Double Strain‐Promoted Macrocyclization for the Rapid Selection of Cell‐Active Stapled Peptides

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.     

Constrained Peptides with Target‐Adapted Cross‐Links as Inhibitors of a Pathogenic Protein–Protein Interaction

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 α‐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.     

Structure‐Based Design of Inhibitors of Protein–Protein Interactions: Mimicking Peptide Binding Epitopes

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.     

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” through the conventional small‐molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure–activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras‐binding and cell‐penetrating properties. These cell‐permeable cyclic peptides inhibit Ras signaling by binding to Ras‐GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein–protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.     

Probing the structural requirements of peptoids that inhibit HDM2-p53 interactions

Many cellular processes are controlled by protein-protein interactions, and selective inhibition of these interactions could lead to the development of new therapies for several diseases. In the area of cancer, overexpression of the protein, human double minute 2 (HDM2), which binds to and inactivates the protein p53, has been linked to tumor aggressiveness and drug resistance. In general, inhibition of protein-protein interactions with synthetic molecules is challenging and currently remains a largely uncharted area for drug development. One strategy to create inhibitors of protein-protein interactions is to recreate the three-dimensional arrangement of side chains that are involved in the binding of one protein to another, using a nonnatural scaffold as the attachment point for the side chains. In this study, we used oligomeric peptoids as the scaffold to begin to develop a general strategy in which we could rationally design synthetic molecules that can be optimized for inhibition of protein-protein interactions. Structural information on the HDM2-p53 complex was used to design our first class of peptoid inhibitors, and we provide here, in detail, the strategy to modify peptoids with the appropriate side chains that are effective inhibitors of HDM2-p53 binding. While we initially tried to develop rigid, helical peptoids as HDM2 binders, the best inhibitors were surprisingly peptoids that lacked any helix-promoting groups. These results indicate that starting with rigid peptoid scaffolds may not always be optimal to develop new inhibitors.     

An Epitope‐Imprinted Biointerface with Dynamic Bioactivity for Modulating Cell–Biomaterial Interactions

In this study, an epitope‐imprinting strategy was employed for the dynamic display of bioactive ligands on a material interface. An imprinted surface was initially designed to exhibit specific affinity towards a short peptide (i.e., the epitope). This surface was subsequently used to anchor an epitope‐tagged cell‐adhesive peptide ligand (RGD: Arg‐Gly‐Asp). Owing to reversible epitope‐binding affinity, ligand presentation and thereby cell adhesion could be controlled. As compared to current strategies for the fabrication of dynamic biointerfaces, for example, through reversible covalent or host–guest interactions, such a molecularly tunable dynamic system based on a surface‐imprinting process may unlock new applications in in situ cell biology, diagnostics, and regenerative medicine.     

Preparation of poly(styrene‐co‐acrylonitrile)‐grafted multiwalled carbon nanotubes via surface‐initiated atom transfer radical polymerization

The in situ grafting‐from approach via atom transfer radical polymerization was successfully applied to polystyrene, poly(styrene‐co‐acrylonitrile), and polyacrylonitrile grafted onto the convex surfaces of multiwalled carbon nanotubes (MWCNTs) with (2‐hydroxyethyl 2‐bromoisobutyrate) as an initiator. Thermogravimetric analysis showed that effective functionalization was achieved with the grafting approach. The grafted polymers on the MWCNT surface were characterized and confirmed with Fourier transform infrared spectroscopy and nuclear magnetic resonance. Raman and near‐infrared spectroscopy revealed that the grafting of polystyrene, poly(styrene‐co‐acrylonitrile), and polyacrylonitrile slightly affected the side‐wall structures. Field emission scanning electron microscopy showed that the carbon nanotube surface became rough because of the grafting of the polymers. Differential scanning calorimetry results indicated that the polymers grafted onto MWCNTs showed higher glass‐transition temperatures. The polymer‐grafted MWCNTs exhibited relatively good dispersibility in an organic solvent such as tetrahydrofuran. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 460–470, 2007     

Polyoxometalate Clusters Integrated into Peptide Chains and as Inorganic Amino Acids: Solution‐ and Solid‐Phase Approaches

General synthetic methods for the grafting of peptide chains onto polyoxometalate clusters by the use of general activated precursors have been developed. Using a solution‐phase approach, pre‐synthesized peptides can be grafted to a metal oxide cluster to produce hybrids of unprecedented scale (up to 30 residues). An adapted solid‐phase method allows the incorporation of these clusters, which may be regarded as novel hybrid unnatural amino acids, during the peptide synthesis itself. These methods may open the way for the automated synthesis of peptides and perhaps even proteins that contain “inorganic” amino acids.     

Ile‐Lys‐Val‐ala‐Val (IKVAV) peptide for neuronal tissue engineering

Despite the great advances in microsurgery, some neural injuries cannot be treated surgically. Stem cell therapy is a potential approach for treating neuroinjuries and neurodegenerative disease. Researchers have developed various bioactive scaffolds for tissue engineering, exhibiting enhanced cell viability, attachment, migration, neurite elongation, and neuronal differentiation, with the aim of developing functional tissue grafts that can be incorporated in vivo. Facilitating the appropriate interactions between the cells and extracellular matrix is crucial in scaffold design. Modification of scaffolds with biofunctional motifs such as growth factors, drugs, or peptides can improve this interaction. In this review, we focus on the laminin‐derived Ile‐Lys‐Val‐Ala‐Val peptide as a biofunctional epitope for neuronal tissue engineering. Inclusion of this bioactive peptide within a scaffold is known to enhance cell adhesion as well as neuronal differentiation in both 2‐dimensional and 3‐dimensional environments. The in vivo application of this peptide is also briefly described.     

Bicyclic stapled peptides based on p53 as dual inhibitors for the interactions of p53 with MDM2 and MDMX

In recent years, the strategy of inhibiting the interactions of p53 with murine double minute 2(MDM2)and murine double minute X(MDMX) has been proved to be a promising approach for tumor therapy.However, the poor proteolytical stability and low intracellular delivery efficiency of peptide inhibitors limit their clinical application. Here, we designed and synthesized the bicyclic stapled peptides based on p53 by combining all-hydrocarbon stapling and lactam stapling strategies. We demonstrated th...     

Terphenyl-Based Bak BH3 alpha-helical proteomimetics as low-molecular-weight antagonists of Bcl-xL

We describe a general method for the mimicry of one face of an alpha-helix based on a terphenyl scaffold that spatially projects functionality in a manner similar to that of two turns of an alpha-helix. The synthetic scaffold reduces the flexibility and molecular weight of the mimicked protein secondary structure. We have applied this design to the development of antagonists of the alpha-helix binding protein Bcl-x(L). Using a sequential synthetic strategy, we have prepared a library of terphenyl derivatives to mimic the helical region of the Bak BH3 domain that binds Bcl-x(L). Fluorescence polarization assays were carried out to evaluate the ability of terphenyl derivatives to displace the Bcl-x(L)-bound Bak peptide. Terphenyl 14 exhibited good in vitro affinity with a K(i) value of 0.114 muM. These terphenyl derivatives were more selective at disrupting the Bcl-x(L)/Bak over the HDM2/p53 interaction, which involves binding of the N-terminal alpha-helix of p53 to HDM2. Structural studies using NMR spectroscopy and computer-aided docking simulations suggested that the helix binding area on the surface of Bcl-x(L) is the target for the synthetic ligands. Treatment of human embryonic kidney 293 (HEK293) cells with terphenyl derivatives resulted in the disruption of the binding of Bcl-x(L) to Bax in intact cells.     

An Epitope‐Specific Respiratory Syncytial Virus Vaccine Based on an Antibody Scaffold

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in children. We have generated an epitope‐specific RSV vaccine by grafting a neutralizing epitope (F‐epitope) in its native conformation into an immunoglobulin scaffold. The resulting antibody fusion exhibited strong binding affinity to Motavizumab, an RSV neutralizing antibody, and effectively induced potent neutralizing antibodies in mice. This work illustrates the potential of the immunoglobulin molecule as a scaffold to present conformationally constrained B‐cell epitopes.     

An Optimised Small‐Molecule Stabiliser of the 14‐3‐3–PMA2 Protein–Protein Interaction

Modulation of protein–protein interactions (PPIs) is a highly demanding, but also a very promising approach in chemical biology and targeted drug discovery. In contrast to inhibiting PPIs with small, chemically tractable molecules, stabilisation of these interactions can only be achieved with complex natural products, like rapamycin, FK506, taxol, forskolin, brefeldin and fusicoccin. Fusicoccin stabilises the activatory complex of the plant H⁺‐ATPase PMA2 and 14‐3‐3 proteins. Recently, we have shown that the stabilising effect of fusicoccin could be mimicked by a trisubstituted pyrrolinone (pyrrolidone1, 1 ). Here, we report the synthesis, functional activity and crystal structure of derivatives of 1 that stabilise the 14‐3‐3–PMA2 complex. With a limited compound collection three modifications that are important for activity enhancement could be determined: 1) conversion of the pyrrolinone scaffold into a pyrazole, 2) introduction of a tetrazole moiety to the phenyl ring that contacts PMA2, and 3) addition of a bromine to the phenyl ring that exclusively contacts the 14‐3‐3 protein. The crystal structure of a pyrazole derivative of 1 in complex with 14‐3‐3 and PMA2 revealed that the more rigid core of this molecule positions the stabiliser deeper into the rim of the interface, enlarging especially the contact surface to PMA2. Combination of the aforementioned features gave rise to a molecule ( 37 ) that displays a threefold increase in stabilising the 14‐3‐3–PMA2 complex over 1 . Compound 37 and the other active derivatives show no effect on two other important 14‐3‐3 protein–protein interactions, that is, with CRaf and p53. This is the first study that describes the successful optimisation of a PPI stabiliser identified by screening.     

In Vivo Probe of Lipid II‐Interacting Proteins

β‐Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of penicillin‐binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP–Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d ‐amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.     

Bioorthogonal Probes for the Study of MDM2‐p53 Inhibitors in Cells and Development of High‐Content Screening Assays for Drug Discovery

To study the behavior of MDM2‐p53 inhibitors in a disease‐relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high‐content screening assays. By using automated image analysis with single‐cell resolution, we could visualize the intracellular target binding of compounds by co‐localization and quantify target upregulation upon MDM2‐p53 inhibition in an osteosarcoma model. Additionally, we developed a high‐throughput assay to quantify target occupancy of non‐tagged MDM2‐p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications.     

Grafting of polymers onto and/or from silica surface during the polymerization of vinyl monomers in the presence of γ‐ray‐irradiated silica

The effects of radicals on silica surface, which were formed by γ‐ray irradiation, on the polymerization of vinyl monomers were investigated. It was found that the polymerization of styrene was remarkably retarded in the presence of γ‐ray‐irradiated silica above 60 °C, at which thermal polymerization of styrene is readily initiated. During the polymerization, a part of polystyrene formed was grafted onto the silica surface but percentage of grafting was very small. On the other hand, no retardation of the polymerization of styrene was observed in the presence of γ‐ray‐irradiated silica below 50 °C; the polymerization tends to accelerate and polystyrene was grafted onto the silica surface. Poly(vinyl acetate) and poly(methyl methacrylate) (MMA) were also grafted onto the surface during the polymerization in the presence of γ‐ray‐irradiated silica. The grafting of polymers onto the silica surface was confirmed by thermal decomposition GC‐MS. It was considered that at lower temperature, the grafting based on the propagation of polystyrene from surface radical (“grafting from” mechanism) preferentially proceeded. On the contrary, at higher temperature, the coupling reaction of propagating polymer radicals with surface radicals (“grafting onto” mechanism) proceeded to give relatively higher molecular weight polymer‐grafted silica. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 2972–2979, 2006     

Active Targeting of Tumors through Conformational Epitope Imprinting

Inspired by the knowledge that most antibodies recognize a conformational epitope because of the epitope’s specific three‐dimensional shape rather than its linear structure, we combined scaffold‐based peptide design and surface molecular imprinting to fabricate a novel nanocarrier harboring stable binding sites that captures a membrane protein. In this study, a disulfide‐linked α‐helix‐containing peptide, apamin, was used to mimic the extracellular, structured N‐terminal part of the protein p32 and then serve as an imprinting template for generating a sub‐40 nm‐sized polymeric nanoparticle that potently binds to the target protein, recognizes p32‐positive tumor cells, and successfully mediates targeted photodynamic therapy in vivo. This could provide a promising alternative for currently used peptide‐modified nanocarriers and may have a broad impact on the development of polymeric nanoparticle‐based therapies for a wide range of human diseases.     

Stimulus‐Induced Conformational Transformation of a Cyclic Peptide for Selective Cell‐Targeting On–Off Gatekeeper for Mesoporous Nanocarriers

α_vβ₃ Integrin is upregulated on many cancer cells. We designed a dual functional cyclic peptide gatekeeper with a capability of stimuli‐responsive conformational transformation which could serve as a selective cell‐targeting on–off gatekeeper for mesoporous nanocarriers. The advantage of employing the motif of stimuli‐induced conformational transformation of cyclic peptides is that they could be utilized not only as an on–off gatekeeper for the triggered release of cargo drugs but also as a targeting ligand of the carriers to desired cells with their respective binding receptors. The peptide gatekeepers on the surface of nanocarriers exhibited on–off gatekeeping via conformational transformation triggered by intracellular glutathione levels of the cancer cells. The cyclic RGD sequence of the peptide gatekeepers enhanced the intracellular uptake into tumor cells (A549) and the therapeutic efficacy of the nanocarrier.     

Selective and Potent Proteomimetic Inhibitors of Intracellular Protein–Protein Interactions

Inhibition of protein–protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α‐Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed “proteomimetics”, which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N‐alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified.     

Molecular recognition of protein surfaces: high affinity ligands for the CBP KIX domain

Potent and specific inhibitors of protein.protein interactions have potential both as therapeutic compounds and biological tools, yet discovery of such molecules remains a challenge. Our laboratory has recently described a strategy, called protein grafting, for the identification of miniature proteins that bind protein surfaces with high affinity and specificity and inhibit the formation of protein.protein complexes. In protein grafting, those residues that comprise a functional alpha-helical binding epitope are stabilized on the solvent-exposed alpha-helical face of the small yet stable protein avian pancreatic polypeptide (aPP). Here we use protein grafting in combination with molecular evolution by phage display to identify phosphorylated peptide ligands that recognize the shallow surface of CBP KIX with high nanomolar to low micromolar affinity. Furthermore, we show that grafting of the CBP KIX-binding epitope of CREB KID onto the aPP scaffold yields molecules capable of high affinity recognition of CBP KIX even in the absence of phosphorylation. Importantly, both classes of designed ligands exhibit high specificity for the target CBP KIX domain over carbonic anhydrase and calmodulin, two unrelated proteins that bind hydrophobic or alpha-helical molecules that might be encountered in vivo.