HDM2 Interaction Research Articles

Grafting Hydrophobic Amino Acids Critical for Inhibition of Protein-Protein Interactions on a Cell-Penetrating Peptide Scaffold.

Stapled peptides are a promising class of conformationally restricted peptides for modulating protein-protein interactions (PPIs). However, the low membrane permeability of these peptides is an obstacle to their therapeutic applications. It is common that only a few hydrophobic amino acid residues are mandatory for stapled peptides to bind to their target proteins. Hoping to create a novel class of membrane-permeable PPI inhibitors, the phenylalanine, tryptophan, and leucine residues that play a critical role in inhibiting the p53-HDM2 interaction were grafted into the framework of CADY2─a cell-penetrating peptide (CPP) having a helical propensity. Two analogues (CADY-3FWL and CADY-10FWL) induced apoptotic cell death but lacked the intended HDM2 interaction. Pull-down experiments followed by proteomic analysis led to the elucidation of nesprin-2 as a candidate binding target. Nesprin-2 is considered to play a role in the nuclear translocation of β-catenin upon activation of the Wnt signaling pathway, which leads to the expression of antiapoptosis proteins and cell survival. Cells treated with the two analogues showed decreased nuclear localization of β-catenin and reduced mRNA expression of related antiapoptotic proteins. These data suggest inhibition of β-catenin nuclear translocation as a possible mode of action of the described cell-penetrating stapled peptides.

LncRNA SAMD12-AS1 promotes cell proliferation and inhibits apoptosis by interacting with NPM1

Chronic hepatitis B virus infection is a major risk factor for hepatocellular carcinoma. HBV infection affects lncRNA expression in infected cells, but the detailed mechanism and biological significance are not yet clear. In this study, we focused on exploring the function of the HBV-upregulated lncRNA SAMD12-AS1 in cell proliferation. We found that there is a higher level of SAMD12-AS1 expression in tumors than in adjacent nontumorous liver tissues. We showed that ectopic expression of SAMD12-AS1 promotes cell growth and blocks apoptosis, while knockdown of SAMD12-AS1 inhibits cell proliferation and enhances etoposide-induced apoptosis. Using RNA immunoprecipitation and mass spectrometry, we determined that SAMD12-AS1 interacts with NPM1 and confirmed that SAMD12-AS1(1-350) is required for the interaction with NPM1. As it is known that NPM1 interacts with the E3 ligase HDM2 and reduces HDM2-mediated p53 degradation, we examined whether SAMD12-AS1 can affect p53 stability. Overexpression of SAMD12-AS1 caused a reduction in p53 protein levels by shortening its half-life. Conversely, knockdown of SAMD12-AS1 prolonged the half-life of p53. We further demonstrated that SAMD12-AS1 increased the interaction of HDM2 and p53 and enhanced p53 ubiquitination. Our findings reveal that HBV-upregulated SAMD12-AS1 regulates cell proliferation and apoptosis via the NPM1-HDM2-p53 axis.

Abstract 257: Targeting AML through apoptosis activation using Bcl-2/Mcl-1 or Bcl-2/Hdm2 inhibitor combination therapies

Abstract Acute myeloid leukemia (AML) is an aggressive and heterogeneous hematologic malignancy, characterized by uncontrolled proliferation and impaired differentiation of myeloid cells. With the exception of certain subtypes, the average long-term survival rate remains low, thus underlining the need to further improve the outcome of AML patients. Since AML is one of the least mutated cancer types, the majority of AML patients may not carry targetable genetic alterations. However, the anti-apoptotic proteins of the Bcl-2 family, such as Bcl-2 and Mcl-1, are often overexpressed in AML, allowing deregulated survival; hence pro-apoptosis priming with small molecule inhibitors of Bcl-2 and Mcl-1 may provide a broader therapeutic benefit across the disease. In addition, a majority of AML patients carry wild-type p53, providing therapeutic opportunity for Hdm2 inhibitors to stabilize p53 and lead to expression of pro-apoptotic molecules (e.g., PUMA & BAX). Therefore, targeting the combined apoptosis mechanisms by inhibiting different anti-apoptotic Bcl-2 family of proteins and activating p53 concomitantly may synergistically enhance apoptotic cell death of AML tumor cells. We tested the combination of Bcl-2 inhibitors (BCL201/S55746 or venetoclax) with either MIK665/S64315, a novel and selective inhibitor of Mcl-1 or HDM201, a selective small molecule inhibitor of p53:Hdm2 interaction, in a series of in vitro and in vivo studies in AML. In vitro, strong combination synergy was observed with a remarkable induction of cell death for both combinations. In vivo, the combination of Bcl-2 inhibitors with MIK665/S64315 or HDM201 lead to complete and durable antitumor responses in a variety of p53wt AML patient-derived xenograft models of heterogeneous genetic profiles. Notably, lowering the dose of HDM201 by 4 fold from its most efficacious dose, resulted in a high degree of tumor regressions while mitigating the toxicity effects on platelets. Taken together, these data demonstrate that a combination of Bcl-2 inhibitor (BCL201/S55746 or venetoclax) with MIK665/S64315 or HDM201 provide therapeutic benefit over the monotherapy, and support a rationale for testing these apoptosis enhancing combination approaches in AML patients. Citation Format: Youzhen Wang, Shumei Qui, Sneha Sanghavi, Iain Mulford, Gaëlle Lysiak, Maïa Chanrion, Prakash Mistry, Ulrike Pfaar, Marie Schoumacher, Audrey Claperon, Laurence Kraus-Berthier, Sébastien Banquet, Alix Derreal, Claire Fabre, Heiko Maacke, Frédéric Colland, Olivier Geneste, Erick Morris, Ensar Halilovic. Targeting AML through apoptosis activation using Bcl-2/Mcl-1 or Bcl-2/Hdm2 inhibitor combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 257.

Identification of selective protein-protein interaction inhibitors using efficient in silico peptide-directed ligand design.

The development of protein-protein interaction (PPI) inhibitors with therapeutic value is of increasing importance as the first clinical agent has now been approved, but PPIs remain difficult targets for the development of small molecule ligands. This article describes a highly efficient approach to the development of inhibitors of the p53/hDMX or hDM2 interaction that involves the design of small molecules in silico based upon a peptide/protein structure. The process for molecule design, starting from a virtual library of just over 1200 fragments, led to the eventual synthesis of twenty compounds, of which ten bound to either hDM2, hDMX or both in in vitro binding assays. This 50% success rate is extremely efficient compared to traditional high throughput screening. The identification of two selective hDMX inhibitors from twenty compounds highlights this efficiency as, to date, only two other hDMX-selective agents exist in the literature. Preliminary biological studies show that 20% of the compounds identified have cellular activity and activate downstream pathways associated with p53 activation.

Siladenoserinols M–P, sulfonated serinol derivatives from a tunicate

Four new sulfonated serinol derivatives, siladenoserinols M–P (1–4), were isolated from a tunicate of the family Didemnidae collected in Indonesia. Their chemical structures were elucidated by the interpretation of NMR and mass spectroscopic data. Two of them (2 and 4) were revealed to be disulfonate serinol derivatives, and the others were monosulfonates. Siladenoserinols A (5) and B (6), which we previously isolated from the same tunicate, inhibited the p53–Hdm2 interaction with an IC50 value of 2.0 μM. However, 1–4 did not inhibit the activity. The result suggested that the acetyl group in the bicyclic ketal unit and/or the glycerophospholipid moiety in 5 and 6 were responsible for the inhibition of the p53–Hdm2 interaction.

P53 promotes its own polyubiquitination by enhancing the HDM2 and HDMX interaction

HDM2 and HDMX are two homologs essential for controlling p53 tumor suppressor activity under normal conditions. Both proteins bind different sites on the p53 N-terminus, and while HDM2 has E3 ubiquitin ligase activity towards p53, HDMX does not. Nevertheless, HDMX is required for p53 polyubiquitination and degradation, but the underlying molecular mechanism remains unclear. Alone, HDMX and HDM2 interact via their respective C-terminal RING domains but here we show that the presence of p53 induces an N-terminal interface under normal cellular conditions. This results in an increase in HDM2-mediated p53 polyubiquitination and degradation. The HDM2 inhibitor Nutlin-3 binds the N-terminal p53 binding pocket and is sufficient to induce the HDM2-HDMX interaction, suggesting that the mechanism depends on allosteric changes that control the multiprotein complex formation. These results demonstrate an allosteric interchange between three different proteins (HDMX-HDM2-p53) and help to explain the molecular mechanisms of HDM2-inhibitory drugs.

Total Synthesis and Biological Evaluation of Siladenoserinol A and its Analogues

AbstractThe total synthesis of siladenoserinol A, an inhibitor of the p53–Hdm2 interaction, has been achieved. AuCl3‐catalyzed hydroalkoxylation of an alkynoate derivative smoothly and regioselectively proceeded to afford a bicycloketal in excellent yield. A glycerophosphocholine moiety was successfully introduced through the Horner–Wadsworth–Emmons reaction using an originally developed phosphonoacetate derivative. Finally, removal of the acid‐labile protecting groups, followed by regioselective sulfamate formation of the serinol moiety afforded the desired siladenoserinol A, and benzoyl and desulfamated analogues were also successfully synthesized. Biological evaluation showed that the sulfamate is essential for biological activity, and modification of the acyl group on the bicycloketal can improve the inhibitory activity against the p53–Hdm2 interaction.

A redox mechanism underlying nucleolar stress sensing by nucleophosmin.

The nucleolus has been recently described as a stress sensor. The nucleoplasmic translocation of nucleolar protein nucleophosmin (NPM1) is a hallmark of nucleolar stress; however, the causes of this translocation and its connection to p53 activation are unclear. Using single live-cell imaging and the redox biosensors, we demonstrate that nucleolar oxidation is a general response to various cellular stresses. During nucleolar oxidation, NPM1 undergoes S-glutathionylation on cysteine 275, which triggers the dissociation of NPM1 from nucleolar nucleic acids. The C275S mutant NPM1, unable to be glutathionylated, remains in the nucleolus under nucleolar stress. Compared with wild-type NPM1 that can disrupt the p53–HDM2 interaction, the C275S mutant greatly compromises the activation of p53, highlighting that nucleoplasmic translocation of NPM1 is a prerequisite for stress-induced activation of p53. This study elucidates a redox mechanism for the nucleolar stress sensing and may help the development of therapeutic strategies.

A Cyclized Helix‐Loop‐Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein–Protein Interactions by Epitope and Arginine Grafting

AbstractThe design of inhibitors of intracellular protein–protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix‐loop‐helix (cHLH) peptide as a scaffold for generating cell‐permeable PPI inhibitors through bifunctional grafting: epitope grafting to provide binding activity, and arginine grafting to endow cell‐permeability. To inhibit p53–HDM2 interactions, the p53 epitope was grafted onto the C‐terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53‐R showed high inhibitory activity for this interaction, and computational analysis suggested a binding mode for HDM2. Confocal microscopy of cells treated with fluorescently labeled cHLHp53‐R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells. This strategy of bifunctional grafting onto a well‐structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs.

Computational identification of novel piperidine derivatives as potential HDM2 inhibitors designed by fragment-based QSAR, molecular docking and molecular dynamics simulations

Tumor suppressor protein p53 maintains integrity of genome and regulates the genes responsible for DNA repair mechanism, apoptosis as well as cell cycle and growth arrest. As with murine double minute 2 (MDM2), the human homolog HDM2 is a principal cellular antagonist of p53. In unstressed cells, cellular levels of p53 and HDM2 are maintained in an autoregulatory manner in which both mutually control cellular levels of each other. About half of the human cancers express wild-type p53 protein that is antagonized by over-expressed HDM2. Restoring p53 function via HDM2 antagonists is a leading therapeutic approach for treating a variety of tumors. In this study, we have developed a novel statistically sound group-based QSAR (GQSAR) model using piperidine-derived compounds that have been validated experimentally to inhibit p53–HDM2 interaction. On the basis of developed GQSAR model, a combinatorial library of molecules was prepared and its activity was predicted. These molecules were then docked to HDM2, and two top-scoring molecules possessing a binding energy of −6.639 and −6.305 kcal/mol were selected for further study. These molecules and their binding poses were analyzed further via molecular dynamic simulations. In this study, we report two lead compounds as potent HDM2 inhibitors and also provide an insight into mechanism of interaction of the lead compounds to HDM2 target.

Unified flexible total synthesis of chlorofusin and artificial Click mimics as antagonists against p53–HDM2 interactions

A practical HPLC-free total synthesis of chlorofusin has been successfully accomplished in this work. The new synthesis showed great flexibility and convenience in generating artificial natural product-like mimics through Click chemistry, and enabled us to further investigate the biological importance of the chromophore and the spiro-aminal functionality of the natural product. The entire skeleton is believed to be essential for satisfactory biological activities of both natural chlorofusin and unnatural mimics. Two artificial Click hybrids were found to exhibit improved inhibitory activity against p53–HDM2 bindings over the natural product.

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.

Visualization and targeted disruption of protein interactions in living cells

Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species.

Aromatic Oligoamide Foldamers with a “Wet Edge” as Inhibitors of the α‐Helix‐Mediated p53–hDM2 Protein–Protein Interaction

AbstractThis paper describes the design, synthesis and structural analysis of a 3‐O‐alkylated aromatic oligoamide that incorporates an additional hydrophilic 6‐O‐alkyl substituent in the central monomer. This oligomer exhibits low μM inhibitory potency against the p53–hDM2 interaction compared with its unfunctionalised counterpart and significantly improved solubility.

Siladenoserinols A–L: New Sulfonated Serinol Derivatives from a Tunicate as Inhibitors of p53–Hdm2 Interaction

Siladenoserinols A-L were isolated from a tunicate as inhibitors of p53-Hdm2 interaction, a promising target for cancer chemotherapy. Their structures including the absolute configurations were elucidated to be new sulfonated serinol derivatives, each of which contains a 6,8-dioxabicyclo[3.2.1]octane unit and either glycerophosphocholine or glycerophosphoethanolamine moiety. They inhibited p53-Hdm2 interaction with IC(50) values of 2.0-55 μM. Among them, siladenoserinol A and B exhibited the strongest inhibition with an IC(50) value of 2.0 μM.

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.

P14ARF inhibits human glioblastoma–induced angiogenesis by upregulating the expression of TIMP3

Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%-80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology.

P38 phosphorylates Rb on Ser567 by a novel, cell cycle-independent mechanism that triggers Rb–Hdm2 interaction and apoptosis

The retinoblastoma protein (Rb) inhibits both cell division and apoptosis, but the mechanism by which Rb alternatively regulates these divergent outcomes remains poorly understood. Cyclin dependent kinases (Cdks) promote cell division by phosphorylating and reversibly inactivating Rb by a hierarchical series of phosphorylation events and sequential conformational changes. The stress-regulated mitogen activated protein kinase (MAPK) p38 also phosphorylates Rb, but it does so in a cell cycle-independent manner that is associated with apoptosis rather than with cell division. Here, we show that p38 phosphorylates Rb by a novel mechanism that is distinct from that of Cdks. p38 bypasses the cell cycle-associated hierarchical phosphorylation and directly phosphorylates Rb on Ser567, which is not phosphorylated during the normal cell cycle. Phosphorylation by p38, but not Cdks, triggers an interaction between Rb and the human homologue of murine double minute 2 (Hdm2), leading to degradation of Rb, release of E2F1 and cell death. These findings provide a mechanistic explanation for how Rb regulates cell division and apoptosis through different kinases, and reveal how Hdm2 may functionally link the tumor suppressors Rb and p53.

Further Insight into Substrate Recognition by USP7: Structural and Biochemical Analysis of the HdmX and Hdm2 Interactions with USP7

Ubiquitin-specific protease 7 (USP7) catalyzes the deubiquitination of several substrate proteins including p53 and Hdm2. We have previously shown that USP7, and more specifically its amino-terminal domain (USP7–NTD), interacts with distinct regions on p53 and Hdm2 containing P/AxxS motifs. The ability of USP7 to also deubiquitinate and control the turnover of HdmX was recently demonstrated. We utilized a combination of biochemistry and structural biology to identify which domain of USP7 interacts with HdmX as well as to identify regions of HdmX that interact with USP7. We showed that USP7–NTD recognized two of six P/AxxS motifs of HdmX (8AQCS11 and 398AHSS401). The crystal structure of the USP7–NTD:HdmXAHSS complex was determined providing the molecular basis of complex formation between USP7–NTD and the HdmXAHSS peptide. The HdmX peptide interacted within the same residues of USP7–NTD as previously demonstrated with p53, Hdm2, and EBNA1 peptides. We also identified an additional site on Hdm2 (397PSTS400) that interacts with USP7–NTD and determined the crystal structure of this complex. Finally, analysis of USP7-interacting peptides on filter arrays confirmed the importance of the serine residue at the fourth position for the USP7–NTD interaction and showed that phosphorylation of serines within the binding sequence prevents this interaction. These results lead to a better understanding of the mechanism of substrate recognition by USP7–NTD.

Stereochemical studies of hexylitaconic acid, an inhibitor of p53–HDM2 interaction

Hexylitaconic acid (1) is an intriguing natural product possessing a chiral carbon, and both its enantiomers have been found in nature. Enantiomeric pure (+)-(1) and (−)-(1) were successfully prepared by racemic synthesis followed by enantiomeric separation in a chiral HPLC system. Their absolute configurations were clarified by the vibrational circular dichroism technique using their methyl esters 2 and lactones 3. Their inhibitory activities against the interaction of p53–HDM2 were also examined.