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The pharmacodynamic and pharmacokinetic properties of bioactive peptides can be modulated by introducing conformational constraints such as intramolecular macrocyclizations, which can involve either the backbone and/or side chains. Herein, we aimed at increasing the α-helicity content of temporin L, an isoform of an intriguing class of linear antimicrobial peptides (AMPs), endowed with a wide antimicrobial spectrum, by the employment of diverse side-chain tethering strategies, including lactam, 1,4-substituted [1,2,3]-triazole, hydrocarbon, and disulfide linkers. Our approach resulted in a library of cyclic temporin L analogues that were biologically assessed for their antimicrobial, cytotoxic, and antibiofilm activities, leading to the development of the first-in-class cyclic peptide related to this AMP family. Our results allowed us to expand the knowledge regarding the relationship between the α-helical character of temporin derivatives and their biological activity, paving the way for the development of improved antibiotic cyclic AMP analogues.

Biomolecular assemblies composed of proteins and oligonucleotides play a central role in biological processes. While in nature, oligonucleotides and proteins usually assemble via non‐covalent interactions, synthetic conjugates have been developed which covalently link both modalities. The resulting peptide‐oligonucleotide conjugates have facilitated novel biological applications as well as the design of functional supramolecular systems and materials. However, despite the importance of concerted protein/oligonucleotide recognition in nature, conjugation approaches have barely utilized the synergistic recognition abilities of such complexes. Herein, the structure‐based design of peptide‐DNA conjugates that bind RNA through Watson‐Crick base pairing combined with peptide‐mediated major groove recognition is reported. Two distinct conjugate families with tunable binding characteristics have been designed to adjacently bind a particular RNA sequence. In the resulting ternary complex, their peptide elements are located in proximity, a feature that was used to enable an RNA‐templated click reaction. The introduced structure‐based design approach opens the door to novel functional biomolecular assemblies., DNA, Protein, RNA: The structure‐based design of biomolecular conjugates composed of protein and DNA fragments that synergistically bind RNA via Watson‐Crick base pairing and peptide‐mediated major groove recognition is reported. Two distinct conjugate families with tunable binding characteristics were designed whereby two conjugates can simultaneously assemble on a single RNA scaffold. Through ternary complex formation, the peptide elements of each conjugate are drawn into close proximity, and this feature was exploited to enable an RNA‐templated, double click reaction.

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 for example, 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., Starting from a 52 amino acid protein binding epitope, a bicyclic β‐hairpin structure was developed to bind the transcriptional coactivator β‐catenin. Our structure‐based design approach was supported by screening a focused library of bicyclic mimetics which was generated via late‐stage diversification. The most active bicyclic β‐hairpin shows cell‐penetration and inhibits Wnt signaling in a cell‐based assay.

Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein–protein interactions (PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators., Targeting the interaction between transcription factor TEAD and its co-repressor VGL4 is an attractive strategy to chemically modulate Hippo signaling. Here, the authors develop a proteomimetic with stabilized tertiary structure that inhibits the TEAD:VGL4 interaction in vitro and in cells.

Tuberculosis (TB) is a globally significant infective disease that is caused by a single infectious agent, Mycobacterium tuberculosis (Mtb). Because of the rise in the number of multidrug-resistant (MDR) TB strains, identification of alternative drug targets for the development of drugs with different mechanism of actions is desired. CYP121A1, one of the twenty cytochrome P450 enzymes encoded in the Mtb genome, was previously shown to be essential for bacterial growth. This enzyme catalyzes the intramolecular C–C crosslinking reaction of the cyclopeptide cyclo(L-tyr-L-tyr) (cYY) yielding the metabolite mycocyclosin. In the present study, acetylene-substituted cYY-analogs were synthesized and evaluated as potential mechanism-based inhibitors of CYP121A1. The acetylene-substituted cYY-analogs were capable of binding to CYP121A1 with affinities comparable with cYY, and exhibited a Type I binding mode, indicative of a substrate-like binding, mandatory for metabolism. Only the cYY-analogs which contain an acetylene-substitution at one (2a) or both (3) para-positions of cYY showed mechanism-based inhibition of CYP121A1 activity. The values of KI and kinact were 236 µM and 0.045 min−1, respectively, for compound 2a, and 145 µM and 0.015 min−1, repectively, for compound 3 The inactivation could neither be reversed by dialysis nor be prevented by including glutathione. LC-MS analysis demonstrated that the inactivation results from covalent binding to the apoprotein, whereas the heme was unmodified. Interestingly, the mass increment of the CYP121A1 apoprotein was significantly smaller than was expected from the ketene formed by oxidation of the acetylene-group, indicative for a secondary cleavage reaction in the active site of CYP121A1. Although the two acetylene-containing cYY-analogs showed significant mechanism-based inhibition, growth inhibition of the Mtb strains was only observed at millimolar concentrations. This low efficacy may be due to insufficient irreversible inactivation of CYP121A1 and/or insufficient cellular uptake. Although the identified mechanism-based inhibitors have no perspective for Mtb-treatment, this study is the first proof-of-principle that mechanism-based inhibition of CYP121A1 is feasible and may provide the basis for new strategies in the design and development of compounds against this promising therapeutic target.

Proteins have evolved as a variable platform that provides access to molecules with diverse shapes, sizes, and functions. These features have inspired chemists for decades to seek artificial mimetics of proteins with improved or novel properties. Such work has focused primarily on small protein fragments, often isolated secondary structures; however, there has lately been a growing interest in the design of artificial molecules that mimic larger, more complex tertiary folds. In this Perspective, we define these agents as “proteomimetics” and discuss the recent advances in the field. Proteomimetics can be divided into three categories: (1) protein domains with side chain functionality that alters the native linear chain topology, (2) protein domains in which the chemical composition of the polypeptide backbone has been partially altered, and (3) protein-like folded architectures that are composed entirely of non-natural monomer units. We give an overview of these proteomimetic approaches and outline remaining challenges facing the field.

A combination of free energy perturbations and molecular dynamics simulations were applied to investigate large macrocyclic ligands and their receptor binding., 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.

"Hot loop" protein segments have variable structure and conformation and contribute crucially to protein-protein interactions. We describe a new hot loop mimicking modality, termed PepNats, in which natural product (NP)-inspired structures are incorporated as conformation-determining and -restricting structural elements into macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were synthesized on solid support employing macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors. NP-inspired fragment absolute configuration determines the conformation of the peptide part responsible for binding. These results demonstrate that combination of NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop mimics with conformationally constrained and biologically relevant structure.

Protein complex formation depends on the interplay between preorganization and flexibility of the binding epitopes involved. The design of epitope mimetics typically focuses on stabilizing a particular bioactive conformation, often without considering conformational dynamics, which limits the potential of peptidomimetics against challenging targets such as transcription factors. We developed a peptide‐derived inhibitor of the NF‐Y transcription factor by first constraining the conformation of an epitope through hydrocarbon stapling and then fine‐tuning its flexibility. In the initial set of constrained peptides, a single non‐interacting α‐methyl group was observed to have a detrimental effect on complex stability. Biophysical characterization revealed how this methyl group affects the conformation of the peptide in its bound state. Adaption of the methylation pattern resulted in a peptide that inhibits transcription factor assembly and subsequent recruitment to the target DNA., Set Me free: A peptide‐derived inhibitor of the NF‐Y transcription factor was developed by constraining the conformation of an epitope through hydrocarbon stapling and then fine‐tuning its flexibility. In the initial set of constrained peptides, a non‐interacting α‐methyl group was observed to have a detrimental effect on complex stability. Adaption of the methylation pattern gave a peptide that inhibits transcription factor assembly and subsequent DNA binding.

The structure-based design of constrained alpha-helical peptides derived from the viral suppressor of RNA silencing TAV2b is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased alpha-helicity and protease stability. One of these modified peptides (B3) shows high affinity for different double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry.

The understanding of protein folding and assembly is of central importance for the design of proteins and enzymes with novel or improved functions. Minimalistic model systems, such as coiled‐coils, provide an excellent platform to improve this understanding and to construct novel molecular devices. Along those lines, we designed a conformational switch that is composed of two coiled‐coil forming peptides and a central binding epitope. In the absence of a binding partner, this switch adopts a hairpin‐like conformation that opens upon receptor binding. Variation of the coiled‐coil length modulates the strength of the intramolecular constraint. The two conformational states of this switch have been linked with characteristic fluorescent properties, which enables the detection of the receptor in real‐time.

Structural information about protein–RNA complexes supports the understanding of crucial recognition processes in the cell, and it can allow the development of high affinity ligands to interfere with these processes. In this respect, the identification of amino acid hotspots is particularly important. In contrast to protein–protein interactions, in silico approaches for protein–RNA interactions lag behind in their development. Herein, we report an analysis of available protein–RNA structures. We assembled a data set of 322 crystal and NMR structures and analyzed them regarding interface properties. In addition, we describe a computational alanine-scanning approach which provides interaction scores for interface amino acids, allowing the identification of potential hotspots in protein–RNA interfaces. We have made the computational approach available as an online tool, which allows interaction scores to be calculated for any structure of a protein–RNA complex by uploading atomic coordinates to the PRI HotScore web server (https://pri-hotscore.labs.vu.nl).

Increased tolerance of enzymes towards thermal and chemical stress is required for many applications and can be achieved by macrocyclization of the enzyme resulting in the stabilizing of its tertiary structure. Thus far, macrocyclization approaches utilize a very limited structural diversity, which complicates the design process. Herein, we report an approach that enables cyclization through the installation of modular crosslinks into native proteins composed entirely of proteinogenic amino acids. Our stabilization procedure involves the introduction of three surface‐exposed cysteine residues, which are reacted with a triselectrophile, resulting in the in situ cyclization of the protein (INCYPRO). A bicyclic version of sortase A was designed that exhibits increased tolerance towards thermal as well as chemical denaturation, and proved to be efficient in protein labeling under denaturing conditions. In addition, we applied INCYPRO to the KIX domain, resulting in up to 24 °C increased thermal stability.

Macrocyclic peptides can interfere with challenging biomolecular targets including protein-protein interactions. Whereas there are various approaches that facilitate the identification of peptide-derived ligands, their evolution into higher affinity binders remains a major hurdle. We report a virtual screen based on molecular docking that allows the affinity maturation of macrocyclic peptides taking non-natural amino acids into consideration. These macrocycles bear large and flexible substituents that usually complicate the use of docking approaches. A virtual library containing more than 1400 structures was screened against the target focusing on docking poses with the core structure resembling a known bioactive conformation. Based on this screen, a macrocyclic peptide 22 involving two non-natural amino acids was evolved showing increased target affinity and biological activity. Predicted binding modes were verified by X-ray crystallography. The presented workflow allows the screening of large macrocyclic peptides with diverse modifications thereby expanding the accessible chemical space and reducing synthetic efforts. OA hybrid

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture., The N-end rule pathway targets substrate proteins for proteasomal degradation. Here, White et al. show that Arabidopsis PLANT CYSTEINE OXIDASEs show dioxygenase activity producing Cys-sulfinic acid at the N-terminus of target proteins, which then act as direct substrates for arginyl transferase.

Ligand-directed reactions allow chemical transformations at very low reactant concentrations and can thus provide access to efficient approaches for the post-translational modification of proteins. The development of these proximity-induced reactions is hampered by the number of appropriate ligands and the lack of design principles. Addressing these limitations, we report a proximity-induced labeling system which applies a moderate affinity peptide ligand. The design process was structure-guided and supported by molecular dynamics simulations. We show that selective protein labeling can be performed inside living cells enabling the subcellular translocation of a protein via ligand-directed chemistry for the first time.

The acyl‐binding UNC119 proteins mediate the activation and transport of various N‐myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α‐helical peptides that target UNC119a. By using the hydrocarbon peptide‐stapling approach, cell‐permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis., Targeting molecular chaperones: Cell‐permeable peptidomimetics of a myristoylated peptide were obtained by hydrocarbon peptide stapling. These agents target the human acyl‐binding protein UNC119a, which plays a crucial role in cytokinesis, inducing the accumulation of cytokinetic and binucleated cells. This suggests UNC119a as a potential target for the inhibition of cytokinesis.

Most bacteria and archaea use similar proteins within their cell division machinery, which uses the tubulin homologue FtsZ as its central organiser. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. In biochemical assays we show that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the β domain of FtsQ shows that only residues 64-87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ:FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ:FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalising view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.ImportanceCells in most bacteria and archaea divide through a cell division process that is characterised through its filamentous organiser, FtsZ protein. FtsZ forms a ring structure at the division site and starts the recruitment of 10-20 downstream proteins that together form an elusive multi-protein complex termed divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.

Protein Engineering. In their Communication on page 11164 ff., T. N. Grossmann et al. report on the generation of a bicyclic enzyme with a triselectrophilic linker, which leads to strongly increased stability towards thermal denaturation.

Non‐natural oligonucleotides represent important (bio)chemical tools and potential therapeutic agents. Backbone modifications altering hybridization properties and biostability can provide useful analogues. Here, we employ an artificial nucleosyl amino acid (NAA) motif for the synthesis of oligonucleotides containing a backbone decorated with primary amines. An oligo‐T sequence of this cationic DNA analogue shows significantly increased affinity for complementary DNA. Notably, hybridization with DNA is still governed by Watson–Crick base pairing. However, single base pair mismatches are tolerated and some degree of sequence‐independent interactions between the cationic NAA backbone and fully mismatched DNA are observed. These findings demonstrate that a high density of positive charges directly connected to the oligonucleotide backbone can affect Watson–Crick base pairing. This provides a paradigm for the design of therapeutic oligonucleotides with altered backbone charge patterns.

Advances in protein engineering have made the industrial use of enzymes feasible, though, a more wide-spread application is hampered by the limited stability of enzymes under often required harsh conditions. We developed a protein stabilization technology that involves the in situ cyclization of proteins (INCYPRO) and facilitates the design and semi-synthesis of multicyclic enzymes. These synthetically enhanced durable enzymes add to the current efforts towards a sustainable and green economy.

PTEN is a dual-specificity protein tyrosine phosphatase. As one of the central tumor suppressors, a thorough regulation of its activity is essential for proper cellular homeostasis. The precise implications of PTEN inhibition by reactive oxygen species (e.g. H2 O2 ) and the subsequent structural consequences remain elusive. To study the effects of PTEN inhibition, bisperoxidovanadium (bpV) complexes serve as important tools with the potential for the treatment of nerve injury or cardiac ischemia. However, their mode of action is unknown, hampering further optimization and preventing therapeutic applications. Based on protein crystallography, mass spectrometry, and NMR spectroscopy, we elucidate the molecular basis of PTEN inhibition by H2O2 and bpV complexes. We show that both molecules inhibit PTEN via oxidative mechanisms resulting in the formation of the same intramolecular disulfide, therefore enabling the reactivation of PTEN under reductive conditions.

PTEN ist eine dualspezifische Protein-Tyrosin-Phosphatase. Sie ist einer der zentralen Tumorsuppressoren, weshalb eine genaue Regulierung ihrer Aktivitat essenziell fur die richtige zellulare Homoostase ist. Die genauen Auswirkungen der Inhibition von PTEN durch reaktive Sauerstoffspezies (z. B. H2O2) und deren strukturelle Folgen sind bisher wenig verstanden. Wichtige Instrumente fur das Studium der Wirkung von PTEN-Inhibition sind Bisperoxidovanadium(bpV)-Komplexe, mit dem Potenzial fur die Behandlung von Nervenverletzungen oder kardialer Ischamie. Allerdings ist ihr Wirkmechanismus unbekannt, was weitere Optimierungen und therapeutische Anwendungen erschwert. Mithilfe der Proteinkristallographie, Massenspektrometrie und NMR-Spektroskopie untersuchen wir hier die molekularen Grundlagen der PTEN-Inhibition durch H2O2 und bpV-Komplexe. Dabei zeigen wir, dass PTEN in beiden Fallen oxidativ inhibiert wird und sich die gleiche intramolekulare Disulfidbrucke bildet. Dies ermoglicht die Reaktivierung von PTEN unter reduzierenden Bedingungen.

Peptide-based drug discovery has experienced a significant upturn within the past decade since the introduction of chemical modifications and unnatural amino acids has allowed for overcoming some of the drawbacks associated with peptide therapeutics. Strengthened by such features, modified peptides become capable of occupying a niche that emerges between the two major classes of today's therapeutics-small molecules (500 Da) and biologics (5000 Da). Stabilized α-helices have proven particularly successful at impairing disease-relevant PPIs previously considered "undruggable." Among those, hydrocarbon stapled α-helical peptides have emerged as a novel class of potential peptide therapeutics. This review provides a comprehensive overview of the development and applications of hydrocarbon stapled peptides discussing the benefits and limitations of this technique.

Hydrocarbon-stapled α-helical peptides are a new class of targeting molecules capable of penetrating cells and engaging intracellular targets formerly considered intractable. This technology has been applied to the development of cell-permeable ligands targeting key intracellular protein–protein interactions. However, the properties governing cell penetration of hydrocarbon-stapled peptides have not yet been rigorously investigated. Herein we report our studies to systematically probe cellular uptake of stapled peptides. We developed a high-throughput epifluorescence microscopy assay to quantitatively measure stapled peptide intracellular accumulation and demonstrated that this assay yielded highly reproducible results. Using this assay, we analyzed more than 200 peptides with various sequences, staple positions and types, and found that cell penetration ability is strongly related to staple type and formal charge, whereas other physicochemical parameters do not appear to have a significant effect. We next investigated the mechanism(s) involved in stapled peptide internalization and have demonstrated that stapled peptides penetrate cells through a clathrin- and caveolin-independent endocytosis pathway that involves, in part, sulfated cell surface proteoglycans, but that also seems to exploit a novel, uncharacterized pathway. Taken together, staple type and charge are the key physical properties in determining the cell penetration ability of stapled peptides, and anionic cell surface proteoglycans might serve as receptors to mediate stapled peptide internalization. These findings improve our understanding of stapled peptides as chemical probes and potential targeted therapeutics, and provide useful guidelines for the design of next-generation stapled peptides with enhanced cell permeability.

Chemical modifications are used to stabilize bioactive conformations of peptides thereby increasing their target affinity and selectivity. Such modified peptides proved particularly useful as inhibitors of protein–protein interactions. Most of these strategies aim at the stabilization of α-helices and β-sheets. The use of peptides with irregular secondary structure is hampered by a lack of appropriate stabilization approaches. Herein, we highlight a recent contribution from our group that uses ring-closing metathesis for the macrocyclization of peptides with irregular secondary structure. The peptide precursors are cyclized on solid support to provide cross-linked architectures that bind the ­human protein 14-3-3 thereby inhibiting its interaction with virulence factor exoenzyme S from Pseudomonas aeruginosa.

Summary Objective The canonical Wnt signaling pathway has been shown to be involved in regulating chondrocyte hypertrophic differentiation during Osteoarthritis (OA). The aim of this study was to test the therapeutic potential of two stapled peptide canonical Wnt inhibitors – SAH-Bcl9 and StAx-35R – in preventing Wnt induced cartilage changes in OA. Methods Primary neonatal murine chondrocytes and cartilage explants from OA patients undergoing total joint replacement for knee OA, were used for microscopy to determine matrix and cell penetrating capacity of fluorescein isothiocyanate FITC-tagged SAH-Bcl9 and StAx-35R peptides. T cell factor/lymphoid enhancer-binding factor (TCF/LEF) reporter assays were used to monitor the inhibition of Wnt3a induced β-catenin signaling by each peptide. Changes in chondrocyte phenotypic marker gene expression were analyzed by qRT PCR. Results Both peptides localized intercellular in primary murine chondrocytes and cartilage explants. They inhibited Wnt3a induced TCF/LEF promoter activity in primary murine chondrocytes. Both inhibitors did not rescue Wnt3a altered expression of chondrocyte phenotypic genes (Sox9, Col2a1, Acan) and hypertrophy marker gene (Col10a1) at high doses (100 ng/ml). Upon application of 10 ng/ml Wnt3a, StAx-35R partially reversed the Wnt effect on Sox9 and Col2a1 gene expression. Both peptides, however, reversed the downregulation of SOX9 and aggrecan (ACAN), and decrease of COL10A1 gene expression in preserved human OA cartilage explants. Conclusion These data indicate that blockade of canonical Wnt signaling might be a therapeutic strategy to treat early OA cases and protect further cartilage degradation by preventing chondrocyte hypertrophic differentiation.

An ever increasing understanding of biological systems is providing a range of exciting novel biological targets whose modulation may enable novel therapeutic options in many diseases. These targets include protein-protein and protein-nucleic acid interactions, which are, however, often refractory to classical small molecule approaches. Other types of molecules, or modalities, are therefore required to address these targets, which has led several academic research groups and pharmaceutical companies to increasingly use the concept of so-called 'New Modalities'. This review defines for the first time the scope of this term, which includes novel peptidic scaffolds, oligonucleotides, hybrids, molecular conjugates as well as new uses of classical small molecules. We provide herein a journey through the most representative examples of these modalities to target large binding surface areas such as those found in protein-protein interactions and for biological processes at the center of cell regulation.

Constraining a peptide in its bioactive conformation by macrocyclization represents a powerful strategy to design modulators of challenging biomolecular targets. This holds particularly true for the development of inhibitors of protein-protein interactions which often involve interfaces lacking defined binding pockets. Such flat surfaces are demanding targets for traditional small molecules rendering macrocyclic peptides promising scaffolds for novel therapeutics. However, the contribution of peptide dynamics to binding kinetics is barely understood which impedes the design process. Herein, we report unexpected trends in the binding kinetics of two closely related macrocyclic peptides that bind their receptor protein with high affinity. Isothermal titration calorimetry, 19F NMR experiments and molecular dynamics simulations reveal that increased conformational flexibility of the macrocycle–receptor complex reduces dissociation rates and contributes to complex stability. This observation has impact on macrocycle design strategies that have so far mainly focused on the stabilization of bioactive ligand conformations.

Bioactive conformations of peptides can be stabilized by macrocyclization, resulting in increased target affinity and activity. Such macrocyclic peptides proved useful as modulators of biological functions, in particular as inhibitors of protein–protein interactions (PPI). However, most peptide-derived PPI inhibitors involve stabilized a-helices, leaving a large number of secondary structures unaddressed. Herein, we present a rational approach towards stabilization of an irregular peptide structure, using hydrophobic cross-links that replace residues crucially involved in target binding. The molecular basis of this interaction was elucidated by X-ray crystallography and isothermal titration calorimetry. The resulting cross-linked peptides inhibit the interaction between human adaptor protein 14-3-3 and virulence factor exoenzyme S. Taking into consideration that irregular peptide structures participate widely in PPIs, this approach provides access to novel peptide-derived inhibitors.

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Macrocyclization can be used to constrain peptides in their bioactive conformations, thereby supporting target affinity and bioactivity. In particular, for the targeting of challenging protein–protein interactions, macrocyclic peptides have proven to be very useful. Available approaches focus on the stabilization of α-helices, which limits their general applicability. Here we report for the first time on the use of ring-closing alkyne metathesis for the stabilization of an irregular peptide secondary structure. A small library of alkyne-crosslinked peptides provided a number of derivatives with improved target affinity relative to the linear parent peptide. In addition, we report the crystal structure of the highest-affinity derivative in a complex with its protein target 14-3-3ζ. It can be expected that the alkyne-based macrocyclization of irregular binding epitopes should give rise to new scaffolds suitable for targeting of currently intractable proteins. OA hybrid

Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets., Bicyclic peptides can inhibit biological targets hard to address with small molecules. Here, the authors combine two orthogonal ring-closing reactions to produce bicyclic peptides with improved bioactivity thereby providing a strategy that can greatly improve the structural diversity of such peptides.

The use of peptide nucleic acids (PNAs) is steadily increasing in biochemistry and diagnostics. So far, PNAs have mostly been investigated using cationic conditions in mass spectrometry. Furthermore, the use of fragmentation techniques developed for peptides and proteins like infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) has barely been examined. However, especially the fragmentation behavior of PNA oligomers in negative ion mode is of high importance, due to the ability to interact with nucleic acids which are almost exclusively analyzed in the negatively charged state. In the current study PNA fragmentations under cationic and anionic conditions were investigated and different fragmentation techniques like collision-induced dissociation (CID), IRMPD and ECD were applied. Especially when using CID and IRMPD, amide bonds were broken, whereas ECD resulted in the elimination of nucleobases. Differences were also observed between positive and negative ionization, while the sequence coverage for the negative ions was superior to positive ions. The fragmentation behavior using IRMPD led to almost complete sequence coverage. Additionally, in anions the interesting effect of multiple eliminations of HNCO was found.

The uncharged DNA-analogue peptide nucleic acid (PNA) can invade into dsDNA by displacing the non-complementary DNA strand. The formed strand displacement complexes can create a sterical hindrance to block access of enzymes such as nucleases and polymerases. Due to the high stability of DNA.PNA duplexes it is usually not possible to displace the PNA strand by ssDNA or ssRNA. We herein report that the polycationic, comb-type copolymer alphaPLL-g-Dex can induce such a replacement of PNA in DNA.PNA duplexes by ssDNA. The influence of the copolymer on strand exchange highly depends on the nature of the oligonucleotides. Acceleration has only been observed when both the starting duplex and the single-stranded exchanger strand were negatively charged. The presented approach should allow the withdrawal of PNA induced sterical hindrance of DNA by rehybridisation with ssDNA.

Protein–protein interactions (PPIs) are involved at all levels of cellular organization, thus making the development of PPI inhibitors extremely valuable. The identification of selective inhibitors is challenging because of the shallow and extended nature of PPI interfaces. Inhibitors can be obtained by mimicking peptide binding epitopes in their bioactive conformation. For this purpose, several strategies have been evolved to enable a projection of side chain functionalities in analogy to peptide secondary structures, thereby yielding molecules that are generally referred to as peptidomimetics. Herein, we introduce a new classification of peptidomimetics (classes A–D) that enables a clear assignment of available approaches. Based on this classification, the Review summarizes strategies that have been applied for the structure-based design of PPI inhibitors through stabilizing or mimicking turns, β-sheets, and helices.