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The regulation of the p53 tumor suppressor pathway is critically dependent on the activity of Murine Double Minute 2 (MDM2) and Murine Double Minute X (MDMX) proteins. In certain types of cancer cells, excessive amount of MDMX can poly-ubiquitinate p53, which can result in its degradation, leading to a subsequent reduction in the levels of this protein. Therefore, the design of small-molecule inhibitors targeting the MDMX-p53 interaction has emerged as a promising strategy for cancer therapy. In this study, we employed computational techniques including pharmacophore modeling and molecular docking to identify three potential small molecule inhibitors (CID_25094615, CID_137634453, and CID_25094344) of the MDMX-p53 interaction from a PubChem database. Molecular dynamics of 100000 ps were conducted to assess the stability of the MDMX-inhibitor complexes. Our results showed that all three compounds exhibit stable binding with MDMX, with significantly lower root mean square deviation (RMSD) and fluctuation (RMSF) values than the control ligand, indicating superior stability. Additionally, the three compounds exhibit stronger intermolecular hydrogen bond (HBOND) interactions compared to the control, suggesting stronger stability. Overall, our findings highlight the potential of these compounds as lead candidates for the development of novel anticancer agents that target the MDMX-p53 interaction.Communicated by Ramaswamy H. Sarma.

Intrinsically disordered proteins (IDPs) lack a well-defined tertiary structure but are essential players in various biological processes. Their ability to undergo a disorder-to-order transition upon binding to their partners, known as the folding-upon-binding process, is crucial for their function. One classical example is the intrinsically disordered transactivation domain (TAD) of the tumor suppressor protein p53, which quickly forms a structured α-helix after binding to its partner MDM2, with clinical significance for cancer treatment. However, the contribution of nonnative interactions between the IDP and its partner to the rapid binding kinetics, as well as their interplay with native interactions, is not well understood at the atomic level. Here, we used molecular dynamics simulation and Markov state model (MSM) analysis to study the folding-upon-binding mechanism between p53-TAD and MDM2. Our results suggest that the system progresses from the nascent encounter complex to the well-structured encounter complex and finally reaches the native complex, following an induced-fit mechanism. We found that nonnative hydrophobic and hydrogen bond interactions, combined with native interactions, effectively stabilize the nascent and well-structured encounter complexes. Among the nonnative interactions, Leu25 p53 -Leu54 MDM2 and Leu25 p53 -Phe55 MDM2 are particularly noteworthy, as their interaction strength is close to the optimum. Evidently, strengthening or weakening these interactions could both adversely affect the binding kinetics. Overall, our findings suggest that nonnative interactions are evolutionarily optimized to accelerate the binding kinetics of IDPs in conjunction with native interactions.

The thermodynamics of secondary p53 binding sites on MDM2 and MDMX were evaluated using p53 peptides containing residues 16-29, 17-35, and 1-73. All the peptides had large, negative heat capacity (ΔC p ), consistent with the burial of p53 residues F19, W23, and L26 in the primary binding sites of MDM2 and MDMX. MDMX has a higher affinity and more negative ΔC p than MDM2 for p53 17-35 , which is due to MDMX stabilization and not additional interactions with the secondary binding site. ΔC p measurements show binding to the secondary site is inhibited by the disordered tails of MDM2 for WT p53 but not a more helical mutant where proline 27 is changed to alanine. This result is supported by all-atom molecular dynamics simulations showing that p53 residues 30-35 turn away from the disordered tails of MDM2 in P27A 17-35 and make direct contact with this region in p53 17-35 . Molecular dynamics simulations also suggest that an intramolecular methionine-aromatic motif found in both MDM2 and MDMX structurally adapts to support multiple p53 binding modes with the secondary site. ΔC p measurements also show that tighter binding of the P27A mutant to MDM2 and MDMX is due to increased helicity, which reduces the energetic penalty associated with coupled folding and binding. Our results will facilitate the design of selective p53 inhibitors for MDM2 and MDMX., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The interaction between murine double minute 2 (MDM2) and p53, marked by transcriptional induction and feedback inhibition, orchestrates a functional loop dictating cellular fate. The functional loop comprising p53-MDM2 axis is made up of an interactome consisting of approximately 81 proteins, which are spatio-temporally regulated and involved in DNA repair mechanisms. Biochemical and genetic alterations of the interactome result in dysregulation of the p53-mdm2 axis that leads to gastrointestinal (GI) cancers. A large subset of interactome is well known and it consists of proteins that either stabilize p53 or MDM2 and proteins that target the p53-MDM2 complex for ubiquitin-mediated destruction. Upstream signaling events brought about by growth factors and chemical messengers invoke a wide variety of posttranslational modifications in p53-MDM2 axis. Biochemical changes in the transactivation domain of p53 impact the energy landscape, induce conformational switching, alter interaction potential and could change solubility of p53 to redefine its co-localization, translocation and activity. A diverse set of chemical compounds mimic physiological effectors and simulate biochemical modifications of the p53-MDM2 interactome. p53-MDM2 interactome plays a crucial role in DNA damage and repair process. Genetic aberrations in the interactome, have resulted in cancers of GI tract (pancreas, liver, colorectal, gastric, biliary, and esophageal). We present in this article a review of the overall changes in the p53-MDM2 interactors and the effectors that form an epicenter for the development of next-generation molecules for understanding and targeting GI cancers., (© 2024 John Wiley & Sons Ltd.)

The tumor suppressor p53 plays a crucial role in cellular responses to various stresses, regulating key processes such as apoptosis, senescence, and DNA repair. Dysfunctional p53, prevalent in approximately 50 % of human cancers, contributes to tumor development and resistance to treatment. This study employed deep learning-based protein design and structure prediction methods to identify novel high-affinity peptide binders (Pep1 and Pep2) targeting MDM2, with the aim of disrupting its interaction with p53. Extensive all-atom molecular dynamics simulations highlighted the stability of the designed peptide in complex with the target, supported by several structural analyses, including RMSD, RMSF, Rg, SASA, PCA, and free energy landscapes. Using the steered molecular dynamics and umbrella sampling simulations, we elucidate the dissociation dynamics of p53, Pep1, and Pep2 from MDM2. Notable differences in interaction profiles were observed, emphasizing the distinct dissociation patterns of each peptide. In conclusion, the results of our umbrella sampling simulations suggest Pep1 as a higher-affinity MDM2 binder compared to p53 and Pep2, positioning it as a potential inhibitor of the MDM2-p53 interaction. Using state-of-the-art protein design tools and advanced MD simulations, this study provides a comprehensive framework for rational in silico design of peptide binders with therapeutic implications in disrupting MDM2-p53 interactions for anticancer interventions., Competing Interests: Declaration of competing interest None declared., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Various studies have established that molecules specific for MDMX inhibition or optimized for dual inhibition of p53-MDM2/MDMX interaction signaling are more suitable for activating the Tp53 gene in tumor cells. Nevertheless, there are sparse numbers of approved molecules to treat the health consequences brought by the lost p53 functions in tumor cells. Consequently, this study explored the potential of a small molecule ligand containing 1, 8-naphthyridine scaffold to act as a dual inhibitor of p53-MDM2/X interactions using computational methods. The results obtained from quantum mechanical calculations revealed our studied compound entitled CPO is more stable but less reactive compared to standard dual inhibitor RO2443. Like RO2443, CPO also exhibited good non-linear optical properties. The results of molecular docking studies predicted that CPO has a higher potential to inhibit MDM2/MDMX than RO2443. Furthermore, CPO was stable over 50 ns molecular dynamics (MD) simulation in complex with MDM2 and MDMX respectively. On the whole, CPO also exhibited good drug-likeness and pharmacokinetics properties compared to RO2443 and was found with more anti-cancer activity than RO2443 in bioactivity prediction. CPO is anticipated to elevate effectiveness and alleviate drug resistance in cancer therapy. Ultimately, our results provide an insight into the mechanism that underlay the inhibition of p53-MDM2/X interactions by a molecule containing 1, 8-naphthyridine scaffold in its molecular structure., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

HDM2 negatively regulates the activity of the tumor suppressor p53. Previous NMR studies have shown that apo-HDM2 interconverts between an "open" state in which the N-terminal "lid" is disordered and a "closed" state in which the lid covers the p53-binding site in the core region. Molecular dynamics (MD) simulation studies have been performed to elucidate the conformational dynamics of HDM2, but the direct relevance of the experimental and computational analyses is unclear. In addition, how the phosphorylation of S17 in the lid contributes to the inhibition of p53 binding remains controversial. Here, we used both NMR and MD simulations to investigate the conformational dynamics of apo-HDM2. The NMR analysis revealed that apo-HDM2 exists in a fast-exchanging equilibrium within two closed states, closed 1 and closed 2, in addition to a previously demonstrated slow-exchanging "open-closed" equilibrium. MD simulations visualized two characteristic closed states, where the spatial orientation of the key residues corresponds well to the chemical shift changes of the NMR spectra. Furthermore, the phosphorylation of S17 induced an equilibrium shift toward closed 1, thereby suppressing the binding of p53 to HDM2. This study reveals a multi-state equilibrium of apo-HDM2 and provides new insights into the regulation mechanism of HDM2-p53 interactions.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 μM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of "functional affinity" in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions., Competing Interests: Conflict of interest: The authors declare that they have no conflict of interest with the contents of this article., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Cancer is one of the leading causes of mortality in the world. Despite the existence of diverse antineoplastic treatments, these do not possess the expected efficacy in many cases. Knowledge of the molecular mechanisms involved in tumor processes allows the identification of a greater number of therapeutic targets employed in the study of new anticancer drugs. In the last decades, peptide-based therapy design using computational chemistry has gained importance in the field of oncology therapeutics. This work aims to evaluate the electronic structure, physicochemical properties, stability, and inhibition of ETFS amino acids and peptides derived from the p53-MDM2 binding domain with action in cancer cells; by means of chemical descriptors at the DFT-BHandHLYP level in an aqueous solution, and its intermolecular interactions through molecular docking studies. The results show that The ETFS fragment plays a critical role in the intermolecular interactions. Thus, the amino acids E17, T18 and S20 increase intermolecular interactions through hydrogen bonds and enhance structural stability. F19, W23 and V25 enhance the formation of the alpha-helix. The hydrogen bonds formed by the backbone atoms for PNC-27, PNC-27-B and PNC-28 stabilize the α-helices more than hydrogen bonds formed by the side chains atoms. Also, molecular docking indicated that the PNC27B-MDM2, PNC28B-MDM2, PNC27-MDM2 and PNC28A-MDM2 complexes show the best binding energy. Therefore, DFT and molecular docking studies showed that the proposed peptides: PNC-28B, PNC-27B and PNC-28A could inhibit the binding of MDM2 to the p53 protein, decreasing the translocation and degradation of p53 native protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: Therapeutic peptides are a significant class of drugs in the treatment of a wide range of diseases. To enhance their properties, such as stability or binding affinity, they are usually chemically modified. This includes, among other techniques, cyclization of the peptide chain by bridging, modifications to the backbone, and incorporation of unnatural amino acids. One approach previously established, is the use of halogenated aromatic amino acids. In principle, they are thereby enabled to form halogen bonds (XB). In this study, we focus on the -R-CF 2 X moiety (R = O, NHCO; X = Cl, Br) as an uncommon halogen bond donor. These groups enable more spatial variability in protein-protein interactions. The chosen approach via Fmoc-protected building blocks allows for the incorporation of these modified amino acids in peptides using solid-phase peptide synthesis., Results and Discussion: Using a competitive fluorescence polarization assay to monitor binding to Mdm4, we demonstrate that a p53-derived peptide with Lys24Nle(εNHCOCF 2 X) exhibits an improved inhibition constant K i compared to the unmodified peptide. Decreasing K i values observed with the increasing XB capacity of the halogen atoms (F ≪ Cl < Br) indicates the formation of a halogen bond. By reducing the side chain length of Nle(εNHCOCF 2 X) to Abu(γNHCOCF 2 X) as control experiments and through quantum mechanical calculations, we suggest that the observed affinity enhancement is related to halogen bond-induced intramolecular stabilization of the α-helical binding mode of the peptide or a direct interaction with His54 in human Mdm4., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Vaas et al.)

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In the vast majority of malignancies, the p53 tumor suppressor pathway is compromised. In some cancer cells, high levels of MDM2 polyubiquitinate p53 and mark it for destruction, thereby leading to a corresponding downregulation of the protein. MDM2 interacts with p53 via its hydrophobic pocket, and chemical entities that block the dimerization of the protein-protein complex can restore p53 activity. Thus far, only a few chemical compounds have been reported as potent arsenals against p53-MDM2. The Protein Data Bank has crystallogaphic structures of MDM2 in complex with certain compounds. Herein, we have exploited one of the complexes in the identification of new p53-MDM2 antagonists using a hierarchical virtual screening technique. The initial stage was to compile a targeted library of structurally appropriate compounds related to a known effective inhibitor, Nutlin 2, from the PubChem database. The identified 57 compounds were subjected to virtual screening using molecular docking to discover inhibitors with high binding affinity for MDM2. Consequently, five compounds with higher binding affinity than the standard emerged as the most promising therapeutic candidates. When compared to Nutlin 2, four of the drug candidates (CID&#95;140017825, CID&#95;69844501, CID&#95;22721108, and CID&#95;22720965) demonstrated satisfactory pharmacokinetic and pharmacodynamic profiles. Finally, MD simulation of the dynamic behavior of lead-protein complexes reveals the stability of the complexes after a 100,000 ps simulation period. In particular, when compared to the other three leads, overall computational modeling found CID&#95;140017825 to be the best pharmacological candidate. Following thorough experimental trials, it may emerge as a promising chemical entity for cancer therapy., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The human MDMX protein, also known as MDM4, plays a pivotal role in regulating the activity of the tumor suppressor protein p53 by restricting p53 transcriptional activity and stimulating the E3 ubiquitin ligase activity of another key regulatory protein, MDM2, to promote p53 degradation. MDMX is ubiquitously expressed in most tissue types and overexpression of MDMX has been implicated in many forms of cancer. MDMX has been shown to require an intact N-terminal p53-binding domain and C-terminal RING domain to exert inhibitory effects on p53. The presence of a tryptophan-rich sequence in the central acidic domain of MDMX has also been implicated in regulating the interaction between MDMX and p53, directly interacting with the p53 DNA-binding domain. To date, little structural information has been obtained for this acidic region of MDMX that encompasses the Trp-rich sequence. In order to gain insight into the structure and function of this region, we have carried out solution-state NMR spectroscopy studies utilizing the segment of MDMX spanning residues 181-300-with bounds specifically chosen through multiple sequence alignment-which encompasses nearly 25% of MDMX. Here, we report the 1 H, 15 N and 13 C backbone chemical shift assignments of the acidic domain of MDMX and show that it exhibits hallmarks of intrinsic disorder and localized variation in inferred secondary structure propensity., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Stapled peptides with an enforced α-helical conformation have been shown to overcome major limitations in the development of short peptides targeting protein-protein interactions (PPIs). While the growing arsenal of methodologies to staple peptides facilitates their preparation, stapling methodologies are not broadly embraced in synthetic library screening. Herein, we report a strategy leveraged on hybridization of short PNA-peptide conjugates wherein nucleobase driven assembly facilitates ligation of peptide fragments and constrains the peptide's conformation into an α-helix. Using native chemical ligation, we show that a mixture of peptide fragments can be combinatorially ligated and used directly in affinity selection against a target of interest. This approach was exemplified with a focused library targeting the p-53/MDM2 interaction. One hundred peptides were obtained in a one-pot ligation reaction, selected by affinity against MDM2 immobilized on beads, and the best binders were identified by mass spectrometry.

Identification of low-dose, low-molecular-weight, drug-like inhibitors of protein-protein interactions (PPIs) is a challenging area of research. Despite the challenges, the therapeutic potential of PPI inhibition has driven significant efforts toward this goal. Adding to recent success in this area, we describe herein our efforts to optimize a novel purine carboxylic acid-derived inhibitor of the HDM2-p53 PPI into a series of low-projected dose inhibitors with overall favorable pharmacokinetic and physical properties. Ultimately, a strategy focused on leveraging known binding hot spots coupled with biostructural information to guide the design of conformationally constrained analogs and a focus on efficiency metrics led to the discovery of MK-4688 (compound 56 ), a highly potent, selective, and low-molecular-weight inhibitor suitable for clinical investigation.

The study of protein-protein interactions is of great interest. Several early studies focused on the murine double minute 2 (Mdm2)-tumor suppressor protein p53 interactions. However, the effect of plasma treatment on Mdm2 and p53 is still absent from the literature. This study investigated the structural changes in Mdm2, p53, and the Mdm2-p53 complex before and after possible plasma oxidation through molecular dynamic (MD) simulations. MD calculation revealed that the oxidized Mdm2 bounded or unbounded showed high flexibility that might increase the availability of tumor suppressor protein p53 in plasma-treated cells. This study provides insight into Mdm2 and p53 for a better understanding of plasma oncology.

Protein-protein interactions (PPIs) are often mediated by helical, strand and/or coil secondary structures at the interface regions. We previously showed that non-naturally occurring, stable helical trimers of bicyclic β-amino acids (Abh) with all-trans amide bonds can block the p53-MDM2/MDMX α-helix-helix interaction, which plays a role in regulating p53 function. Here, we conducted docking and molecular dynamics calculations to guide the structural optimization of our reported compounds, focusing on modifications of the C-terminal/N-terminal residues. We confirmed that the modified peptides directly bind to MDM2 by means of thermal shift assay, isothermal titration calorimetry, and enzyme-linked immunosorbent assay (ELISA) experiments. Biological activity assay in human osteosarcoma cell line SJSA-1, which has wild-type p53 and amplification of the Mdm2 gene, indicated that these peptides are membrane-permeable p53-MDM2/MDMX interaction antagonists that can rescue p53 function in the cells.

Protein-protein interactions (PPIs) intimately govern various biological processes and disease states and therefore have been identified as attractive therapeutic targets for small-molecule drug discovery. However, the development of highly potent inhibitors for PPIs has proven to be extremely challenging with limited clinical success stories. Herein, we report irreversible inhibitors of the human double minute 2 (HDM2)/p53 PPI, which employ a reactive N -acyl- N -alkyl sulfonamide (NASA) group as a warhead. Mass-based analysis successfully revealed the kinetics of covalent inhibition and the modification sites on HDM2 to be the N-terminal α-amine and Tyr67, both rarely seen in traditional covalent inhibitors. Finally, we demonstrated prolonged p53-pathway activation and more effective induction of the p53-mediated cell death in comparison to a noncovalent inhibitor. This study highlights the potential of the NASA warhead as a versatile electrophile for the covalent inhibition of PPIs and opens new avenues for the rational design of potent covalent PPI inhibitors.

As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2's ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)-Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Epigallocatechin gallate (EGCG) from green tea can induce apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Using SPR and NMR, here we report a direct, μM interaction between EGCG and the tumor suppressor p53 (K D  = 1.6 ± 1.4 μM), with the disordered N-terminal domain (NTD) identified as the major binding site (K D  = 4 ± 2 μM). Large scale atomistic simulations (>100 μs), SAXS and AUC demonstrate that EGCG-NTD interaction is dynamic and EGCG causes the emergence of a subpopulation of compact bound conformations. The EGCG-p53 interaction disrupts p53 interaction with its regulatory E3 ligase MDM2 and inhibits ubiquitination of p53 by MDM2 in an in vitro ubiquitination assay, likely stabilizing p53 for anti-tumor activity. Our work provides insights into the mechanisms for EGCG's anticancer activity and identifies p53 NTD as a target for cancer drug discovery through dynamic interactions with small molecules.

Designing peptide inhibitors of the p53 -MDM2 interaction against cancer is of wide interest. Computational modeling and virtual screening are a well established step in the rational design of small molecules. But they face challenges for binding flexible peptide molecules that fold upon binding. We look at the ability of five different peptides, three of which are intrinsically disordered, to bind to MDM2 with a new Bayesian inference approach (MELD × MD). The method is able to capture the folding upon binding mechanism and differentiate binding preferences between the five peptides. Processing the ensembles with statistical mechanics tools depicts the most likely bound conformations and hints at differences in the binding mechanism. Finally, the study shows the importance of capturing two driving forces to binding in this system: the ability of peptides to adopt bound conformations (ΔGconformation) and the interaction between interface residues (ΔGinteraction).

Molecular dynamics simulations can now routinely access the microsecond timescale, making feasible direct sampling of ligand association events. While Markov State Model (MSM) approaches offer a useful framework for analyzing such trajectory data to gain insight into binding mechanisms, accurate modeling of ligand association pathways and kinetics must be done carefully. We describe methods and good practices for constructing MSMs of ligand binding from unbiased trajectory data and discuss how to use time-lagged independent component analysis (tICA) to build informative models, using as an example recent simulation work to model the binding of phenylalanine to the regulatory ACT domain dimer of phenylalanine hydroxylase. We describe a variety of methods for estimating association rates from MSMs and discuss how to distinguish between conformational selection and induced-fit mechanisms using MSMs. In addition, we review some examples of MSMs constructed to elucidate the mechanisms by which p53 transactivation domain (TAD) and related peptides bind the oncoprotein MDM2.

Why proteins are fuzzy? Constant adaptation to the cellular environment requires a wide range of changes in protein structure and interactions. Conformational ensembles of disordered proteins in particular exhibit large shifts to activate or inhibit alternative pathways. Fuzziness is critical for liquid-liquid phase separation and conversion of biomolecular condensates into fibrils. Interpretation of these phenomena presents a challenge for the classical structure-function paradigm. Here I discuss a multi-valued formalism, based on fuzzy logic, which can be applied to describe complex cellular behavior of proteins., (© 2020 The Author(s).)

Peptidomimetics have gained great attention for their function as protein-protein interaction (PPI) inhibitors. Herein, we report the design and investigation of a series of right-handed helical heterogeneous 1:1 α/Sulfono-γ-AA peptides as unprecedented inhibitors for p53-MDM2 and p53-MDMX. The most potent helical heterogeneous 1:1 α/Sulfono-γ-AA peptides were shown to bind tightly to MDM2 and MDMX, with K d of 19.3 and 66.8 nM, respectively. Circular dichroism spectra, 2D-NMR spectroscopy, and the computational simulations suggested that these helical sulfono-γ-AA peptides could mimic the critical side chains of p53 and disrupt p53/MDM2 PPI effectively. It was noted that these 1:1 α/Sulfono-γ-AA peptides were completely resistant to proteolytic degradation, boosting their potential for biomedical applications. Furthermore, effective cellular activity is achieved by the stapled 1:1 α/Sulfono-γ-AA peptides, evidenced by significantly enhanced p53 transcriptional activity and much more induced level of MDM2 and p21. The 1:1 α/Sulfono-γ-AA peptides could be an alternative strategy to antagonize a myriad of PPIs.

Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein-protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the "open" configuration, but up to eight times larger for the corresponding "closed" isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the "closed" isomers is mostly enthalpy-driven, whereas the "open" photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.

p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cell-cycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction., Competing Interests: Declaration of Interests L.D.W. is a scientific co-founder and shareholder in Aileron Therapeutics. The compositions of stapled peptides used in the study have been patented by the Dana-Farber Cancer Institute., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

About 50% of human cancers across the globe arise due to a mutation in the p53 gene which gives rise to its functional inactive form, and in the rest of the cancer the efficacy of active p53 (wild-type) is hindered by MDM2-mediated degradation. Breakdown of the p53-MDM2 association may constitute an effective strategy to stimulate or reinstate the activity of wild type p53, thereby reviving the p53 tumor suppressor capability. S100A1 has been revealed to associate with the N-terminal domain of MDM2 and p53 protein. We utilized NMR spectroscopy to study the interface amongst the S100A1 and N-terminal domain of MDM2. Additionally, the S100A1-MDM2 complex generated through the HADDOCK program was then superimposed with the p53 (peptide) -MDM2 complex reported earlier. The overlay indicated that a segment of S100A1 could block the interaction of p53 (peptide) -MDM2 complex significantly. To further justify our assumption, we performed HSQC-NMR titration for the S100A1 and p53 N-terminal domain (p53-TAD). The data obtained indicated that the S100A1 segment comprising nearly 17 residues have some common residues that interact with both MDM2 and p53-TAD. Further, we synthesized the 17-residue peptide derived from the S100A1 protein and attached it to the cell-penetrating HIV-TAT peptide. The HSQC-NMR competitive binding experiment revealed that Peptide 1 could successfully interfere with the p53-MDM2 interaction. Furthermore, functional effects of the peptide was validated in cancer cells. The results showed that Peptide 1 effectively inhibited cell proliferation, and increased the protein levels of p53 and its downstream p21 in MCF-7 cells. Treatment of Peptide 1 resulted in cell cycle arrest at G2/M phase, and also induced apoptotic cell death at higher concentration. Taken together, the results suggest that disruption of the interaction of p53 and MDM2 by Peptide 1 could activate normal p53 functions, leading to cell cycle arrest and apoptotic cell death in cancer cells. We proposed here that S100A1 could influence the p53-MDM2 interaction credibly and possibly reactivates the wild type p53 pathway., Competing Interests: The authors have declared that no competing interests exist.

Inactivation of the tumor suppressor p53 resulting from the binding with a negative regulator HDM2 is among the predominant defects in human cancers. p53-mimicking peptides whose conformational and proteolytic stability is enhanced by an all-hydrocarbon staple are being recognized as promising anticancer agents for disrupting the p53-HDM2 binding and reactivating p53. Herein, we conduct a computational modeling and thermodynamic characterization of stapled p53/HDM2 complex via molecular docking, simulations, and binding free energy analysis. The binding thermodynamics analysis is done based on the end-point calculation of the effective binding energy-a sum of the direct peptide-protein interaction energy and the dehydration penalty-and on its decomposition into contributions from specific groups constituting the complex. This allows us to investigate how individual amino acids in the stapled p53 and HDM2 contribute to the binding affinity. We find that not only the epitope residues (F19, W23 and L26), but also the hydrocarbon linker of the stapled p53 impart significant contributions. Our computational approach will be useful in designing new stapled peptides in which the staple location is also optimized to improve the binding affinity., Competing Interests: The authors have declared that no competing interests exist.

Here, we investigate the association and dissociation mechanisms of a typical intrinsically disordered region (IDR), transcriptional activation subdomain of tumor suppressor protein p53 (TAD-p53), with murine double-minute clone 2 protein (MDM2). Using a combination of cycles of association and dissociation parallel cascade molecular dynamics, multiple standard molecular dynamics (MD), and the Markov state model, we were successful in obtaining the lowest free energy structure of the MDM2/TAD-p53 complex as the structure closest to the crystal structure without prior knowledge of the crystal structure. This method also reproduced the experimentally measured standard binding free energy, and the association and dissociation rate constants, requiring only an accumulated MD simulation cost of 11.675 μs even though that actual dissociation occurs on the order of seconds. We identified few complex intermediates with similar free energies; yet TAD-p53 first binds MDM2 as the second lowest free energy intermediate kinetically with >90% of the flux, adopting a conformation similar to that of one of these few intermediates in its monomeric state. Even though the mechanism of the first step has a conformational-selection-type aspect, the second step shows induced-fit-like features and occurs as concomitant dehydration of the interface, side-chain π-π stacking, and main-chain hydrogen-bond formation to complete binding as an α-helix. In addition, dehydration is a key process for the final relaxation process around the complex interface. These results demonstrate that TAD-p53 kinetically selects its initial binding form and then relaxes to complete the binding.

Stapled peptides are chemical entities in-between biologics and small molecules, which have proven to be the solution to high affinity protein-protein interaction antagonism, while keeping control over pharmacological performance such as stability and membrane penetration. We demonstrate that the multicomponent reaction-based stapling is an effective strategy for the development of α-helical peptides with highly potent dual antagonistic action of MDM2 and MDMX binding p53. Such a potent inhibitory activity of p53-MDM2/X interactions was assessed by fluorescence polarization, microscale thermophoresis, and 2D NMR, while several cocrystal structures with MDM2 were obtained. This MCR stapling protocol proved efficient and versatile in terms of diversity generation at the staple, as evidenced by the incorporation of both exo- and endo-cyclic hydrophobic moieties at the side chain cross-linkers. The interaction of the Ugi-staple fragments with the target protein was demonstrated by crystallography., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of peptidomimetic scaffolds is a promising strategy for the inhibition of protein-protein interactions (PPIs). Herein, we demonstrate that sulfono-γ-AApeptides can be rationally designed to mimic the p53 α-helix and inhibit p53-MDM2 PPIs. The best inhibitor, with K d and IC 50 values of 26 nM and 0.891 μM toward MDM2, respectively, is among the most potent unnatural peptidomimetic inhibitors disrupting the p53-MDM2/MDMX interaction. Using fluorescence polarization assays, circular dichroism, nuclear magnetic resonance spectroscopy, and computational simulations, we demonstrate that sulfono-γ-AApeptides adopt helical structures resembling p53 and competitively inhibit the p53-MDM2 interaction by binding to the hydrophobic cleft of MDM2. Intriguingly, the stapled sulfono-γ-AApeptides showed promising cellular activity by enhancing p53 transcriptional activity and inducing expression of MDM2 and p21. Moreover, sulfono-γ-AApeptides exhibited remarkable resistance to proteolysis, augmenting their biological potential. Our results suggest that sulfono-γ-AApeptides are a new class of unnatural helical foldamers that disrupt PPIs.

Based on structural optimization work, probes 9 - 11 with practical activity and selectivity in tissue as well as living cell lines are well designed and synthesized. All the probes showed potent inhibitory and acceptable cell toxicity compared with the commercially available p53-MDM2 inhibitor Nutlin-3, and can increase the protein expression level of p53 and MDM2 in the A549 cell line; in particular, probes 10 and 11 can increase the protein expression level of p53 better than Nutlin-3. Moreover, their application in imaging and detecting wild-type p53-MDM2 protein-protein interactions have been well demonstrated in at the cell and tissue levels. Overall, these environmentally sensitive fluorescent turn-on probes are affordable and rapid for imaging, which is expected for applications in target drug screening as well as in pathologic diagnosis.

Targeting of MDM2-p53 protein-protein interaction is a current approach for the development of potent anticancer agents. The classical pharmacophore hypothesis for the design of such molecules describes the three point binding of a small molecule inhibitor to the MDM2 protein. However, this hypothesis is not confirmed when considering the activity of a number of known potent MDM2 inhibitors. Here we demonstrate the important role of the flexible N-terminal region of the MDM2 protein in the binding with small molecule compounds, which contributes to the transition from three point binding to four point binding during the development of new anticancer agents. To evaluate the contribution of the MDM2 N-terminal region to the structure-activity relationship of known MDM2 inhibitors, compounds of nutlin series, whose spatial configuration was shown to dramatically affect the target activity, were used as objects of the study. The key amino acid residues within the N-terminal region involved in the interaction with small molecule ligands were determined by means of molecular dynamics. The conformational stability of the flexible MDM2 fragment was simulated under different conditions. The effects of point mutations on the N-terminal region stability were also demonstrated.

The p53 protein, also called guardian of the genome, plays a critical role in the cell cycle regulation and apoptosis. This protein is frequently inactivated in several types of human cancer by abnormally high levels of its negative regulator, mouse double minute 2 (MDM2). As a result, restoration of p53 function by inhibiting p53-MDM2 protein-protein interaction has been pursued as a compelling strategy for cancer therapy. To date, a limited number of small-molecules have been reported as effective p53-MDM2 inhibitors. X-ray structures of MDM2 in complex with some ligands are available in Protein Data Bank and herein, these data have been exploited to efficiently identify new p53-MDM2 interaction antagonists through a hierarchical virtual screening strategy. For this purpose, the first step was aimed at compiling a focused library of 686,630 structurally suitable compounds, from PubChem database, similar to two known effective inhibitors, Nutlin-3a and DP222669. These compounds were subjected to the subsequent structure-based approaches (quantum polarized ligand docking and molecular dynamics simulation) to select potential compounds with highest binding affinity for MDM2 protein. Additionally, ligand binding energy, ADMET properties and PAINS analysis were also considered as filtering criteria for selecting the most promising drug-like molecules. On the basis of these analyses, three top-ranked hit molecules, CID&#95;118439641, CID&#95;60452010 and CID&#95;3106907, were found to have acceptable pharmacokinetics properties along with superior in silico inhibitory ability towards the p53-MDM2 interaction compared to known inhibitors. Molecular docking and molecular dynamics results well confirmed the interactions of the final selected compounds with critical residues within p53 binding site on the MDM2 hydrophobic clefts with satisfactory thermodynamics stability. Consequently, the new final scaffolds identified by the presented computational approach could offer a set of guidelines for designing promising anti-cancer agents targeting p53-MDM2 interaction., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Proteins that limit the activity of the tumour suppressor protein p53 are increasingly being targeted for inhibition in a variety of cancers. In addition to the development of small molecules, there has been interest in developing constrained (stapled) peptide inhibitors. A stapled peptide ALRN&#95;6924 that activates p53 by preventing its interaction with its negative regulator Mdm2 has entered clinical trials. This stapled peptide mimics the interaction of p53 with Mdm2. The chances that this peptide could bind to other proteins that may also interact with the Mdm2-binding region of p53 are high; one such protein is the CREB binding protein (CBP)/p300. It has been established that phosphorylated p53 is released from Mdm2 and binds to p300, orchestrating the transcriptional program. We investigate whether molecules such as ALRN&#95;6924 would bind to p300 and, to do so, we used molecular simulations to explore the binding of ATSP&#95;7041, which is an analogue of ALRN&#95;6924. Our study shows that ATSP&#95;7041 preferentially binds to Mdm2 over p300; however, upon phosphorylation, it appears to have a higher affinity for p300. This could result in attenuation of the amount of free p300 available for interacting with p53, and hence reduce its transcriptional efficacy. Our study highlights the importance of assessing off-target effects of peptide inhibitors, particularly guided by the understanding of the networks of protein-protein interactions (PPIs) that are being targeted.

We present the first application of hydrophobic tagging to a non-covalent inhibitor of protein-protein interactions. Nutlin-3a-HT, created by fusing the hydrophobic tag HyT13 to the MDM2-p53 interaction inhibitor Nutlin-3a, prevented cellular accumulation of MDM2 upon p53 reactivation, and had a stronger effect on cell viability and the induction of apoptosis than Nutlin-3a.

Intrinsically disordered proteins are an emerging class of proteins without a folded structure and currently disorder-based drug targeting remains a challenge. p53 is the principal regulator of cell division and growth whereas MDM2 consists its main negative regulator. The MDM2-p53 recognition is a dynamic and multistage process that amongst other, employs the dissociation of a transient α-helical N-terminal ''lid'' segment of MDM2 from the proximity of the p53-complementary interface. Several small molecule inhibitors have been reported to inhibit the formation of the p53-MDM2 complex with the vast majority mimicking the p53 residues Phe19, Trp23 and Leu26. Recently, we have described the transit from the 3-point to 4-point pharmacophore model stabilizing this intrinsically disordered N-terminus by increasing the binding affinity by a factor of 3. Therefore, we performed a thorough SAR analysis, including chiral separation of key compound which was evaluated by FP and 2D NMR. Finally, p53-specific anti-cancer activity towards p53-wild-type cancer cells was observed for several representative compounds., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Phosphorylation serves as an important post-translational modification implicated in cellular signaling and regulation. In this work, real-time monitoring of site-specific phosphorylation of p53 protein by several protein kinases, followed by its interaction with MDM2 protein was conducted using surface plasmon resonance (SPR). The binding of phosphorylated p53 to MDM2 yields a smaller SPR signal in comparison with that in the case of unphosphorylated p53 protein. Three specific protein kinases were involved in the in situ phosphorylation of the surface-confined p53 protein, and the binding kinetics between the phosphorylated p53 and MDM2 protein was monitored. The results indicate that phosphorylation of Ser15 and Ser37 at the p53 transactivation domain 1 (TAD1) by DNA-dependent protein kinase (DNA-PK) is critical for inhibiting the p53-MDM2 interaction, and the weaker binding affinity is most likely caused by the hydrophobicity change in the vicinity of the MDM2-binding motif or phosphorylation-induced p53 conformational change. In contrast, phosphorylation of Ser46 at the p53 TAD2 domain by c-Jun NH 2 -terminal kinase 2α2 (JNK2α2) exerts a weaker influence on the binding affinity, whereas phosphorylation of Ser376 and Ser378 at the C-terminus of p53 by protein kinase C (PKC) appears to have little effect. The feasibility of the method for the screening of the DNA-PK inhibitor and the inhibitor of p53-MDM2 interaction has been demonstrated and the half-maximal inhibitory concentration (IC 50 ) values of wortmannin and Nutlin-3 (21 nM and 83 nM, respectively) were highly comparable with those obtained by other methods. The proposed method holds great promise for monitoring protein phosphorylation and unraveling the post-translational modification mechanism.

Cellular senescence plays both beneficial and detrimental roles in embryonic development and tissue regeneration, while the underlying mechanism remains elusive. Recent studies disclosed the emerging roles of heat-shock proteins in regulating muscle regeneration and homeostasis. Here, we found that Hsp90β, but not Hsp90α isoform, was significantly upregulated during muscle regeneration. RNA-seq analysis disclosed a transcriptional elevation of p21 in Hsp90β-depleted myoblasts, which is due to the upregulation of p53. Moreover, knockdown of Hsp90β in myoblasts resulted in p53-dependent cellular senescence. In contrast to the notion that Hsp90 interacts with and protects mutant p53 in cancer, Hsp90β preferentially bound to wild-type p53 and modulated its degradation via a proteasome-dependent manner. Moreover, Hsp90β interacted with MDM2, the chief E3 ligase of p53, to regulate the stability of p53. In line with these in vitro studies, the expression level of p53-p21 axis was negatively correlated with Hsp90β in aged mice muscle. Consistently, administration of 17-AAG, a Hsp90 inhibitor under clinical trial, impaired muscle regeneration by enhancing injury-induced senescence in vivo. Taken together, our finding revealed a previously unappreciated role of Hsp90β in regulating p53 stability to suppress senescence both in vitro and in vivo., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

An oncoprotein MDM2 binds to the extreme N-terminal peptide region of a tumor suppressor protein p53 (p53NTD) and inhibits its anticancer activity. We recently discovered a peptide named MIP which exhibits much higher binding affinity for MDM2 than p53NTD. Experiments showed that the binding free energy (BFE) of MDM2-MIP is lower than that of MDM2-p53NTD by approximately -4 kcal/mol. Here, we develop a theoretical method which is successful in reproducing this quantitative difference and elucidating its physical origins. It enables us to decompose the BFE into a variety of energetic and entropic components, evaluate their relative magnitudes, and identify the physical factors driving or opposing the binding. It should be applicable also to the assessment of differences among ligands in the binding affinity for a particular receptor, which is a central issue in modern chemistry. In the MDM2 case, the higher affinity of MIP is ascribed to a larger gain of translational, configurational entropy of water upon binding. This result is useful to the design of a peptide possessing even higher affinity for MDM2 as a reliable drug against a cancer.

Imidazoline-based small molecule inhibitors of p53-MDM2 interaction intended for the treatment of p53 wild-type tumors are the promising structures for design of anticancer drugs. Based on fragment approach we have investigated a key role of substituents in cis-imidazoline core for biological activity of nutlin family compounds. Although the necessity of the substituents in the phenyl rings of cis-imidazoline has been shown, there are no studies in which the replacements of a halogen by other substituents have been investigated. A series of simple cis-imidazoline derivatives containing halogen, hydroxy and alkoxy-substituents were synthesized. The biological activity of the compounds was studied using assays of cytotoxicity (MTT) and p53 level. It was found that the hydroxyl-derivatives were not cytotoxic whereas the alkoxy analogues were the same or more active as halogen-substituted compounds in cell viability test. The synthesized alkoxy derivatives induced an increase of p53 level and did not promote necrotic cell death in the concentration up to 40 µM., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

MDM2 can mediate the degradation of tumor suppressor p53 through an autoregulatory feedback loop, in which MDM2 abolishes wild-type p53 function and accelerates malignant transformation. However, the incorporation of MDM2 antagonist Nutlin-3 could reactivate the transcriptional activity of p53, up-regulate caspase-3, and induce apoptosis. In this work, the simultaneous and label-free monitoring of p53-MDM2 complex and caspase-3 levels in cancer cells before and after Nutlin-3 treatment was conducted using dual-channel surface plasmon resonance (SPR). The p53-MDM2 complex was captured in one fluidic channel covered with consensus double-stranded (ds)-DNA, while the other channel was pre-immobilized with caspase-3-specific biotinylated DEVD-containing peptides. To amplify the SPR signals, the attachment of streptavidin (SA)-conjugated anti-MDM2 antibody in both channels was achieved. The signal diversity before and after Nutlin-3 treatment is indicative of the difference in the levels of the intracellular p53-MDM2 complex and caspase-3. The limit of detection for p53-MDM2 and caspase-3 down to 4.54 pM and 0.03 ng mL -1 , respectively, was attained. Upon treatment with Nutlin-3, MCF-7 cancer cells with wild-type p53 showed decreased expression of the p53-MDM2 complex and an increased caspase-3 level, while MDA-MB-231 cancer cells with mutant p53 exhibited an elevated caspase-3 level and unchanged p53-MDM2 complex expression. The apoptosis of MCF-7 and MDA-MB-231 cancer cells upon Nutlin-3 treatment follows a p53-dependent and a p53-independent pathway, respectively. The proposed method is sensitive, selective and label-free, holding great promise for assaying intracellular p53-MDM2 complex and caspase-3 levels and differentiating Nutlin-3-mediated p53-dependent or p53-independent apoptotic pathways.

MDM2 is a well-known oncoprotein overexpressed in a variety of cancers, and the identification of inhibitors that disrupt the MDM2/p53 interaction is of great interest in anticancer drug development. Here we designed a platform for the facile and visualizable identification of inhibitors of MDM2 using co-expressed protein complexes of MDM2/p53. A hexahistidine-tag on MDM2 allows the binding of the protein complex to the Ni-NTA affinity resin, while the fluorescent protein fused to p53 enables the direct visualization of the interaction of p53 with MDM2. Hence, the inhibition of the MDM2/p53 interaction can be observed with the naked eye. The assay can be set up by directly loading cell lysate to the Ni-NTA affinity resin, and no chemical modification of proteins is needed. In addition to the qualitative analyses, the binding affinity of inhibitors to the MDM2 protein can be quantified by fluorescence titration. The applications of this system have been verified using small molecules and peptide inhibitors. As a proof of concept, we screened a small library using this platform. Interestingly, two types of novel inhibitors of MDM2, including cyclohexyl-triphenylamine derivatives and platinum complexes, were identified and their binding affinities were obtained. Quantitative measurements show that these new types of inhibitors demonstrate a high binding affinity (up to K d = 51.9 nM) to MDM2.

The recently developed MM/GBSA&#95;IE method is applied to computing hot and warm spots in p53/PMI-MDM2/MDMX protein-protein interaction systems. Comparison of the calculated hot (>2 kcal/mol) and warm spots (>1 kcal/mol) in P53 and PMI proteins interacting with MDM2 and MDMX shows a good quantitative agreement with the available experimental data. Further, our calculation predicted hot spots in MDM2 and MDMX proteins in their interactions with P53 and PMI and they help elucidate the interaction mechanism underlying this important PPI system. In agreement with the experimental result, the present calculation shows that PMI has more hot and warm spots and binds stronger to MDM2/MDMX. The analysis of these hot and warm spots helps elucidate the fundamental difference in binding between P53 and PMI to the MDM2/MDMX systems. Specifically, for p53/PMI-MDM2 systems, p53 and PMI use essentially the same residues (L54, I61, Y67, Q72, V93, H96, and I99) of MDM2 for binding. However, PMI enhanced interactions with residues L54, Y67, and Q72 of MDM2. For the p53/PMI-MDMX system, p53 and PMI use similar residues (M53, I60, Y66, Q71, V92, and Y99) of MDMX for binding. However, PMI exploited three extra residues (M61, K93, and L98) of MDMX for enhanced binding. In addition, PMI enhanced interaction with four residues (M53, Y66, Q71, and Y99) of MDMX. These results gave quantitative explanation on why the binding affinities of PMI-MDM2/MDMX interactions are stronger than that of p53-MDM2/MDMX although their binding modes are similar. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Background: The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing key roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); this domain, SAMp73, formed by five helices (α1 to α5), is thought to mediate in protein-protein interactions. The E3-ligase MDM2 binds to p73 at its N terminus transactivation domain (TA), but it does not promote its degradation via ubiquitination; however, the details of such MDM2/p73 interaction are not fully known., Methods: We studied the binding of SAMp73 with N-terminal MDM2, by several biophysical techniques, namely, fluorescence, far-UV circular dichroism (CD), NMR and bio-layer interferometry (BLI)., Results: Our results obtained by fluorescence, T 2 -relaxation measurements and BLI show that there was binding between both proteins with a dissociation constant of ~10 μM. Furthermore, the binding region of SAMp73 involved mainly residues in the major α-helix, α5, and the nearby α4, as shown by HSQC-NMR. The binding was so specific that an isolated peptide comprising α4 and α5 helices of SAMp73, α4α5, did also bind to the N terminus of MDM2, although with weaker affinity than the entire domain., Conclusions: A new interaction between MDM2 and SAMp73 has been found, which could have potential therapeutic applications in cancers involving inactivated p53., General Significance: A novel interaction between the C-terminal SAM of p73 and N-terminal MDM2 is described. The interaction could be used to modulate the functions where the wild-type, intact p73 is involved., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Recently, we efficiently generated dissociation pathways of a protein-ligand complex without applying force bias with parallel cascade selection molecular dynamics (PaCS-MD) and showed that PaCS-MD in combination with the Markov state model (MSM) yielded a binding free energy comparable to experimental values. In this work, we applied the same procedure to a complex of MDM2 protein and the transactivation domain of p53 protein (TAD-p53), the latter of which is known to be very flexible in the unbound state. Using 30 independent MD simulations in PaCS-MD, we successfully generated 25 dissociation pathways of the complex, which showed complete or partial unfolding of the helical region of TAD-p53 during the dissociation process within an average simulation time of 154.8 ± 46.4 ns. The standard binding free energy obtained in combination with one-dimensional-, three-dimensional (3D)- or C α -MSM was in good agreement with those determined experimentally. Using 3D-MSM based on the center of mass position of TAD-p53 relative to MDM2, the dissociation rate constant was calculated, which was comparable to those measured experimentally. C α -MSM based on all C α coordinates of TAD-p53 reproduced the experimentally measured standard binding free energy, and dissociation and association rate constants. We conclude that the combination of PaCS-MD and MSM offers an efficient computational procedure to calculate binding free energies and kinetic rates.

Therapeutic peptides suffer from major drawbacks such as peptide degradation in vivo due to proteolysis. Gold nanoparticles (AuNPs) are an effective carrier for therapeutic peptides that improve their stability in vivo, while also enabling nonspecific adsorption of complementary proteins to enhance their effectiveness. Using p53 peptide as a model known to disrupt the intracellular MDM2-p53 protein-protein interaction which tags the endogenous p53 proteins for degradation, we conjugated p53 peptides to AuNPs (AuNP-p53) and examined the functionality of AuNP-p53 to release the endogenous p53 proteins from being tagged for degradation, thereby increasing the level of stable p53 proteins in acute myeloid leukemia 2 (AML2) cells. We found that AuNPs did not just protect conjugated p53 peptides from trypsin degradation, but also helped to recruit 56.5% and 26.4% of total MDM2 and p53 proteins in the cells to form a protein corona around AuNP-p53. The proximity of MDM2/p53 complexes and p53 peptide on the surface of AuNP-p53 facilitated the action of p53 peptides to cause a sustained elevation of the p53 level in AML2 cells up to 6 h, which was not possible with free p53 peptide alone at the same concentration. Even a 20-fold higher concentration of free p53 peptide caused only a short-lived elevated p53 level of 1 h. The outcome of this study highlights the utility of combining conjugated ligands and complementary protein adsorption on nanoparticles to improve the biological functionality of the therapeutic ligands.

Cervical cancer is the second most common cancer in women worldwide. Human papillomavirus (HPV) infection appears to be a necessary factor in the development of almost all cases (>95%) of cervical cancer. HPV E6 induces a change of control of p53 stabilization from Hdm2 to E6/E6AP in HPV‑infected cells. It is well known that the LxxLL motif of cellular ubiquitin ligase E6AP binds to the pocket of E6 and causes a conformational change to enable E6 to bind p53 competently. In the ternary complex E6/E6AP/p53, p53 is polyubiquitinated by E6AP and subsequently degraded by a proteasome. Therefore, these cells are deficient in the processes regulated by p53, including apoptosis, damaged DNA repair, and the cell cycle. In the present study, it was demonstrated that quercetin induced G2 phase cell cycle arrest and apoptosis in both HeLa and SiHa cells, accompanied by an increase of p53 and its nuclear signal. It was also observed that quercetin increased the level of the p21 transcript and the pro‑apoptotic Bax protein, which are two p53‑downstream effectors. However, quercetin did not alter the expression of the HPV E6 protein in cervical cancer cells; therefore, the increase in p53 occurred in an E6 expression‑independent manner. Furthermore, molecular docking demonstrated that quercetin binds stably in the central pocket of E6, the binding site of E6AP. These data suggest that quercetin increases the nuclear localization of p53 by interrupting E6/E6AP complex formation in cervical cancer cells.

p53 is frequently mutated in human cancers. Its levels are tightly regulated by the E3 ubiquitin ligase MDM2. The complex between MDM2 and p53 is largely formed by the interaction between the N-terminal domain of MDM2 and the N-terminal transactivation (TA) domain of p53 (residues 15-29). We investigated the kinetic and thermodynamic basis of the MDM2/p53 interaction by using wild-type and mutant variants of the TA domain. We focus on the effects of phosphorylation at positions Thr18 and Ser20 including their substitution with phosphomimetics. Conformational propensities of the isolated peptides were investigated using in silico methods and experimentally by circular dichroism and 1 H-NMR in aqueous solution. Both experimental and computational analyses indicate that the p53 peptides are mainly disordered in aqueous solution, with evidence of nascent helix around the Ser20-Leu25 region. Both phosphorylation and the phosphomimetics at Thr18 result in a decrease in the binding affinity by ten- to twenty-fold when compared to the wild-type. Phosphorylation and phosphomimetics at Ser20 result in a smaller decrease in the affinity. Mutation of Lys24 and Leu25 also disrupts the interaction. Our results may be useful for further development of peptide-based drugs targeting the MDM2/p53 interaction.