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Protein-protein interactions (PPIs), many of which are dominated by α-helical recognition domains, play key roles in many essential cellular processes, and the dysregulation of these interactions can cause detrimental effects. For instance, aberrant PPIs involving the Bcl-2 protein family can lead to several diseases including cancer, neurodegenerative diseases, and diabetes. Interactions between Bcl-2 pro-life proteins, such as Mcl-1, and pro-death proteins, such as Bim, regulate the intrinsic pathway of apoptosis. p53, a tumor-suppressor protein, also has a pivotal role in apoptosis and is negatively regulated by its E3 ubiquitin ligase HDM2. Both Mcl-1 and HDM2 are upregulated in numerous cancers, and, interestingly, there is crosstalk between both protein pathways. Recently, synergy has been observed between Mcl-1 and HDM2 inhibitors. Towards the development of new anticancer drugs, we herein describe a polypharmacology approach for the dual inhibition of Mcl-1 and HDM2 by employing three densely functionalized isoxazoles, pyrazoles, and thiazoles as mimetics of key α-helical domains of their partner proteins., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Abundant phosphorylation events control the activity of nuclear proteins involved in gene regulation and DNA repair. These occur mostly on disordered regions of proteins, which often contain multiple phosphosites. Comprehensive and quantitative monitoring of phosphorylation reactions is theoretically achievable at a residue-specific level using 1 H- 15 N NMR spectroscopy, but is often limited by low signal-to-noise at pH>7 and T>293 K. We have developed an improved 13 Cα- 13 CO correlation NMR experiment that works equally at any pH or temperature, that is, also under conditions at which kinases are active. This allows us to obtain atomic-resolution information in physiological conditions down to 25 μm. We demonstrate the potential of this approach by monitoring phosphorylation reactions, in the presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2, and Oct4., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.)

Hdm2 (human MDM2, human double minute 2 homologue) counteracts p53 function by direct binding to p53 and by ubiquitin-dependent p53 protein degradation. Activation of p53 by inhibitors of the p53-Hdm2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. In addition, HdmX (human MDMX, human MDM4) was also identified as an important therapeutic target to efficiently reactivate p53, and it is likely that dual inhibition of Hdm2 and HdmX is beneficial. Herein we report four new X-ray structures for Hdm2 and five new X-ray structures for HdmX complexes, involving different classes of synthetic compounds (including the worldwide highest resolutions for Hdm2 and HdmX, at 1.13 and 1.20 Å, respectively). We also reveal the key additive 18-crown-ether, which we discovered to enable HdmX crystallization and show its stabilization of various Lys residues. In addition, we report the previously unpublished details of X-ray structure determinations for eight further Hdm2 complexes, including the clinical trial compounds NVP-CGM097 and NVP-HDM201. An analysis of all compound binding modes reveals new and deepened insight into the possible adaptations and structural states of Hdm2 (e.g., flip of F55, flip of Y67, reorientation of H96) and HdmX (e.g., flip of H55, dimer induction), enabling key binding interactions for different compound classes. To facilitate comparisons, we used the same numbering for Hdm2 (as in Q00987) and HdmX (as in O15151, but minus 1). Taken together, these structural insights should prove useful for the design and optimization of further selective and/or dual Hdm2/HdmX inhibitors., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Progress in drug delivery is hampered by a lack of efficient strategies to target drugs with high specificity and precise spatiotemporal regulation. The remote control of nanoparticles and drugs with light allows regulation of their action site and dosage. Peptide-based drugs are highly specific, non-immunogenic, and can be designed to cross the plasma membrane. In order to combine target specificity and remote control of drug action, here we describe a versatile strategy based on a generalized template to design nanoswitchable peptides that modulate protein-protein interactions upon light activation. This approach is demonstrated to promote photomodulation of two important targets involved in apoptosis (the interactions Bcl-xL-Bak and MDM2-p53), but can be also applied to a large pool of therapeutically relevant protein-protein interactions mediated by α-helical motifs. The template can be adjusted using readily available information about hot spots (residues contributing most to the binding energy) at the protein-protein interface of interest., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mouse double minute 2 (MDM2) is a main and direct inhibitor of the crucial tumor suppressor p53. Reports from initial clinical trials showed that blocking this interaction with a small-molecule inhibitor can have great value in the treatment of cancer for patients with p53 wild-type tumors; however, it also revealed dose-limiting hematological toxicities and drug-induced resistance as main issues. To overcome the former, an inhibitor with superior potency and pharmacokinetic properties to ultimately achieve full efficacy with less-frequent dosing schedules is required. Toward this aim, we optimized our recently reported spiro-oxindole inhibitors by focusing on the crucial interaction with the amino acid side chain of His96 MDM2 . The designed molecules required the targeted synthesis of structurally complex spiro[indole-3,2'-pyrrolo[2,3-c]pyrrole]-2,4'-diones for which we developed an unprecedented intramolecular azomethine ylide cycloaddition and investigated the results by computational methods. One of the new compounds showed superior cellular potency over previously reported BI-0252. This finding is a significant step toward an inhibitor suitable to potentially mitigate hematological on-target adverse effects., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

A growing number of approaches to "staple" α-helical peptides into a bioactive conformation using cysteine cross-linking are emerging. Here, the replacement of l-cysteine with "cysteine analogues" in combinations of different stereochemistry, side chain length and beta-carbon substitution, is explored to examine the influence that the thiol-containing residue(s) has on target protein binding affinity in a well-explored model system, p53-MDM2/MDMX, which is constituted by the interaction of the tumour suppressor protein p53 and proteins MDM2 and MDMX, which regulate p53 activity. In some cases, replacement of one or more l-cysteine residues afforded significant changes in the measured binding affinity and target selectivity of the peptide. Computationally constructed homology models indicate that some modifications, such as incorporating two d-cysteine residues, favourably alter the positions of key functional amino acid side chains, which is likely to cause changes in binding affinity, in agreement with measured surface plasmon resonance data., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The total synthesis of siladenoserinol A, an inhibitor of the p53-Hdm2 interaction, has been achieved. AuCl 3 -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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

p53 is an important tumor-suppressor protein deactivation of which by mdm2 results in cancers. A SUMO-specific protease 4 (SUSP4) was shown to rescue p53 from mdm2-mediated deactivation, but the mechanism is unknown. The discovery by NMR spectroscopy of a "p53 rescue motif" in SUSP4 that disrupts p53-mdm2 binding is presented. This 29-residue motif is pre-populated with two transient helices connected by a hydrophobic linker. The helix at the C-terminus binds to the well-known p53-binding pocket in mdm2 whereas the N-terminal helix serves as an affinity enhancer. The hydrophobic linker binds to a previously unidentified hydrophobic crevice in mdm2. Overall, SUSP4 appears to use two synergizing modules, the p53 rescue motif described here and a globular-structured SUMO-binding catalytic domain, to stabilize p53. A p53 rescue motif peptide exhibits an anti-tumor activity in cancer cell lines expressing wild-type p53. A pre-structures motif in the intrinsically disordered proteins is thus important for target recognition., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The 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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A recent therapeutic strategy in oncology is based on blocking the protein-protein interaction between the murine double minute (MDM) homologues MDM2/X and the tumor-suppressor protein p53. Inhibiting the binding between wild-type (WT) p53 and its negative regulators MDM2 and/or MDMX has become an important target in oncology to restore the antitumor activity of p53, the so-called guardian of our genome. Interestingly, based on the multiple disclosed compound classes and structural analysis of small-molecule-MDM2 adducts, the p53-MDM2 complex is perhaps the best studied and most targeted protein-protein interaction. Several classes of small molecules have been identified as potent, selective, and efficient inhibitors of the p53-MDM2/X interaction, and many co-crystal structures with the protein are available. Herein we review the properties as well as preclinical and clinical studies of these small molecules and peptides, categorized by scaffold type. A particular emphasis is made on crystallographic structures and the observed binding modes of these compounds, including conserved water molecules present., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Inhibitors of the p53-MDM2 protein-protein interaction are emerging as a new and validated approach to treating cancer. Herein, we describe the synthesis and inhibitory evaluation of a series of isoquinolin-1-one analogues, and highlight the utility of an initial growth-rates saturation-transfer difference (STD) NMR approach supported by protein-ligand docking to investigate p53-MDM2 inhibition. The approach is illustrated by the study of compound 1, providing key insights into the binding mode of this kind of MDM2 ligands and, more importantly, readily unveiling the previously proposed three-finger pharmacophore requirement for p53-MDM2 inhibition., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new analytical method to study the dissociation of the complexes between the oncosuppressor p53 and its negative modulators murine double-minute protein 2 (MDM2) or MDMX, is proposed. This technique is reliable to determine the dissociative power exerted by small molecules on the complex taking advantage of the appearance of migrating MDM2 or MDMX in a native polyacrylamide gel, when inhibitors are added to the complex mixture. Therefore, we propose this new approach to easily screen library of compounds, with potential pharmacological anticancer activity., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In an approach to design drugs with higher affinity for π-π stacking and electrostatic interactions with targeted biomolecules, complexes of the type [{cis-Pt(A)2 (L)}2 -μ-{trans-1,4-dach}](NO3 )4 ((A)2 =(NH3 )2 or ethylenediamine (en), L=quinoline (quin) or benzothiazole (bztz), dach=trans-1,4-diaminocyclohexane) were synthesized. The quinoline complex, [{cis-Pt(en)(quin)}2 -μ-(dach)](NO3 )4 (9) was synthesized from the precursor K[PtCl3 (quin)] (1), while the benzothiazole complexes, [{cis-Pt(A)2 (bztz)}2 -μ-(dach)](NO3 )4 ((A)2 =(NH3 )2 (10) and (A)2 =en (11)) were synthesized from the precursors cis-[Pt(A)2 Cl(bztz)] ((A)2 =(NH3 )2 (7) and (A)2 =en (8)). Their interactions with N-acetyltryptophan and a model pentapeptide (N-Ac-WLDSW-OH), modeled on the pentapeptide recognition sequence (FSDLW) of p53-mdm2 interaction, were examined by fluorescence spectroscopy. The dinuclear complexes were found to be significantly stronger at quenching the fluorescence of tryptophan than their mononuclear Pt-based analogues indicating stronger binding. Molecular modeling suggests a "sandwich" mode of binding, and the flexibility of the dinuclear motif can allow the design of more selective and stronger-binding complexes. Based on these results a further prototype, [{Pt(en)(9-EtGua)}2 μ-H2 N(CH2 )6 NH2 ](4+) , incorporating the purine 9-ethylguanine (9-EtG) as a stacking moiety, was prepared which showed good cytotoxicity in A2780 and OsACL tumor cell lines., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

MDM2 is a multidomain protein that functions as an E3 ubiquitin ligase, transcription repressor, mRNA-binding protein, translation factor, and molecular chaperone. The small molecule Nutlin-3 has been engineered to bind to the N-terminal hydrophobic pocket domain of MDM2. This binding of Nutlin-3 has two consequences: (i) antagonistic effects through competitive disruption of the MDM2-p53 complex and (ii) agonist effects that allosterically stabilize MDM2 protein-protein interactions that increase p53 ubiquitination as well as nucleophosmin deoligomerization. We present a methodology using a hydrogen/deuterium (H/D) exchange platform that measures Nutlin-3 binding to the N-terminal domain of MDM2 (MDM2(1-126)) in order to begin to develop dynamic assays that evaluate MDM2 allostery. In order to localize the regions in MDM2 being suppressed by Nutlin-3, MDM2 was incubated with the ligand and H/D amide exchange was measured after pepsin digestion. One dynamic segment containing amino acids 55-60 exhibited slower deuterium exchange after Nutlin-3 binding, reflecting ligand binding within the hydrophobic pocket. However, another dominant suppression of H/D exchange was observed in a motif from amino acids 103-107 that reflects surface hydrophobic residues surrounding the hydrophobic pocket of MDM2. In order to explore the consequences of this latter Nutlin-3 interaction site on MDM2, the Y104G and L107G mutant series was constructed. The MDM2(Y104G) and MDM2(L107G) mutants were fully active in p53 binding. However, the authentic p53-derived peptide:MDM2(Y104G) complex exhibited partial resistance to Nutlin-3 inhibition, while the p53-mimetic 12.1 peptide:MDM2(Y104G) complex retained normal Nutlin-3 responsiveness. These data reveal the existence of a second functional Nutlin-3-binding site in a surface hydrophobic patch of MDM2, flanking the hydrophobic pocket. This reveals two modes of peptide binding by MDM2 and highlights the utility of H/D exchange as an assay for measuring allosteric effects in MDM2., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

TARGETING PPIS: A novel strategy for designing libraries targeting protein-protein interfaces enabled us to identify diverse chemical entry points to interact with therapeutic targets for which conventional screening libraries delivered no or only few hit structures. The concept was experimentally validated by early hit evaluation in biochemical screens and early ADMET profiling., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)