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Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Bicyclic β‐sheet mimetics that target the transcriptional coactivator β‑catenin and inhibit Wnt signaling Mathias Wendt , Rosa Bellavita , Alan Gerber , Nina-Louisa Efrém , Thirza Van Ramshorst , Nicholas M. Pearce , Paul R. J. Davey , Isabel Everard , Mercedes Vazquez-Chantada , Elisabetta Chiarpari , Paolo Grieco , Sven Hennig , Tom N. Grossmann Angewandte Chemie International Edition DOI: 10.1002/anie.202102082 Diamond Proposal Number(s): [19800] Open Access Show Abstract ▼ Abstract: Protein complexes are defined by the three‐dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied e.g. as inhibitors of protein‐protein interactions (PPIs). Even though β‐sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure‐based design of β‐sheet mimetics targeting the intracellular protein β‐catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of β‐catenin, a macrocyclic peptide was designed and its crystal structure in complex with β‐catenin obtained. Using this structure, we designed a library of bicyclic β‐sheet mimetics employing a late‐stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to β‐catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other β‐sheet‐mediated PPIs.	Mar 2021
I04-Macromolecular Crystallography	MMP activation associated Aminopeptidase N reveals a bivalent 14-3-3 binding motif Sebastian Kiehstaller , Christian Ottmann , Sven Hennig Journal Of Biological Chemistry DOI: 10.1074/jbc.RA120.014708 Diamond Proposal Number(s): [19800] Show Abstract ▼ Abstract: Aminopeptidase N (APN, CD13) is a trans-membrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction, and is involved in the activation of Matrix Metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases such as fibrosis, rheumatoid arthritis, tumor angiogenesis and metastasis as well as viral infections. However, the exact mechanism that leads to APN driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins binding APN induces the transcription of MMPs. As a first step, we sought to identify potential 14‑3‑3 binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3 binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding Fluorescence Polarization (FP) assays and thermodynamic analysis (ITC). In addition, we identified a secondary, non-canonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14‑3‑3σ bound to mono- and bis‑phosphorylated APN derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and opens the field for further investigation of this important signaling pathway.	Oct 2020
I04-Macromolecular Crystallography	Adapting free energy perturbation simulations for large macrocyclic ligands: how to dissect contributions from direct binding and free ligand flexibility Kerstin Wallraven , Fredrik L. Holmelin , Adrian Glas , Sven Hennig , Andrey I. Frolov , Tom N. Grossmann Chemical Science 11, 2269 - 2276 DOI: 10.1039/C9SC04705K Diamond Proposal Number(s): [19800] Open Access Show Abstract ▼ Abstract: Large and flexible ligands gain increasing interest in the development of bioactive agents. They challenge the applicability of computational ligand optimization strategies originally developed for small molecules. Free energy perturbation (FEP) is often used for predicting binding affinities of small molecule ligands, however, its use for more complex ligands remains limited. Herein, we report the structure-based design of peptide macrocycles targeting the protein binding site of human adaptor protein 14-3-3. We observe a surprisingly strong dependency of binding affinities on relatively small variations in substituent size. FEP was performed to rationalize observed trends. To account for insufficient convergence of FEP, restrained calculations were performed and complemented with extensive REST MD simulations of the free ligands. These calculations revealed that changes in affinity originate both from altered direct interactions and conformational changes of the free ligand. In addition, MD simulations provided the basis to rationalize unexpected trends in ligand lipophilicity. We also verified the anticipated interaction site and binding mode for one of the high affinity ligands by X-ray crystallography. The introduced fully-atomistic simulation protocol can be used to rationalize the development of structurally complex ligands which will support future ligand maturation efforts.	Feb 2020

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