p53 activity further slows down cell cycle progression and leads to cell growth inhibition. Conversely, the level of p53 is dramatically decreased upon the depletion of RPL6, indicating that RPL6 is essential for p53 stabilization. We also found that RPL6 translocalizes from the nucleolus to nucleoplasm under ribosomal stress, which facilitates its binding with HDM2. The interaction of RPL6 and HDM2 drives HDM2-mediated RPL6 polyubiquitination and proteasomal degradation. Longer treatment of actinomycin D increases RPL6 ubiquitination and destabilizes RPL6, and thereby putatively attenuates p53 response until the level of L6 subsides. Therefore, RPL6 and HDM2 form an autoregulatory feedback loop to monitor the level of p53 in response to ribosomal stress. Together, our study identifies the crucial function of RPL6 in regulating HDM2-p53 pathway, which highlights the importance of RPL6 in human genetic diseases and cancers.